Biochimica et Biophysica Acta

A second class of nuclear receptors for oxysterols: Regulation of RORalpha and RORgamma activity by 24S-Hydroxycholesterol (Cerebrosterol).

Publication Date: 2010 Mar 4 PMID: 20211758
Authors: Wang, Y. - Kumar, N. - Crumbley, C. - Griffin, P. R. - Burris, T. P.
Journal: Biochim Biophys Acta

The retinoic acid receptor-related orphan receptor alpha and gamma (RORalpha [NR1F1] and RORgamma [NR1F3]) are members of the nuclear hormone receptor superfamily. These 2 receptors regulate many physiological processes including development, metabolism and immunity. We recently found that certain oxysterols, namely the 7-substituted oxysterols, bound to the ligand binding domains (LBDs) of RORalpha and RORgamma with high affinity, altered the LBD conformation and reduced coactivator binding resulting in suppression of the constitutive transcriptional activity of these two receptors. Here, we show that another oxysterol, 24S-hydroxycholesterol (24S-OHC), is also a high affinity ligand for RORalpha and RORgamma (K(i) approximately 25nM). 24S-OHC is also known as cerebrosterol due to its high level in the brain where it plays an essential role as an intermediate in cholesterol elimination from the CNS. 24S-OHC functions as a RORalpha/gamma inverse agonist suppressing the constitutive transcriptional activity of these receptors in cotransfection assays. Additionally, 24S-OHC suppressed the expression of several RORalpha target genes including BMAL1 and REV-ERBalpha in a ROR-dependent manner. We also demonstrate that 24S-OHC decreases the ability of RORalpha to recruit the coactivator SRC-2 when bound to the BMAL1 promoter. We also noted that 24(S), 25-epoxycholesterol selectively suppressed the activity of RORgamma. These data indicate that RORalpha and RORgamma may serve as sensors of oxsterols. Thus, RORalpha and RORgamma display an overlapping ligand preference with another class of oxysterol nuclear receptors, the liver X receptors (LXRalpha [NR1H3] and LXRbeta [NR1H2]).

post to: CiteULike

Docosahexaenoic acid and synaptic protection in Alzheimer's disease mice.

Publication Date: 2010 Mar 4 PMID: 20211757
Authors: Oster, T. - Pillot, T.
Journal: Biochim Biophys Acta

Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Amyloid-beta (Abeta) oligomers are considered the proximate effectors in the early stages of AD. AD-related cognitive impairment, synaptic loss and neurodegeneration result from interactions of Abeta oligomers with the synaptic membrane and subsequent activation of pro-apoptotic signalling pathways. Therefore, membrane structure and lipid status appear determinant in Abeta-induced toxicity. Numerous epidemiological studies have highlighted the beneficial influence of docosahexaenoic acid (DHA, C22:6 n-3) on the preservation of synaptic function and memory capacities in aged individuals or upon Abeta exposure, whereas its deficiency is presented as a risk factor for AD. An elevated number of studies have been reporting the beneficial effects of dietary DHA supplementation on cognition and synaptic integrity in various AD models. In this review, we describe the important potential of DHA to preserve neuronal and brain functions and classified its numerous molecular and cellular effects from impact on membrane lipid content and organisation to activation of signalling pathways sustaining synaptic function and neuronal survival. DHA appears as one of the most valuable diet ingredients whose neuroprotective properties could be crucial for designing nutrition-based strategies able to prevent AD as well as other lipid- and age-related diseases whose prevalence is progressing in elderly populations.

post to: CiteULike

Signaling mechanisms that link salt retention to hypertension: Endogenous ouabain, the Na(+) Pump, the Na(+)/Ca(2+) exchanger and TRPC proteins.

Publication Date: 2010 Mar 4 PMID: 20211726
Authors: Blaustein, M. P. - Hamlyn, J. M.
Journal: Biochim Biophys Acta

Salt retention as a result of chronic, excessive dietary salt intake, is widely accepted as one of the most common causes of hypertension. In a small minority of cases, enhanced Na(+) reabsorption by the kidney can be traced to specific genetic defects of salt transport, or pathological conditions of the kidney, adrenal cortex, or pituitary. Far more frequently, however, the salt retention may be the result of minor renal injury or small genetic variation in renal salt transport mechanisms. How the salt retention actually leads to the increase in peripheral vascular resistance (the hallmark of hypertension) and the elevation of blood pressure remain an enigma. Here we review the evidence that endogenous ouabain (an adrenocortical hormone), arterial smooth muscle alpha2 Na(+) pumps, type-1 Na/Ca exchangers, and receptor- and store-operated Ca(2+) channels play key roles in the pathway that links salt to hypertension. We discuss cardenolide structure-function relationships in an effort to understand why prolonged administration of ouabain, but not digoxin, induces hypertension, and why digoxin is actually anti-hypertensive. Finally, we summarize recent observations which indicate that ouabain upregulates arterial myocyte Ca(2+) signaling mechanisms that promote vasoconstriction, while simultaneously downregulating endothelial vasodilator mechanisms. In sum, the reports reviewed here provide novel insight into the molecular mechanisms by which salt retention leads to hypertension.

post to: CiteULike

Sir2-dependent asymmetric segregation of damaged proteins in ubp10 null mutants is independent of genomic silencing.

Publication Date: 2010 Mar 4 PMID: 20211662
Authors: Orlandi, I. - Bettiga, M. - Alberghina, L. - Nystrom, T. - Vai, M.
Journal: Biochim Biophys Acta

Carbonylation of proteins is irreversible oxidative damage that increases during both chronological and replicative yeast aging. In the latter, a spatial protein quality control system that relies on Sir2 is responsible for the asymmetrical damage segregation in the mother cells. Proper localization of Sir2 on chromatin depends on the deubiquitinating enzyme Ubp10, whose loss of function deeply affects the recombination and gene-silencing activities specific to Sir2. Here, we have analyzed the effects of SIR2 and UBP10 inactivations on carbonylated proteins patterns obtained in two aging models such as stationary phase cells and size-selected old mother ones. In line with the endogenous situation of higher oxidative stress resulting from UBP10 inactivation, an increase of protein carbonylation has been found in the ubp10 stationary phase cells compared with sir2 ones. Moreover, Calorie Restriction had a salutary effect for both mutants by reducing carbonylated proteins accumulation. Remarkably, in the replicative aging model, whereas SIR2 inactivation resulted in a failure to establish damage asymmetry, the Sir2-dependent damage inheritance is maintained in the ubp10 mutant which copes with the increased oxidative damage by retaining it in the mother cells. This indicates that both Ubp10 and a correct association of Sir2 with the silenced chromatin are not necessary in such a process but also suggests that additional Sir2 activities on non-chromatin substrates are involved in the establishment of damage asymmetry.

post to: CiteULike

Identification of two distinct cell binding sequences in the vitamin d binding protein.

Publication Date: 2010 Mar 4 PMID: 20211661
Authors: Zhang, J. - Habiel, D. M. - Ramadass, M. - Kew, R. R.
Journal: Biochim Biophys Acta

The vitamin D binding protein (DBP) is a multifunctional, albumin-like plasma protein that often requires cell surface binding to mediate some of its diverse functions. DBP binds to several different molecules on the external face of the plasma membrane indicating that it may possess distinct cell binding sequences. In this report, surface plasmon resonance was utilized to evaluate the relative binding of the human myeloid cell line U937 to immobilized recombinant expressed DBP in order to identify cell localization sequences. U937 cells showed robust binding to immobilized native DBP, but essentially no interaction when sensor chips were coated with b(2)-microglobulin or BSA. The cell-DBP interaction was completely eliminated if cells were pretreated with soluble DBP. Recombinant DBP domains and truncated domains were next evaluated to determine the location of cell binding regions. Domains I (amino acids 1-191) and III (379-458), but not domain II (192-378), could support cell binding. Further evaluation of domain I, using truncated proteins and overlapping peptides, demonstrated that a single amino acid sequence, residues 150-172 (NYGQAPLSLLVSYTKSYLSMVGS), mediated cell binding. The domain III cell binding region was investigated using truncated versions of domain III fused to full-length domain II that served as a scaffold. These experiments indicated that the cell binding sequence is located in the first portion of that domain (379-402: ELSSFIDKGQELCADYSENTFTEY). Overlapping peptides spanning this sequence could partially block cell binding only when used in combination. We conclude that DBP contains two cell localization sequences that may be required for some of the multiple functions of this protein.

post to: CiteULike

Colocalization of mineralocorticoid and EGF receptor at the plasma membrane.

Publication Date: 2010 Mar 4 PMID: 20211660
Authors: Grossmann, C. - Husse, B. - Mildenberger, S. - Schreier, B. - Schuman, K. - Gekle, M.
Journal: Biochim Biophys Acta

The mineralocorticoid receptor (MR), a ligand-activated transcription factor expressed in various cell types (e.g. epithelial cells, neurons, smooth muscle cells, immune cells), plays important roles in neurohumoral, neuronal, cardiovascular, renal and intestinal function. Pathophysiological relevant signaling mechanisms include nongenomic pathways involving the EGF receptor (EGFR). We investigated whether a MR-EGFR colocalization may underlie the functional MR-EGFR interaction by coimmunoprecipitation, fluorescence resonance energy transfer (FRET) and confocal microscopy in a heterologous expression system. EGFR and a small fraction of MR colocalize at the cell membrane, independently of short time exposure (
post to: CiteULike

Sodium-coupled electrogenic transport of pyroglutamate (5-oxoproline) via SLC5A8, a monocarboxylate transporter.

Publication Date: 2010 Mar 4 PMID: 20211600
Authors: Miyauchi, S. - Gopal, E. - Babu, E. - Srinivas, S. R. - Kubo, Y. - Umapathy, N. S. - Thakkar, S. V. - Ganapathy, V. - Prasad, P. D.
Journal: Biochim Biophys Acta

Pyroglutamate, also known as 5-oxoproline, is a structural analog of proline. This amino acid derivative is a byproduct of glutathione metabolism, and is reabsorbed efficiently in kidney by Na(+)-coupled transport mechanisms. Previous studies have focused on potential participation of amino acid transport systems in renal reabsorption of this compound. Here we show that it is not the amino acid transport systems but instead the Na(+)-coupled monocarboxylate transporter SLC5A8 that plays a predominant role in this reabsorptive process. Expression of cloned human and mouse SLC5A8 in mammalian cells induces Na(+)-dependent transport of pyroglutamate that is inhibitable by various SLC5A8 substrates. SLC5A8-mediated transport of pyroglutamate is saturable with a Michaelis constant of 0.36+/-0.04mM. Na(+)-activation of the transport process exhibits sigmoidal kinetics with a Hill co-efficient of 1.8+/-0.4, indicating involvement of more than one Na(+) in the activation process. Expression of SLC5A8 in X. laevis oocytes induces Na(+)-dependent inward currents in the presence of pyroglutamate under voltage-clamp conditions. The concentration of pyroglutamate necessary for induction of half-maximal current is 0.19+/-0.01mM. The Na(+)-activation kinetics is sigmoidal with a Hill coefficient of 2.3+/-0.2. Ibuprofen, a blocker of SLC5A8, suppressed pyroglutamate-induced currents in SLC5A8-expressing oocytes; the concentration of the blocker necessary for causing half-maximal inhibition is 14+/-1muM. The involvement of SLC5A8 can be demonstrated in rabbit renal brush border membrane vesicles by showing that the Na(+)-dependent uptake of pyroglutamate in these vesicles is inhibitable by known substrates of SLC5A8. The Na(+) gradient-driven pyroglutamate uptake was stimulated by an inside-negative K(+) diffusion potential induced by valinomycin, showing that the uptake process is electrogenic.

post to: CiteULike

Protein-protein and protein-lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles.

Publication Date: 2010 Mar 4 PMID: 20211599
Authors: Kahya, N.
Journal: Biochim Biophys Acta

Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid-lipid and lipid-protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains.

post to: CiteULike

Genotype-phenotype correlations in Leber Hereditary Optic Neuropathy.

Publication Date: 2010 Mar 4 PMID: 20211598
Authors: Tonska, K. - Kodron, A. - Bartnik, E.
Journal: Biochim Biophys Acta

Leber hereditary optic neuropathy (LHON), acute or subacute vision loss due to retinal ganglion cell death which in the long run leads to optic nerve atrophy is one of the most widely studied maternally inherited diseases caused by mutations in mitochondrial DNA. Although three common mutations, 11778G>A, 14484T>C or 3460G>A are responsible for over 90 per cent of cases and affect genes encoding complex I subunits of the respiratory chain, their influence on bioenergetic properties of the cell is marginal and cannot fully explain the pathology of the disease. The following chain of events was proposed, based on biochemical and anatomical properties of retinal ganglion cells whose axons form the optic nerve: mitochondrial DNA mutations increase reactive oxygen species production in these sensitive cells, leading to caspase independent apoptosis. As LHON is characterized by low penetrance and sex bias (men are affected about 5 times more frequently than women) the participation of the other factors - genetic and environmental - beside mtDNA mutations was studied. Mitochondrial haplogroups and smoking are some of the factors involved in the complex etiology of this disease.

post to: CiteULike

Human Mitochondrial mRNAs - Like Members of all Families, Similar but Different.

Publication Date: 2010 Mar 4 PMID: 20211597
Authors: Temperley, R. J. - Wydro, M. - Lightowlers, R. N. - Chrzanowska-Lightowlers, Z. M.
Journal: Biochim Biophys Acta

The messenger RNAs containing the thirteen protein coding sequences of the human mitochondrial genome have frequently been regarded as a single functional category, alike in arrangement and hence in mode of expression. The 'generic' mitochondrial mRNA is perceived as having i) an arrangement within the polycistronic unit that permits its liberation following mt-tRNA processing, ii) no 5'cap structure or introns, iii) essentially no untranslated regions, and iv) a poly(A) tail of approximately fifty nucleotides that is required in part to complete the termination codon. Closer inspection reveals that only two molecules fit this pattern. This article examines the extent to which human mitochondrial mRNA species differ from one another.

post to: CiteULike

The regulation and turnover of mitochondrial uncoupling proteins.

Publication Date: 2010 Mar 4 PMID: 20211596
Authors: Azzu, V. - Jastroch, M. - Divakaruni, A. S. - Brand, M. D.
Journal: Biochim Biophys Acta

Uncoupling proteins (UCP1, UCP2 and UCP3) are important in regulating cellular fuel metabolism and as attenuators of reactive oxygen species production, through strong or mild uncoupling. The generic function and broad tissue distribution of the uncoupling protein family means that they are increasingly implicated in a range of pathophysiological processes including obesity, insulin resistance and diabetes mellitus, neurodegeneration, cardiovascular disease, immunity and cancer. The significant recent progress describing the turnover of novel uncoupling proteins, as well as current views on the physiological roles and regulation of UCPs, is outlined.

post to: CiteULike

Plasticity of the mitoproteome to nitrogen sources (nitrate and ammonium) in Chlamydomonas reinhardtii: The logic of Aox1 gene localization.

Publication Date: 2010 Mar 4 PMID: 20211595
Authors: Gerin, S. - Mathy, G. - Blomme, A. - Franck, F. - Sluse, F. E.
Journal: Biochim Biophys Acta

Nitrate and ammonium constitute primary inorganic nitrogen sources that can be incorporated into carbon skeletons in photosynthetic eukaryotes. In Chlamydomonas, previous studies and the present one showed that the mitochondrial AOX is up-regulated in nitrate-grown cells in comparison with ammonium-grown cells. In this work we have performed a comparative proteomic analysis of the soluble mitochondrial proteome of Chlamydomonas cells growth either on nitrate or ammonium. Our results highlight important proteomics modifications mostly related to primary metabolism in cells grown on nitrate. We could note an up-regulation of some TCA cycle enzymes and a down-regulation of cytochrome c(1) together with an up-regulation of L-arginine and purine catabolism enzymes and of ROS scavenging systems. Hence, in nitrate-grown cells, AOX may play a dual role: (1) lowering the ubiquinone pool reduction level and (2) permitting the export of mitochondrial reducing power under the form of malate for nitrate and nitrite reduction. This role of AOX in the mitochondrial plasticity makes logical the localization of Aox1 in a nitrate assimilation gene cluster.

post to: CiteULike

Complex III-dependent superoxide production of brain mitochondria contributes to seizure-related ROS formation.

Publication Date: 2010 Mar 5 PMID: 20211146
Authors: Malinska, D. - Kulawiak, B. - Kudin, A. P. - Kovacs, R. - Huchzermeyer, C. - Kann, O. - Szewczyk, A. - Kunz, W. S.
Journal: Biochim Biophys Acta

Brain seizure activity is characterised by intense activation of mitochondrial oxidative phosphorylation. This stimulation of oxidative phosphorylation is in the low magnesium model of seizure-like events accompanied by substantial increase in formation of reactive oxygen species (ROS). However, it has remained unclear which ROS-generating sites can be attributed to this phenomenon. Here, we report stimulatory effects of calcium ions and uncouplers, mimicking mitochondrial activation, on ROS generation of isolated rat and mouse brain mitochondria. Since these stimulatory effects were visible with superoxide sensitive dyes, but with hydrogen peroxide sensitive dyes only in the additional presence of SOD, we conclude that the complex redox properties of the 'Qo' center at respiratory chain complex III are very likely responsible for these observations. In accordance with this hypothesis redox titrations of the superoxide production of antimycin-inhibited submitochondrial particles with the succinate/fumarate redox couple confirmed for brain tissue a bell shaped dependency with a maximal superoxide production rate at +10 mV (pH=7.4). This reflects the complex redox properties of a semiquinone species which is the direct electron donor for oxygen reduction in complex III-dependent superoxide production. Therefore, we conclude that under conditions of increased energy load the complex III site can contribute to superoxide production of brain mitochondria, which might be relevant for epilepsy-related seizure activity.

post to: CiteULike

A surface view on membrane structure, dynamics and applications.

Publication Date: 2010 Apr PMID: 20206732
Authors: Perez-Gil, J.
Journal: Biochim Biophys Acta

post to: CiteULike

Differences in RNA processing underlie the tissue specific phenotype of ISCU myopathy.

Publication Date: 2010 Mar 3 PMID: 20206689
Authors: Sanaker, P. S. - Toompuu, M. - Hogan, V. E. - He, L. - Tzoulis, C. - Chrzanowska-Lightowlers, Z. M. - Taylor, R. W. - Bindoff, L. A.
Journal: Biochim Biophys Acta

Hereditary myopathy with lactic acidosis, or Myopathy with exercise intolerance, Swedish type (OMIM #255125) is caused by mutations in the iron-sulfur cluster scaffold (ISCU) gene. The g.7044G>C ISCU mutation induces a splicing error in the pre-mRNA that strengthens a weak intronic splice site leading to inclusion of a new exon and subsequent loss of mRNA and protein. While ISCU is widely expressed, homozygosity for this particular intronic mutation gives rise to a pure myopathy. In order to investigate tissue specificity and disease mechanism, we studied muscle, myoblasts, fibroblasts and blood cells from the first non-Swedish case of this disease. Consistent with the recognised role of ISCU, we found abnormal activities of respiratory chain complexes containing iron-sulfur clusters in patient muscle. We confirmed that, in the presence of the g.7044G>C mutation, splicing produces both abnormally and normally spliced mRNA in all tissues. The ratio of these products varies dramatically between tissues, being most abnormal in mature skeletal muscle that also has the lowest relative starting levels of ISCU mRNA compared with other tissues. Myoblasts and fibroblasts have more of the normally spliced variant as well as higher starting levels of ISCU mRNA. Up-regulation of mtDNA copy number was found in skeletal muscle and myoblasts, but not fibroblasts, and is thought to represent a compensatory response. Tissue specificity in this disorder appears therefore to be dependant on the mRNA starting level, the amount of remaining normally spliced RNA, and the degree to which compensatory mechanisms can respond.

post to: CiteULike

Modulation of intracellular chloride channels by ATP and Mg(2+).

Publication Date: 2010 Mar 3 PMID: 20206596
Authors: Kominkova, V. - Malekova, L. - Tomaskova, Z. - Slezak, P. - Szewczyk, A. - Ondrias, K.
Journal: Biochim Biophys Acta

We report the effects of ATP and Mg(2+) on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n=112) possessed wide variation in single channel parameters and sensitivities to ATP. ATP (0.5-2mmol/l) modulated and/or inhibited the chloride channel activities (n=38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n=5/93) or reversible (n=15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n=6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5-2mmol/l) without the presence of Mg(2+) decreased the chloride channel current (n=12/14), whereas Mg(2+) significantly reversed the effect (n=4/4). We suggest that ATP-intracellular chloride channel interactions and Mg(2+) modulation of these interactions may regulate different physiological and pathological processes.

post to: CiteULike

The alternative complex iii of Rhodothermus marinus and its structural and functional association with caa(3) oxygen reductase.

Publication Date: 2010 Mar 3 PMID: 20206595
Authors: Refojo, P. N. - Teixeira, M. - Pereira, M. M.
Journal: Biochim Biophys Acta

An alternative complex III (ACIII) is a respiratory complex with quinol: electron acceptor oxidoreductase activity. It is the only example of an enzyme performing complex III function that does not belong to bc(1) complex family. ACIII from Rhodothermus (R.) marinus was the first enzyme of this type to be isolated and characterized, and in this work we deepen its characterization. We addressed its interaction with quinol substrate and with the caa(3) oxygen reductase, whose coding gene cluster follows that of the ACIII. There is at least, one quinone binding site present in R. marinus ACIII as observed by fluorescence quenching titration of HQNO, a quinone analogue inhibitor. Furthermore, electrophoretic and spectroscopic evidence, taken together with mass spectrometry revealed a structural association between ACIII and caa(3) oxygen reductase. The association was also shown to be functional, since quinol: oxygen oxidoreductase activity was observed when the two isolated complexes were put together. This work is thus a step forward in the recognition of the structural and functional diversities of prokaryotic respiratory chains.

post to: CiteULike

Quinolinate phosphoribosyl transferase, a key enzyme in de novo NAD(+) synthesis, suppresses spontaneous cell death by inhibiting overproduction of active-caspase-3.

Publication Date: 2010 Mar 2 PMID: 20206212
Authors: Ishidoh, K. - Kamemura, N. - Imagawa, T. - Oda, M. - Sakurai, J. - Katunuma, N.
Journal: Biochim Biophys Acta

Quinolinate phosphoribosyl transferase (QPRT) is a key enzyme in de novo NAD(+) synthesis. QPRT enzyme activity has a restricted tissue distribution, although QPRT mRNA is expressed ubiquitously. This study was designed to elucidate the functions of QPRT protein in addition to NAD(+) synthesis. QPRT was identified as a caspase-3 binding protein using double layer fluorescent zymography, but was not a substrate for caspase-3. Surface plasmon resonance analysis using recombinant proteins showed interaction of QPRT with active-caspase-3 in a dose dependent manner at 55 nM of the dissociation constant. The interaction was also confirmed by immunoprecipitation analysis of actinomycin D-treated QPRT-FLAG expressing cells using anti-FLAG-agarose. QPRT-depleted cells showed increased sensitivity to spontaneous cell death, upregulated caspase-3 activity and strong active-caspase-3 signals. Considered together, the results suggested that QPRT protein acts as an inhibitor of spontaneous cell death by suppressing overproduction of active-caspase-3.

post to: CiteULike

Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice.

Publication Date: 2010 Mar 2 PMID: 20206124
Authors: Bevilacqua, L. - Seifert, E. L. - Estey, C. - Gerrits, M. F. - Harper, M. E.
Journal: Biochim Biophys Acta

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocas to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT mediated 'energy-wasting' process during CR.

post to: CiteULike

Oscillations in energy metabolism.

Publication Date: 2010 Mar 2 PMID: 20206123
Authors: Iotti, S. - Borsari, M. - Bendahan, D.
Journal: Biochim Biophys Acta

Organisation of mitochondrial metabolism is a quintessential example of a complex dissipative system which can display dynamic instabilities. Several findings have indicated that the conditions inducing instabilities are within the physiological range and that mild perturbations could elicit oscillations. Different mathematical models have been put forth in order to explain the genesis of oscillations in energy metabolism. One model considers mitochondria as an organised network of oscillators and indicates that communication between mitochondria involves mitochondrial reactive oxygen species (ROS) production acting as synchronisers of the energy status of the whole population of mitochondria. An alternative model proposes that extra-mitochondrial pH variations could lead to mitochondrial oscillations. Oscillatory phenomena in energy metabolism have also been investigated in vivo on the basis of (31)P magnetic resonance spectroscopy (MRS) measurements of phosphocreatine post-exercise recovery in human and animal skeletal muscle. The corresponding results provide experimental evidences about the role exerted by cytosolic pH on oscillations. Finally a new simple non linear mathematical model describing the overall chemical reaction of phosphocreatine recovery predicting oscillatory recovery pattern under certain experimental conditions is presented and discussed in the light of the experimental results reported so far.

post to: CiteULike

Substitution of Chloride by Bromide Modifies the Low-Temperature Tyrosine Z Oxidation in Active Photosystem II.

Publication Date: 2010 Mar 2 PMID: 20206122
Authors: Ren, Y. - Zhang, C. - Zhao, J.
Journal: Biochim Biophys Acta

Chloride is an essential cofactor for photosynthetic water oxidation. However, its location and functional roles in active photosystem II are still a matter of debate. We have investigated this issue by studying the effects of Cl(-) replacement by Br(-) in active PSII. In Br(-) substituted samples, Cl(-) is effectively replaced by Br(-) in the presence of 1.2 M NaBr under room light with protection of anaerobic atmosphere followed by dialysis. Following results have been obtained. i) The oxygen evolving activities of the Br(-) -PSII samples are significantly lower than that of the Cl(-) -PSII samples; ii) The same S(2) multiline EPR signals are observed in both Br(-) and Cl(-) -PSII samples; iii) The amplitudes of the visible light induced S(1)Tyr(Z)() and S(2)Tyr(Z)() EPR signals are significantly decreased after Br(-) substitution; the S(1)Tyr(Z)() EPR signal is up-shifted about 8 Gauss, whereas the S(2)Tyr(Z)() signal is down-shifted about 12 Gauss after Br- substitution. These results imply that the redox properties of Tyr(Z) and spin interactions between Tyr(Z)() and Mn-cluster could be significantly modified due to Br(-) substitution. It is suggested that Cl(-)/Br(-) probably coordinates to the Ca(2+) ion of the Mn-cluster in active photosystem II.

post to: CiteULike

Modulation of transcription factor function by O-GlcNAc modification.

Publication Date: 2010 Mar 1 PMID: 20202486
Authors: Ozcan, S. - Andrali, S. S. - Cantrell, J. E.
Journal: Biochim Biophys Acta

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytoplasmic proteins is important for many cellular processes, and the number of proteins that contain this modification is steadily increasing. This modification is dynamic and reversible, and in some cases competes for phosphorylation of the same residues. O-GlcNAc modification of proteins is regulated by cell cycle, nutrient metabolism, and other extracellular signals. Compared to protein phosphorylation, which is mediated by a large number of kinases, O-GlcNAc modification is catalyzed only by one enzyme called O-linked N-acetylglucosaminyl transferase or OGT. Removal of O-GlcNAc from proteins is catalyzed by the enzyme beta-N-acetylglucosaminidase (O-GlcNAcase or OGA). Altered O-linked GlcNAc modification levels contribute to the establishment of many diseases, such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Many transcription factors have been shown to be modified by O-linked GlcNAc modification, which can influence their transcriptional activity, DNA binding, localization, stability, and interaction with other co-factors. This review focuses on modulation of transcription factor function by O-linked GlcNAc modification.

post to: CiteULike

Involvement of ADAMs in tumorigenesis and progression of hepatocellular carcinoma: Is it merely fortuitous or a real pathogenic link?

Publication Date: 2010 Mar 1 PMID: 20197081
Authors: Mazzocca, A. - Giannelli, G. - Antonaci, S.
Journal: Biochim Biophys Acta

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and one of the most frequent types of cancer worldwide. It normally develops in patients with chronic liver disease, especially cirrhosis, although some cases without an apparent underlying liver disease have been reported. The pathogenesis of HCC is multi-factorial and complex. Hepatitis viruses are the main factors favoring the development of HCC. In fact, chronic inflammation associated with hepatitis C or B virus infection can lead to progressive liver fibrosis, cirrhosis and ultimately HCC. Chronic inflammation and liver fibrosis cause a continuous remodeling of the extracellular matrix (ECM), a dynamic process that involves several molecules including integrins and matrix processing enzymes. An increasing body of evidence indicates that ADAMs are involved in promoting tumor formation and progression of HCC. A Disintegrin And Metalloproteases (ADAMs) are a group of proteins belonging to the zinc protease superfamily. ADAMs are usually transmembrane proteins that contain disintegrin and metalloprotease domains and are, therefore, able to carry out both cell adhesion and protease activities. Soluble isoforms of ADAMs have also been discovered and characterized. In this review, we focus on the contribution of ADAM proteins to HCC tumorigenesis and cancer progression. The potential role of ADAMs as key modulators of tumor-stroma interactions during tumor progression, by means of the activities of their constituent domains, is also discussed.

post to: CiteULike

The role of Cys108 in Trigonopsis variabilisd-amino acid oxidase examined through chemical oxidation studies and point mutations C108S and C108D.

Publication Date: 2010 Mar 1 PMID: 20193780
Authors: Mueller, M. - Kratzer, R. - Schiller, M. - Slavica, A. - Rechberger, G. - Kollroser, M. - Nidetzky, B.
Journal: Biochim Biophys Acta

Oxidative modification of Trigonopsis variabilisd-amino acid oxidase in vivo is traceable as the conversion of Cys108 into a stable cysteine sulfinic acid, causing substantial loss of activity and thermostability of the enzyme. To simulate native and modified oxidase each as a microheterogeneity-resistant entity, we replaced Cys108 individually by a serine (C108S) and an aspartate (C108D), and characterized the purified variants with regard to their biochemical and kinetic properties, thermostability, and reactivity towards oxidation by hypochlorite. Tandem MS analysis of tryptic peptides derived from a hypochlorite-treated inactive preparation of recombinant wild-type oxidase showed that Cys108 was converted into cysteine sulfonic acid, mimicking the oxidative modification of native enzyme as isolated. Colorimetric titration of protein thiol groups revealed that in the presence of ammonium benzoate (0.12mM), the two mutants were not oxidized at cysteines whereas in the wild-type enzyme, one thiol group was derivatized. Each site-directed replacement caused a conformational change in d-amino acid oxidase, detected with an assortment of probes, and resulted in a turnover number for the O(2)-dependent reaction with D-Met which in comparison with the corresponding wild-type value was decreased two- and threefold for C108S and C108D, respectively. Kinetic analysis of thermal denaturation at 50 degrees C was used to measure the relative contributions of partial unfolding and cofactor dissociation to the overall inactivation rate in each of the three enzymes. Unlike wild-type, C108S and C108D released the cofactor in a quasi-irreversible manner and were therefore not stabilized by external FAD against loss of activity. The results support a role of the anionic side chain of Cys108 in the fine-tuning of activity and stability of d-amino acid oxidase, explaining why C108S was a surprisingly poor mimic of the native enzyme.

post to: CiteULike

Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient's primary cells.

Publication Date: 2010 Mar 1 PMID: 20193760
Authors: Foltopoulou, P. F. - Tsiftsoglou, A. S. - Bonovolias, I. D. - Ingendoh, A. T. - Papadopoulou, L. C.
Journal: Biochim Biophys Acta

Mutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with l-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origins (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) l-arginine solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [(35)S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders.

post to: CiteULike

Carbon nanotubes in cancer diagnosis and therapy.

Publication Date: 2010 Feb 26 PMID: 20193746
Authors: Ji, S. R. - Liu, C. - Zhang, B. - Yang, F. - Xu, J. - Long, J. - Jin, C. - Fu, D. L. - Ni, Q. X. - Yu, X. J.
Journal: Biochim Biophys Acta

During the past years, great progress has been made in the field of nanomaterials given their great potential in biomedical applications. Carbon nanotubes (CNTs), due to their unique physicochemical properties, have become a popular tool in cancer diagnosis and therapy. They are considered one of the most promising nanomaterials with the capability of both detecting the cancerous cells and delivering drugs or small therapeutic molecules to these cells. Over the last several years, CNTs have been explored in almost every single cancer treatment modality, including drug delivery, lymphatic targeted chemotherapy, thermal therapy, photodynamic therapy, and gene therapy. In this review, we will show how they have been introduced into the diagnosis and treatment of cancer. Novel SWNT-based tumor-targeted drug delivery systems (DDS) will be highlighted. Furthermore, the in vitro and in vivo toxicity of CNTs reported in recent years will be summarized.

post to: CiteULike

Building a better understanding of the intracellular tyrosine kinasePTK6 - BRK by BRK.

Publication Date: 2010 Feb 26 PMID: 20193745
Authors: Brauer, P. M. - Tyner, A. L.
Journal: Biochim Biophys Acta

Protein tyrosine kinase 6 (PTK6), also referred to as breast tumor kinase BRK, is a member of a distinct family of kinases that is evolutionarily related to the SRC family of tyrosine kinases. While not expressed in the normal mammary gland, PTK6 expression is detected in a large proportion of human mammary gland tumors. In breast tumor cells, PTK6 promotes growth factor signaling and cell migration. PTK6 expression is also increased in a number of other epithelial tumors, including ovarian and colon cancer. In contrast, PTK6 is expressed in diverse normal epithelia, including the linings of the gastrointestinal tract, skin and prostate, where its expression correlates with cell cycle exit and differentiation. Disruption of the mouse Ptk6 gene leads to increased growth and impaired differentiation in the small intestine that is accompanied by increased AKT and Wnt signaling. Following total body irradiation, PTK6 expression is induced in proliferating progenitor cells of the intestine, where it plays an essential role in DNA-damage induced apoptosis. A distinguishing feature of PTK6 is its flexibility in intracellular localization, due to a lack of amino-terminal myristoylation/palmitoylation. Recently a number of substrates of PTK6 have been identified, including nuclear RNA-binding proteins and transcription factors. We discuss PTK6 signaling, its apparent conflicting roles in cancer and normal epithelia, and its potential as a therapeutic target in epithelial cancers.

post to: CiteULike

F(1)F(0)-ATP synthases of alkaliphilic bacteria: Lessons from their adaptations.

Publication Date: 2010 Mar 1 PMID: 20193659
Authors: Hicks, D. B. - Liu, J. - Fujisawa, M. - Krulwich, T. A.
Journal: Biochim Biophys Acta

This review focuses on the ATP synthases of alkaliphilic bacteria and, in particular, those that successfully overcome the bioenergetic challenges of achieving robust H(+)-coupled ATP synthesis at external pH values >10. At such pH values the protonmotive force, which is posited to provide the energetic driving force for ATP synthesis, is too low to account for the ATP synthesis observed. The protonmotive force is lowered at a very high pH by the need to maintain a cytoplasmic pH well below the pH outside, which results in an energetically adverse pH gradient. Several anticipated solutions to this bioenergetic conundrum have been ruled out. Although the transmembrane sodium motive force is high under alkaline conditions, respiratory alkaliphilic bacteria do not use Na(+)- instead of H(+)-coupled ATP synthases. Nor do they offset the adverse pH gradient with a compensatory increase in the transmembrane electrical potential component of the protonmotive force. Moreover, studies of ATP synthase rotors indicate that alkaliphiles cannot fully resolve the energetic problem by using an ATP synthase with a large number of c-subunits in the synthase rotor ring. Increased attention now focuses on delocalized gradients near the membrane surface and H(+) transfers to ATP synthases via membrane-associated microcircuits between the H(+) pumping complexes and synthases. Microcircuits likely depend upon proximity of pumps and synthases, specific membrane properties and specific adaptations of the participating enzyme complexes. ATP synthesis in alkaliphiles depends upon alkaliphile-specific adaptations of the ATP synthase and there is also evidence for alkaliphile-specific adaptations of respiratory chain components.

post to: CiteULike

ATP-dependent proteases in biogenesis and maintenance of plant mitochondria.

Publication Date: 2010 Mar 1 PMID: 20193658
Authors: Janska, H. - Piechota, J. - Kwasniak, M.
Journal: Biochim Biophys Acta

ATP-dependent proteases from three families have been identified experimentally in Arabidopsis mitochondria: four FtsH proteases (AtFtsH3, AtFtsH4, AtFtsH10, and AtFtsH11), two Lon proteases (AtLon1 and AtLon4), and one Clp protease (AtClpP2 with regulatory subunit AtClpX). In this review we discuss their submitochondrial localization, expression profiles and proposed functions, with special emphasis on their impact on plant growth and development. The best characterized plant mitochondrial ATP-dependent proteases are AtLon1 and AtFtsH4. It has been proposed that AtLon1 is necessary for proper mitochondrial biogenesis during seedling establishment, whereas AtFtsH4 is involved in maintaining mitochondrial homeostasis late in rosette development under short-day photoperiod.

post to: CiteULike

Mass spectrometry analysis of complexes formed by myotonic dystrophy protein kinase (DMPK).

Publication Date: 2010 Feb 25 PMID: 20188867
Authors: Forner, F. - Furlan, S. - Salvatori, S.
Journal: Biochim Biophys Acta

Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats at the 3'-UTR of the serine/threonine protein kinase DMPK. Expanded CTG repeats are toxic since they are transcribed into an RNA molecule which is then sequestered within the nucleus in the form of foci. RNA cytotoxicity is linked to the aberrant splicing of several developmentally regulated genes. DMPK transcripts undergo alternative splicing giving rise to many isoforms but do not seem to be involved in the splicing dysregulation of DM1. However, decreased levels of DMPK in DM1 patients and DMPK involvement in muscle weakness and cardiac dysfunction in animal models have been reported. The variability in phenotypic expression of DMPK together with its differential subcellular targeting, suggests that different splicing isoforms may be involved in different signalling pathways, possibly through DMPK-interacting proteins. To gain better insight into the DMPK function, we used mass spectrometry to identify proteins co-segregating with DMPK in soluble complexes isolated from high-speed supernatant of rat muscles. We carried out experiments with native DMPK to preserve the physiological stoichiometry with potential partners. DMPK-containing complexes were isolated and immuno-detected by non-denaturing electrophoresis, gel filtration, ionic-exchange chromatography and immunoprecipitation. DMPK peptides were identified by high-resolution mass spectrometry together with several putative DMPK-binding proteins, including several heat shock proteins such as HSP20/HSPB6, HSP60/CPN60, HSP70 and HSP90. We also obtained evidence of a direct interaction of DMPK with alphaB-crystallin/HSPB5 and HSP25/HSPB1.

post to: CiteULike

Aberrant RNA splicing in RHD 7-9 exons of DEL individuals in Taiwan: A mechanism study.

Publication Date: 2010 Feb 25 PMID: 20188798
Authors: Liu, H. C. - Eng, H. L. - Yang, Y. F. - Wang, Y. H. - Lin, K. T. - Wu, H. L. - Lin, T. M.
Journal: Biochim Biophys Acta

The Rh blood D group provides a clinically important model of aberrant splicing with skipped exons. Approximately 30% of serologically D-negative Chinese individuals have an intact RHD gene (DEL phenotype) and induce allo-immunization in transfusions. The RHD1227G>A polymorphism occurs in >95% DEL phenotype of Asian descent. The effects of RHD 1227A and a novel allele on exon 9 splicing were examined. Amplified DEL RNA products revealed that 3 transcripts involved skipping of exons 8-9, exon 9, or exon 9 with an inserted 170-bp cryptic exon located between exons 7 and 8. A novel, single nucleotide polymorphism was identified in the 7th intron, (IVS7)+923C>T, and present in all DEL patients. The odds ratio of RHD1227G>A allele with DEL phenotype was 2711. Splicing analysis of transcripts from minigenes containing the 1227G>A allele, but not the (IVS7)+923C>T allele, demonstrated aberrant exon 9 skipping. A combined haplotype of 1227G>A and IVS7+923C>T alleles was apparent in >95% DEL Chinese individuals. RHD1227A mutation significantly increased aberrant mRNA splicing, producing a hybrid RHD mRNA lacking exon 9. These results provide a molecular basis of the DEL phenotype in the Chinese population.

post to: CiteULike

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy.

Publication Date: 2010 Feb 24 PMID: 20188699
Authors: Machan, R. - Hof, M.
Journal: Biochim Biophys Acta

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.

post to: CiteULike

P66 porins are present in both Lyme disease and relapsing fever spirochetes: A comparison of the biophysical properties of P66 porins from six Borrelia species.

Publication Date: 2010 Feb 25 PMID: 20188698
Authors: Barcena-Uribarri, I. - Thein, M. - Sacher, A. - Bunikis, I. - Bonde, M. - Bergstrom, S. - Benz, R.
Journal: Biochim Biophys Acta

The genus Borrelia is the cause of the two human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia species are obligate parasites and are dependent on nutrients provided by their hosts. The first step of nutrient uptake across the outer membrane of these Gram-negative bacteria is accomplished by water-filled channels, so-called porins. The knowledge of the porin composition in the outer membranes of the different pathogenic Borrelia species is limited. Only one porin has been described in relapsing fever spirochetes to date, whereas four porins are known to be present in Lyme disease agents. From these, the Borrelia burgdorferi outer membrane channel P66 is known to act as an adhesin and was well studied as a porin. To investigate if P66 porins are expressed and similarly capable of pore formation in other Borrelia causing Lyme disease or relapsing fever three LD species (B. burgdorferi, B. afzelii, B. garinii) and three RF species (B. duttonii, B. recurrentis and B. hermsii) were investigated for outer membrane proteins homologous to P66. A search in current published RF genomes, comprising the ones of B. duttonii, B. recurrentis and B. hermsii, indicated that they all contained P66 homologues. The P66 homologues of the six Borrelia species were purified to homogeneity and their pore-forming abilities as well as the biophysical properties of the pores were analyzed using the black lipid bilayer assay.

post to: CiteULike

PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells.

Publication Date: 2010 Feb 25 PMID: 20188697
Authors: Kurzawa, L. - Pellerano, M. - Morris, M. C.
Journal: Biochim Biophys Acta

Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.

post to: CiteULike

Combined effects of polymers and KL(4) peptide on surface activity of pulmonary surfactant lipids.

Publication Date: 2010 Feb 25 PMID: 20188696
Authors: Lu, K. W. - Taeusch, H. W.
Journal: Biochim Biophys Acta

Addition of ionic and nonionic polymers can improve the function of therapeutic surfactants in vitro and in vivo, especially under conditions that tend to inhibit surfactant activity. Since surfactant proteins also act to reduce surfactant inhibition, we studied the relative effects of a synthetic peptide (that mimics some of the properties of a surfactant protein), polymers, and their combination on function of surfactant phospholipid activity in vitro. We evaluated surface activity after adding polymers-polyethylene glycol or hyaluronan-to a lipid mixture with or without the synthetic peptide, sinapultide (KL(4)). Using a pulsating bubble surfactometer, we measured peptide/polymer effects separately or combined at two peptide concentrations. Phospholipid mixtures, with or without KL(4) or polymers, all demonstrated good surface activity. With serum present as an inhibiting agent, adding either concentration of KL(4) reduced inhibition. Mixtures containing the higher concentration of KL(4) required higher concentrations of serum for inhibition to occur. Adding either polymer to mixtures with KL(4) further decreased susceptibility to inhibition (required higher serum concentrations). In the presence of serum, high molecular weight hyaluronan with KL(4) at 0.4mg/ml improved surface activity to a greater degree than 0.8mg/ml KL(4) without polymer. If the beneficial effects of adding polymer to KL(4)-lipid mixtures are also borne out in the treatment of experimental lung injury, these peptide-polymer surfactant combinations may eventually prove useful in the treatment of some forms of acute lung injury in humans.

post to: CiteULike

The p13 protein of human T cell leukemia virus type 1 (HTLV-1) modulates mitochondrial membrane potential and calcium uptake.

Publication Date: 2010 Feb 25 PMID: 20188695
Authors: Biasiotto, R. - Aguiari, P. - Rizzuto, R. - Pinton, P. - D'Agostino, D. M. - Ciminale, V.
Journal: Biochim Biophys Acta

Human T cell leukemia virus type 1 (HTLV-1) encodes p13, an 87-amino-acid protein that accumulates in the inner mitochondrial membrane. Recent studies performed using synthetic p13 and isolated mitochondria demonstrated that the protein triggers an inward potassium (K(+)) current and inner membrane depolarization. The present study investigated the effects of p13 on mitochondrial inner membrane potential (Deltapsi) in living cells. Using the potential-dependent probe tetramethyl rhodamine methyl ester (TMRM), we observed that p13 induced dose-dependent mitochondrial depolarization in HeLa cells. This effect was abolished upon mutation of 4 arginines in p13's alpha-helical domain that were previously shown to be essential for its activity in in vitro assays. As Deltapsi is known to control mitochondrial calcium (Ca(2+)) uptake, we next analyzed the effect of p13 on Ca(2+) homeostasis. Experiments carried out in HeLa cells expressing p13 and organelle-targeted aequorins revealed that the protein specifically reduced mitochondrial Ca(2+) uptake. These observations suggest that p13 might control key processes regulated through Ca(2+) signaling such as activation and death of T cells, the major targets of HTLV-1 infection.

post to: CiteULike

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Publication Date: 2010 Feb 25 PMID: 20188694
Authors: Cano-Estrada, A. - Vazquez-Acevedo, M. - Villavicencio-Queijeiro, A. - Figueroa-Martinez, F. - Miranda-Astudillo, H. - Cordeiro, Y. - Mignaco, J. A. - Foguel, D. - Cardol, P. - Lapaille, M. - Remacle, C. - Wilkens, S. - Gonzalez-Halphen, D.
Journal: Biochim Biophys Acta

Mitochondrial F(1)F(0)-ATP synthase of chlorophycean algae is a dimeric complex of 1600kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.

post to: CiteULike

DNA polymerases: Perfect enzymes for an imperfect world.

Publication Date: 2010 Feb 24 PMID: 20188219
Authors: Berdis, A. J.
Journal: Biochim Biophys Acta

This Special Thematic Issue explores the molecular properties of DNA polymerases as extraordinary biological catalysts. In this short introductory chapter, I briefly highlight some of the most important concepts from the articles contained with this Special Issue. The contents of this Special Issue are arranged into distinct sub-categories corresponding to mechanistic studies of faithful DNA polymerization, studies of "specialized" DNA polymerases that function on damaged DNA, and DNA polymerases that are of therapeutic importance against various diseases. Emphasis is placed on understanding the dynamic cellular roles and biochemical functions of DNA polymerases, and how their structure and mechanism impact their cellular roles.

post to: CiteULike

The role of ATP-binding cassette transporter A1 in Alzheimer's disease and neurodegeneration.

Publication Date: 2010 Feb 24 PMID: 20188211
Authors: Koldamova, R. - Fitz, N. F. - Lefterov, I.
Journal: Biochim Biophys Acta

ATP-binding cassette transporter A1 - ABCA1, is the most extensively studied transporter in human pathology. ABCA1 became a primary subject of research in many academic and pharmaceutical laboratories immediately after the discovery that mutations at the gene locus cause severe familial HDL deficiency and, in the homozygous form - Tangier disease. The protein is the major regulator of intracellular cholesterol efflux which is the initial and essential step in the biogenesis and formation of nascent high density lipoprotein particles. The transcriptional regulation of ABCA1 by Nuclear Liver X Receptors (LXR) provided a starting point for drug discovery and development of synthetic LXR ligands/ABCA1 activators for treatment of arteriosclerosis. A series of reports that revealed the role of ABCA1 in Abeta deposition and clearance, as well the possibility for association of some ABCA1 genetic variants with risk for Alzheimer's disease (AD) brought a new dimension to ABCA1 research. The LXR-ABCA1-APOE regulatory axis is considered now a promising therapeutic target in AD, which includes the only proven risk factor for AD - APOE, at two distinct levels - transcriptional regulation by LXR, and ABCA1 controlled lipidation which can influence Abeta aggregation and amyloid clearance. This review will summarize the results of research on ABCA1, particularly related to AD and neurodegeneration.

post to: CiteULike

T regulatory cells, the evolution of targeted immunotherapy.

Publication Date: 2010 Feb 23 PMID: 20188145
Authors: Nizar, S. - Meyer, B. - Copier, J. - Galustian, C. - Kumar, D. - Dalgleish, A.
Journal: Biochim Biophys Acta

T regulatory cells are able to suppress anti-tumour immunity in pre-clinical models and in patients. This review highlights the important discoveries in Treg immunology critical to the evolution of targeted immunotherapy. We also describe the therapeutic applications that are currently being assessed and future potential.

post to: CiteULike

Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFbeta1.

Publication Date: 2010 Feb 23 PMID: 20188144
Authors: Clarke, C. - Baghdadi, H. - Howie, A. F. - Mason, J. I. - Walker, S. W. - Beckett, G. J.
Journal: Biochim Biophys Acta

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor beta1 (TGFbeta1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated. METHODS: In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFbeta1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells. RESULTS: We found that in response to treatment with TGFbeta1, C3A cells produced mRNA encoding the pro-alphaI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase. CONCLUSIONS: Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFbeta1, possibly as part of an epithelial to mesenchymal transition. GENERAL SIGNIFICANCE: These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.

post to: CiteULike

Antimicrobial peptides bind more strongly to membrane pores.

Publication Date: 2010 Feb 24 PMID: 20188066
Authors: Mihajlovic, M. - Lazaridis, T.
Journal: Biochim Biophys Acta

Antimicrobial peptides (AMPs) are small, usually cationic peptides, which permeabilize bacterial membranes. Understanding their mechanism of action might help design better antibiotics. Using an implicit membrane model, modified to include pores of different shapes, we show that four AMPs (alamethicin, melittin, a magainin analogue, MG-H2, and piscidin 1) bind more strongly to membrane pores, consistent with the idea that they stabilize them. The effective energy of alamethicin in cylindrical pores is similar to that in toroidal pores, whereas the effective energy of the other three peptides is lower in toroidal pores. Only alamethicin intercalates into the membrane core; MG-H2, melittin and piscidin are located exclusively at the hydrophobic/hydrophilic interface. In toroidal pores, the latter three peptides often bind at the edge of the pore, and are in an oblique orientation. The calculated binding energies of the peptides are correlated with their hemolytic activities. We hypothesize that one distinguishing feature of AMPs may be the fact that they are imperfectly amphipathic which allows them to bind more strongly to toroidal pores. An initial test on a melittin-based mutant seems to support this hypothesis.

post to: CiteULike

P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function.

Publication Date: 2010 Feb 23 PMID: 20188065
Authors: Aurade, R. M. - Jayalakshmi, S. K. - Sreeramulu, K.
Journal: Biochim Biophys Acta

Helicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30-40 degrees C, respectively. Kinetic studies indicated that this enzyme had a K(m) value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest.

post to: CiteULike

Realistic modeling approaches of structure-function properties of CPPs in non-covalent complexes.

Publication Date: 2010 Feb 23 PMID: 20188064
Authors: Thomas, A. - Lins, L. - Divita, G. - Brasseur, R.
Journal: Biochim Biophys Acta

Transfers of cargoes into cells by means of carrier peptides are multi-steps biological phenomenon the mechanisms of which are unclear. We here discuss bases of realistic in silico molecular modeling approaches of the formation of non-covalent complexes considering CPPs and cargo diversities.

post to: CiteULike

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors.

Publication Date: 2010 Feb 23 PMID: 20188063
Authors: Gerken, M. - Krippner-Heidenreich, A. - Steinert, S. - Willi, S. - Neugart, F. - Zappe, A. - Wrachtrup, J. - Tietz, C. - Scheurich, P.
Journal: Biochim Biophys Acta

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2x10(-9)cm(2)/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D=3.1x10(-9)cm(2)/s) with a marked reduction after 30min of TNF treatment (D=0.9x10(-9)cm(2)/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.

post to: CiteULike

CLC channels and transporters: Proteins with borderline personalities.

Publication Date: 2010 Feb 24 PMID: 20188062
Authors: Accardi, A. - Picollo, A.
Journal: Biochim Biophys Acta

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl(-) channels and H(+)/Cl(-) exchangers. All CLC's are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes. Key residues for ion binding and selectivity are also highly conserved. Most CLC's have large cytosolic carboxy-terminal domains containing two cystathionine beta-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds. This review focuses on the recent mechanistic insights on the how the structural similarities between CLC channels and transporters translate in unexpected mechanistic analogies between these two sub-classes.

post to: CiteULike

Stability study of the human G-protein coupled receptor, Smoothened.

Publication Date: 2010 Feb 23 PMID: 20188061
Authors: Nehme, R. - Joubert, O. - Bidet, M. - Lacombe, B. - Polidori, A. - Pucci, B. - Mus-Veteau, I.
Journal: Biochim Biophys Acta

Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-beta-d-maltoside (DDM). The fluorinated surfactant C(8)F(17)TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C(8)F(17)TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.

post to: CiteULike

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

Publication Date: 2010 Feb 24 PMID: 20188060
Authors: Wittig, I. - Meyer, B. - Heide, H. - Steger, M. - Bleier, L. - Wumaier, Z. - Karas, M. - Schagger, H.
Journal: Biochim Biophys Acta

We have studied ATP synthase from human rho(0) (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and shown that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast subunits e and g null mutants detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF(1) inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF(1) still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho(0) cells and not to the dimeric form. This supports previous suggestions that IF(1) plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

post to: CiteULike

The ups and downs of mitochondrial calcium signalling in the heart.

Publication Date: 2010 Feb 24 PMID: 20188059
Authors: Griffiths, E. J. - Balaska, D. - Cheng, W. H.
Journal: Biochim Biophys Acta

Regulation of intramitochondrial free calcium ([Ca(2+)](m)) is critical in both physiological and pathological functioning of the heart. The full extent and importance of the role of [Ca(2+)](m) is becoming apparent as evidenced by the increasing interest and work in this area of the last two decades. However, controversies remain, such as the existence of beat-to-beat mitochondrial Ca(2+) transients; the role of [Ca(2+)](m) in modulating whole-cell Ca(2+) signalling; whether or not an increase in [Ca(2+)](m) is essential to couple ATP supply and demand; and the role of [Ca(2+)](m) in cell death by both necrosis and apoptosis, especially in formation of the mitochondrial permeability transition pore. The role of [Ca(2+)](m) in heart failure is an area that has also recently been highlighted. [Ca(2+)](m) can now be measured reasonably specifically in intact cells and hearts thanks to developments in fluorescent indicators and targeted proteins and more sensitive imaging technology. This has revealed interactions of the mitochondrial Ca(2+) transporters with those of the sarcolemma and sarcoplasmic reticulum, and has gone a long way to bringing the mitochondrial Ca(2+) transporters to the forefront of cardiac research. Mitochondrial Ca(2+) uptake occurs via the ruthenium red sensitive Ca(2+) uniporter (mCU), and efflux via an Na(+)/Ca(2+) exchanger (mNCX). The purification and cloning of the transporters, and development of more specific inhibitors, would produce a step-change in our understanding of the role of these apparently critical but still elusive proteins. In this article I will summarise the key physiological roles of [Ca(2+)](m) in ATP production and cell Ca(2+) signalling in both adult and neonatal hearts, as well as highlighting some of the controversies in these areas. I will also briefly discuss recent ideas on interactions of nitric oxide with [Ca(2+)](m).

post to: CiteULike

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase.

Publication Date: 2010 Feb 23 PMID: 20184858
Authors: Brzezinski, P. - Johansson, A. L.
Journal: Biochim Biophys Acta

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c(2+) and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O(2). In this reaction part of the free energy available from O(2) reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O(2). Our understanding of the molecular design of the machinery that couples O(2) reduction to proton pumping in oxidases has greatly benefited from studies of so called "uncoupled" structural variants of the oxidases. In these uncoupled oxidases the catalytic O(2)-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O(2) is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pK(a) of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pK(a) presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, changes in apparent pK(a) and uncoupling of proton pumping from O(2) reduction.

post to: CiteULike

The NIZP1 KRAB and C2HR domains cross-talk for transcriptional regulation.

Publication Date: 2010 Feb 20 PMID: 20176155
Authors: Losson, R. - Nielsen, A. L.
Journal: Biochim Biophys Acta

The NSD1 histone methyltransferase is involved in the outgrowth disorders Sotos and Weaver syndromes and childhood acute myeloid leukemia. NSD1 is a bona fida transcriptional co-repressor for Nizp1 which is a protein including SCAN, KRAB, C2HR and zinc-finger domains. In this study the Nizp1 KRAB-domain was identified to possess an intrinsic transcriptional activation capacity suppressed in cis by the presence of the C2HR domain. Oppositely, the KRAB-domain supported C2HR domain mediated transcriptional repression. The presence of the KRAB-domain resulted in increased NSD1 co-repressor association with the C2HR domain. This study shows a new function of the KRAB-domain, C2HR-domain, and the associated factors to confer Nizp1 mediated transcriptional regulation.

post to: CiteULike

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G.

Publication Date: 2010 Feb 20 PMID: 20176147
Authors: Suh, M. J. - Clark, D. J. - Parmer, P. P. - Fleischmann, R. D. - Peterson, S. N. - Pieper, R.
Journal: Biochim Biophys Acta

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date. SasG is highly abundant in the cell wall of the vancomycin-intermediate S. aureus strain HIP5827, and was purified and subjected to sequence analysis by MS. Data from MALDI-TOF and LC-MS/MS experiments confirmed the predicted N-terminal signal peptide cleavage site at residue A(51) and the C-terminal cell wall anchor site at residue T(1086). The protein was also derivatized with N-succinimidyloxycarbonyl-methyl-tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) to assess the presence of additional N-terminal sites of mature SasG. TMPP-derivatized SasG peptides featured m/z peaks with a 572Da mass increase over the equivalent underivatized peptides. Multiple N-terminal peptides, all of which were observed in the 150 amino acid segment following the signal peptide cleavage at the residue A(51), were characterized from MS and MS/MS data, suggesting a series of successive N-terminal truncations of SasG. A strategy combining TMPP derivatization, multiple enzyme digestions to generate overlapping peptides and detailed MS analysis will be useful to determine and understand functional implications of PTMs in bacterial cell wall-anchored proteins, which are frequently involved in the modulation of virulence-associated bacterial surface properties.

post to: CiteULike

Function and metabolism of sirtuin metabolite O-acetyl-ADP-ribose.

Publication Date: 2010 Feb 20 PMID: 20176146
Authors: Tong, L. - Denu, J. M.
Journal: Biochim Biophys Acta

Sirtuins catalyze the NAD(+)-dependent deacetylation of target proteins, which are regulated by this reversible lysine modification. During deacetylation, the glycosidic bond of the nicotinamide ribose is cleaved to yield nicotinamide and the ribose accepts the acetyl group from substrate to produce O-acetyl-ADP-ribose (OAADPr), which exists as an approximately 50:50 mixture of 2' and 3' isomers at neutral pH. Discovery of this metabolite has fueled the idea that OAADPr may play an important role in the biology associated with sirtuins, acting as a signaling molecule and/or an important substrate for downstream enzymatic processes. Evidence for OAADPr-metabolizing enzymes indicates that at least three distinct activities exist that could modulate the cellular levels of this NAD(+)-derived metabolite. In Saccharomyces cerevisiae, NUDIX hydrolase Ysa1 cleaves OAADPr to AMP and 2- and 3-O-acetylribose-5-phosphate, lowering the cellular levels of OAADPr. A buildup of OAADPr and ADPr has been linked to a metabolic shift that lowers endogenous reactive oxygen species and diverts glucose towards preventing oxidative damage. In vitro, the mammalian enzyme ARH3 hydrolyzes OAADPr to acetate and ADPr. A third nuclear-localized activity appears to utilize OAADPr to transfer the acetyl-group to another small molecule, whose identity remains unknown. Recent studies suggest that OAADPr may regulate gene silencing by facilitating the assembly and loading of the Sir2-4 silencing complex onto nucleosomes. In mammalian cells, the Trpm2 cation channel is gated by both OAADPr and ADP-ribose. Binding is mediated by the NUDIX homology (NudT9H) domain found within the intracellular portion of the channel. OAADPr is capable of binding the Macro domain of splice variants from histone protein MacroH2A, which is highly enriched at heterochromatic regions. With recently developed tools, the pace of new discoveries of OAADPr-dependent processes should facilitate new molecular insight into the diverse biological processes modulated by sirtuins.

post to: CiteULike

Residue N84 of Yeast Cystathionine beta-Synthase is a Determinant of Reaction Specificity.

Publication Date: 2010 Feb 20 PMID: 20176145
Authors: Lodha, P. H. - Hopwood, E. M. - Manders, A. L. - Aitken, S. M.
Journal: Biochim Biophys Acta

Cystathionine beta-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent condensation of l-serine and l-homocysteine to form l-cystathionine in the first step of the reverse transsulfuration pathway. Residue N84 of yeast CBS (yCBS), predicted to form a hydrogen bond with the hydroxyl moiety of the PLP cofactor, was mutated to alanine, aspartate and histidine. The truncated form of yCBS (ytCBS, residues 1-353) was employed in this study to eliminate any effects of the C-terminal, regulatory domain. The k(cat)/K(m)(l-Ser) of the N84A, N84D and N84H mutants for the beta-replacement reaction are reduced by a factor of 230, 11000 and 640, respectively. Fluorescence resonance energy transfer between tryptophan residue(s) of the enzyme and the PLP cofactor, observed in the wild-type enzyme and N84A mutant, is altered in N84H and absent in N84D. PLP saturation values of 73%, 30% and 67% were observed for the alanine, aspartate and histidine mutants, respectively, compared to 98% for the wild-type enzyme. A marginal beta-elimination activity was detected for N84D (k(cat)/K(m)(l-Ser)=0.23+/-0.02M(-1) s(-1)) and N84H (k(cat)/K(m)(l-Ser)=0.34+/-0.06M(-1) s(-1)), in contrast with wild-type ytCBS and the N84A mutant, which do not catalyze this reaction. The ytCBS-N84D enzyme is also inactivated upon incubation with l-serine, via an aminoacrylate-mediated mechanism. These results demonstrate that residue N84 is essential in maintaining the orientation of the pyridine ring of the PLP cofactor and the equilibrium between the open and closed conformations of the active site.

post to: CiteULike

Standardized evaluation of protein stability.

Publication Date: 2010 Feb 20 PMID: 20176144
Authors: Thomas, A. - Joris, B. - Brasseur, R.
Journal: Biochim Biophys Acta

We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP values that are averaged to -65+/-3kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from -25 for Gly to -115kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of -63.5+/-2.5kcal/aa, i.e., only 1.5kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.

post to: CiteULike

Incorporation and remodeling of phosphatidylethanolamine containing short acyl residues in yeast.

Publication Date: 2010 Feb 20 PMID: 20176132
Authors: Deng, L. - Fukuda, R. - Kakihara, T. - Narita, K. - Ohta, A.
Journal: Biochim Biophys Acta

Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn-2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn-2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.

post to: CiteULike

Systematic construction of a conceptual minimal model of plasma cholesterol levels based on knockout mouse phenotypes.

Publication Date: 2010 Feb 20 PMID: 20176131
Authors: van de Pas, N. C. - Soffers, A. E. - Freidig, A. P. - van Ommen, B. - Woutersen, R. A. - Rietjens, I. M. - de Graaf, A. A.
Journal: Biochim Biophys Acta

Elevated plasma cholesterol, a well-known risk factor for cardiovascular diseases, is the result of the activity of many genes and their encoded proteins in a complex physiological network. We aim to develop a minimal kinetic computational model for predicting plasma cholesterol levels. To define the scope of this model, it is essential to discriminate between important and less important processes influencing plasma cholesterol levels. To this end, we performed a systematic review of mouse knockout strains and used the resulting dataset, named KOMDIP, for the identification of key genes that determine plasma cholesterol levels. Based on the described phenotype of mouse knockout models, 36 of the 120 evaluated genes were marked as key genes that have a pronounced effect on the plasma cholesterol concentration. The key genes include well-known genes, e.g., Apoe and Ldlr, as well as genes hardly linked to cholesterol metabolism so far, e.g., Plagl2 and Slc37a4. Based on the catalytic function of the genes, a minimal conceptual model was defined. A comparison with nine conceptual models from literature revealed that each of the individual published models is less complete than our model. Concluding, we have developed a conceptual model that can be used to develop a physiologically based kinetic model to quantitatively predict plasma cholesterol levels.

post to: CiteULike

Involvements of the lipid peroxidation product, HNE, in the pathogenesis and progression of Alzheimer's disease.

Publication Date: 2010 Feb 20 PMID: 20176130
Authors: Butterfield, D. A. - Bader Lange, M. L. - Sultana, R.
Journal: Biochim Biophys Acta

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. A number of hypotheses have been proposed to explain AD pathogenesis. One such hypothesis proposed to explain AD pathogenesis is the oxidative stress hypothesis. Increased levels of oxidative stress markers including the markers of lipid peroxidation such as acrolein, 4-hydroxy-2-trans-nonenal (HNE), malondialdehyde, etc. are found in brains of AD subjects. In this review, we focus principally on research conducted in the area of HNE in the central nervous system (CNS) of AD and mild cognitive impairment (MCI), and further, we discuss likely consequences of lipid peroxidation with respect to AD pathogenesis and progression. Based on the research conducted so far in the area of lipid peroxidation, it is suggested that lipid accessible antioxidant molecules could be a promising therapeutic approach to treat or slow progression of MCI and AD.

post to: CiteULike

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants.

Publication Date: 2010 Feb 20 PMID: 20176106
Authors: Wang, S. S. - Liu, K. N. - Han, T. C.
Journal: Biochim Biophys Acta

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 degrees C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.

post to: CiteULike

Glucose deprivation causes oxidative stress and stimulates aggresome formation and autophagy in cultured cardiac myocytes.

Publication Date: 2010 Feb 20 PMID: 20176105
Authors: Marambio, P. - Toro, B. - Sanhueza, C. - Troncoso, R. - Parra, V. - Verdejo, H. - Garcia, L. - Quiroga, C. - Munafo, D. - Diaz-Elizondo, J. - Bravo, R. - Gonzalez, J. - Diaz-Araya, G. - Pedrozo, Z. - Chiong, M. - Colombo, M. I. - Lavandero, S.
Journal: Biochim Biophys Acta

Aggresomes are dynamic structures formed when the ubiquitin-proteasome system is overwhelmed with aggregation-prone proteins. In this process, small protein aggregates are actively transported towards the microtubule-organizing center. A functional role for autophagy in the clearance of aggresomes has also been proposed. In the present work we investigated the molecular mechanisms involved on aggresome formation in cultured rat cardiac myocytes exposed to glucose deprivation. Confocal microscopy showed that small aggregates of polyubiquitinated proteins were formed in cells exposed to glucose deprivation for 6h. However, at longer times (18h), aggregates formed large perinuclear inclusions (aggresomes) which colocalized with gamma-tubulin (a microtubule-organizing center marker) and Hsp70. The microtubule disrupting agent vinblastine prevented the formation of these inclusions. Both small aggregates and aggresomes colocalized with autophagy markers such as GFP-LC3 and Rab24. Glucose deprivation stimulates reactive oxygen species (ROS) production and decreases intracellular glutathione levels. ROS inhibition by N-acetylcysteine or by the adenoviral overexpression of catalase or superoxide dismutase disrupted aggresome formation and autophagy induced by glucose deprivation. In conclusion, glucose deprivation induces oxidative stress which is associated with aggresome formation and activation of autophagy in cultured cardiac myocytes.

post to: CiteULike

Deleterious effects of reactive aldehydes and glycated proteins on macrophage proteasomal function: Possible links between diabetes and atherosclerosis.

Publication Date: 2010 Feb 20 PMID: 20176104
Authors: Moheimani, F. - Morgan, P. E. - van Reyk, D. M. - Davies, M. J.
Journal: Biochim Biophys Acta

People with diabetes experience chronic hyperglycemia and are at a high risk of developing atherosclerosis and microvascular disease. Reactions of glucose, or aldehydes derived from glucose (e.g. methylglyoxal, glyoxal, or glycolaldehyde), with proteins result in glycation that ultimately yield advanced glycation end products (AGE). AGE are present at elevated levels in plasma and atherosclerotic lesions from people with diabetes, and previous in vitro studies have postulated that the presence of these materials be deleterious to cell function. This accumulation of AGE and glycated proteins within cells may arise from either increased formation and/or ineffective removal by cellular proteolytic systems, such as the proteasomes, the major multi-enzyme complex that removes proteins within cells. In this study it is shown that whilst high glucose concentrations fail to modify proteasome enzyme activities in J774A.1 macrophage-like cell extracts, reactive aldehydes enhanced proteasomal enzyme activities. In contrast BSA, pre-treated with high glucose for 8weeks, inhibited both the chymotrypsin-like and caspase-like activities. BSA glycated using methylglyoxal or glycolaldehyde, also inhibited proteasomal activity though to differing extents. This suppression of proteasome activity by glycated proteins may result in further intracellular accumulation of glycated proteins with subsequent deleterious effects on cellular function.

post to: CiteULike

Cytosolic and mitochondrial ferritins in the regulation of cellular iron homeostasis and oxidative damage.

Publication Date: 2010 Feb 20 PMID: 20176086
Authors: Arosio, P. - Levi, S.
Journal: Biochim Biophys Acta

BACKGROUND: Ferritin structure is designed to maintain large amounts of iron in a compact and bioavailable form in solution. All ferritins induce fast Fe(II) oxidation in a reaction catalyzed by a ferroxidase center that consumes Fe(II) and peroxides, the reagents that produce toxic free radicals in the Fenton reaction, and thus have anti-oxidant effects. Cytosolic ferritins are composed of the H- and L-chains, whose expression are regulated by iron at a post-transcriptional level and by oxidative stress at a transcriptional level. The regulation of mitochondrial ferritin expression is presently unclear. SCOPE OF REVIEW: The scope of the review is to update recent progress regarding the role of ferritins in the regulation of cellular iron and in the response to oxidative stress with particular attention paid to the new roles described for cytosolic ferritins, to genetic disorders caused by mutations of the ferritin L-chain, and new findings on mitochondrial ferritin. MAJOR CONCLUSIONS: The new data on the adult conditional knockout (KO) mice for the H-chain and on the hereditary ferritinopathies with mutations that reduce ferritin functionality strongly indicate that the major role of ferritins is to protect from the oxidative damage caused by iron deregulation. In addition, the study of mitochondrial ferritin, which is not iron-regulated, indicates that it participates in the protection against oxidative damage, particularly in cells with high oxidative activity. GENERAL SIGNIFICANCE: Ferritins have a central role in the protection against oxidative damage, but they are also involved in non-iron-dependent processes.

post to: CiteULike

Relevance of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) in the structural integrity of the chromatoid body during spermatogenesis.

Publication Date: 2010 Feb 20 PMID: 20176059
Authors: Sato, H. - Tsai-Morris, C. H. - Dufau, M. L.
Journal: Biochim Biophys Acta

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)-a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis.

post to: CiteULike

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: An in situ PM-IRRAS investigation.

Publication Date: 2010 Feb 20 PMID: 20175990
Authors: Nakahara, H. - Lee, S. - Shibata, O.
Journal: Biochim Biophys Acta

In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air-water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500-1700cm(-1)) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the alpha-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the alpha-helix to the extended beta-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the beta-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air-water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.

post to: CiteULike

Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1alpha or PGC-1beta and mitofusin 2 in skeletal muscle in type 2 diabetes.

Publication Date: 2010 Feb 20 PMID: 20175989
Authors: Zorzano, A. - Hernandez-Alvarez, M. I. - Palacin, M. - Mingrone, G.
Journal: Biochim Biophys Acta

Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARdelta, PGC-1alpha or PGC-1beta. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1alpha, PGC-1beta, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1alpha, PGC-1beta, PPARdelta or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease.

post to: CiteULike

Wanderings in bioenergetics and biomembranes.

Publication Date: 2010 Feb 20 PMID: 20175988
Authors: Klingenberg, M.
Journal: Biochim Biophys Acta

Having worked for 55years in the center and at the fringe of bioenergetics, my major research stations are reviewed in the following wanderings: from microsomes to mitochondria, from NAD to CoQ, from reversed electron transport to reversed oxidative phosphorylation, from mitochondrial hydrogen transfer to phosphate transfer pathways, from endogenous nucleotides to mitochondrial compartmentation, from transport to mechanism, from carrier to structure, from coupling by AAC to uncoupling by UCP, and from specific to general transport laws. These wanderings are recalled with varying emphasis paid to the covered science stations.

post to: CiteULike

Redox-optimized ROS balance: A unifying hypothesis.

Publication Date: 2010 Feb 20 PMID: 20175987
Authors: Aon, M. A. - Cortassa, S. - O'Rourke, B.
Journal: Biochim Biophys Acta

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O(2) to superoxide (O(2)()) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD(+)) or ROS scavenging (NADPH/NADP(+), GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, Psi(m)), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low Psi(m)) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.

post to: CiteULike

The quinone-binding and catalytic site of complex II.

Publication Date: 2010 Feb 20 PMID: 20175986
Authors: Maklashina, E. - Cecchini, G.
Journal: Biochim Biophys Acta

The complex II family of proteins includes succinate:quinone oxidoreductase (SQR) and quinol:fumarate oxidoreductase (QFR). In the facultative bacterium Escherichia coli both are expressed as part of the aerobic (SQR) and anaerobic (QFR) respiratory chains. SQR from E. coli is homologous to mitochondrial complex II and has proven to be an excellent model system for structure/function studies of the enzyme. Both SQR and QFR from E. coli are tetrameric membrane-bound enzymes that couple succinate/fumarate interconversion with quinone/quinol reduction/oxidation. Both enzymes are capable of binding either ubiquinone or menaquinone, however, they have adopted different quinone binding sites where catalytic reactions with quinones occur. A comparison of the structures of the quinone binding sites in SQR and QFR reveals how the enzymes have adapted in order to accommodate both benzo- and napthoquinones. A combination of structural, computational, and kinetic studies of members of the complex II family of enzymes has revealed that the catalytic quinone adopts different positions in the quinone-binding pocket. These data suggest that movement of the quinone within the quinone-binding pocket is essential for catalysis.

post to: CiteULike

Mitochondrial research and medicine.

Publication Date: 2010 Mar PMID: 20171500
Authors: Wei, Y. H.
Journal: Biochim Biophys Acta

post to: CiteULike

Expression of functional mammal flavocytochrome b(558) in yeast: Comparison with improved insect cell system.

Publication Date: 2010 Feb 18 PMID: 20171157
Authors: Ostuni, M. A. - Lamanuzzi, L. B. - Bizouarn, T. - Dagher, M. C. - Baciou, L.
Journal: Biochim Biophys Acta

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b(558) (Cytb(558)) which consists of a heterodimer of the gp91(phox) and p22(phox) subunits. The Cytb(558) is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb(558) (rCytb(558)). We expressed the gp91(phox) and p22(phox) subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91(phox) undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb(558) displayed comparable spectral properties to neutrophil Cytb(558). In contrast to rCytb(558) produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.

post to: CiteULike

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase.

Publication Date: 2010 Feb 17 PMID: 20170757
Authors: Gedi, V. - Jayaraman, K. - Kalme, S. - Park, H. Y. - Park, H. C. - La, I. J. - Hahn, H. G. - Yoon, M. Y.
Journal: Biochim Biophys Acta

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichiacoli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0+/-0.02, 1.0+/-0.04, 2.1+/-0.12, and 2.0+/-0.08microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05+/-0.002, 0.153+/-0.004, 1.30+/-0.10, and 1.29+/-0.40microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52kcal/mol and formed a single hydrogen bond with a distance of 1.973A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.

post to: CiteULike

Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions.

Publication Date: 2010 Feb 17 PMID: 20170756
Authors: Hung, Y. C. - Wang, P. W. - Pan, T. L.
Journal: Biochim Biophys Acta

Salvia miltiorrhiza is a Chinese herb widely used for cardiovascular disorder regimens, yet little is known about the cellular mechanisms that contribute to attenuated growth of smooth muscle cells (SMCs) under oxidative stress such as homocysteine (Hcy) treatment. As anticipated, a low dose (0.015 mg/mL) of S.miltiorrhiza aqueous extract (SMAE) significantly inhibited (>60%) the growth of a rat smooth muscle cell line (A10) under Hcy stimulation and the intracellular reactive oxygen species (ROS) concentration obviously decreased after SMAE treatment in terms of reducing p47(phox) translocation and increasing catalase activity. Signaling profile suggests that SMAE inhibited Hcy-induced A10 cell growth via the PKC/MAPK-dependent pathway. Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 14 proteins in response to Hcy and Hcy/SMAE. Meanwhile, SMAE attenuated carbonyl-modification of specific cytoskeleton and chaperone proteins leading to cell type transformation. Moreover, a network analysis using MetaCore shed more light on the molecular basis associated with SMAE efficacy. SMAE exerts its protective effect through the scavenging of ROS and subsequent modulation of protein carbonylation to inhibit cell proliferation. These signature networks and functional proteomics highlighted herein may facilitate the evaluation of potential therapeutic targets and elucidate novel mechanisms through which protein functions can be regulated by the redox status.

post to: CiteULike

Lipid mediators in the nucleus: Their potential contribution to Alzheimer's disease.

Publication Date: 2010 Feb 17 PMID: 20170745
Authors: Farooqui, A. A. - Ong, W. Y. - Farooqui, T.
Journal: Biochim Biophys Acta

Degradation of glycerophospholipids, sphingolipids and cholesterol in the nucleus modulates neural cell proliferation and differentiation, inflammation, apoptosis, migration, cell adhesion, and intracellular trafficking. Extracellular signals from agonists (neurotransmitters, cytokines, and growth factors) regulate the activity of a key set of lipid-metabolizing enzymes, such as phospholipases, sphingomyelinases, and cholesterol hydroxylases. These enzymes and their downstream targets constitute a complex lipid signaling network with multiple nodes of interaction and cross-regulation through their lipid mediators, which include eicosanoids, docosanoids, diacylglycerols, platelet activating factor, lysophosphatidic acid, ceramide and ceramide 1-phosphate, sphingosine and sphingosine 1-phosphate, and hydroxycholesterols. Receptors for above lipid mediators are localized at the neural cell nucleus. Stimulation of isolated nuclei with these lipids and agonists results in changes in transcriptional regulation of major genes, including c-fos, cylooxygenase-2, secretory phospholipase A(2) and endothelial as well as inducible nitric oxide synthases. Imbalances in signaling network involving above genes may contribute to the pathogenesis of human neurological disorders. In this review, we have attempted to integrate available information on above lipid mediators in the nucleus. In addition, attempts have been made to explain cross-talk among glycerophospholipid-, sphingolipid-, and cholesterol-derived lipid mediators in neural cell death in Alzheimer's disease.

post to: CiteULike

Formation and function of apolipoprotein E-containing lipoproteins in the nervous system.

Publication Date: 2010 Feb 17 PMID: 20170744
Authors: Vance, J. E. - Hayashi, H.
Journal: Biochim Biophys Acta

The strongest known genetic risk factor for the development of late-onset Alzheimer disease is inheritance of the apolipoprotein (apo) E4 (epsilon4 allele) although the mechanisms underlying this connection are still not entirely clear. In this review, we shall discuss the role of apo E in the brain, particularly in relation to Alzheimer disease. Cholesterol transport and homeostasis in the central nervous system (CNS) are separated from that in the peripheral circulation by the blood-brain barrier. However, the brain operates its own lipoprotein transport system that is mediated by high density lipoprotein-sized, apo E-containing lipoproteins that are synthesized and secreted by glial cells (primarily astrocytes). Several ATP-binding cassette (ABC) transporters are expressed in the brain, including ABCA1 and ABCG1 which play important roles in the transfer of phospholipids and cholesterol to apo E. The astrocyte-derived apo E-containing lipoproteins can bind to, and be internalized by, receptors of the low density lipoprotein receptor superfamily that are located on the surface of neurons. In addition to these receptors serving as endocytosis receptors for lipoproteins, several of these receptors also act as signaling receptors in neurons and activate pathways involved in axonal growth, as well as neuronal survival. These beneficial pathways appear to be enhanced to a greater extent by apo E3 than by apo E4. Apo E has also been implicated in the deposition of amyloid plaques since apo E3, more readily than apo E4, forms a complex with Ass peptides, and mediates the degradation of amyloid deposits.

post to: CiteULike

Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery.

Publication Date: 2010 Feb 17 PMID: 20170629
Authors: Yu, H. H. - Nakase, I. - Pujals, S. - Hirose, H. - Tanaka, G. - Katayama, S. - Imanishi, M. - Futaki, S.
Journal: Biochim Biophys Acta

Expressed protein ligation (EPL) is a useful method for the native chemical ligation of proteins with other proteins or peptides. This study assessed the practicability of EPL in the preparation of fusion proteins of enhanced green fluorescent protein (EGFP) with chemically synthesized cell-penetrating peptides (CPPs) for intracellular delivery. Using intein-mediated purification with an affinity chitin-binding tag (IMPACT) system, the thioester of EGFP (EGFP-SR) was prepared. Optimization of the ligation of EGFP-SR with arginine 12-mer (R12) produced the fusion protein in high yield. The EPL procedure also allows the preparation of EGFP-R12 containing a low level of endotoxin (ET), via the satisfactory ET removal of EGFP-SR prior to ligation with the R12 peptide. Fusion proteins of EGFP with R12 and the d-isomer of R12 prepared by EPL showed similar levels of cellular uptake compared to the fusion protein directly expressed in Escherichiacoli.

post to: CiteULike

Morpholinos and their peptide conjugates: Therapeutic promise and challenge for Duchenne muscular dystrophy.

Publication Date: 2010 Feb 17 PMID: 20170628
Authors: Moulton, H. M. - Moulton, J. D.
Journal: Biochim Biophys Acta

Exon-skipping efficacy of phosphodiamidate morpholino oligomers (PMOs) or the conjugates of PMOs with cell-penetrating peptides (PPMOs) has been tested in various animal models of Duchenne muscular dystrophy (DMD), including mdx mice, utrophin-dystrophin double-knockout mice, and CXMD dogs, as well as in DMD patients in clinical trials. The studies have shown that PMOs can diffuse into leaky muscle cells, modify splicing of DMD transcripts, induce expression of partially functional dystrophin, and improve function of some skeletal muscles. PMOs are non-toxic, with a report of mdx mice tolerating a 3g/kg dose, and no drug-related safety issue in human has been reported. However, because of their poor cell uptake and rapid renal clearance, large and frequently repeated doses of PMOs are likely required for functional benefit in some skeletal muscles of DMD patients. In addition, PMOs do not enter cardiomyocytes sufficiently to relieve heart pathology, the efficacy of delivery to various muscles varies greatly, and delivery across the tissue of each skeletal muscle tissue is patchy. PPMOs enter cells at far lower doses, enter cardiomyocytes in useful quantities, and deliver more evenly to myocytes both when different muscles are compared and when assessed at the level of single muscle tissue sections. Compared to PMOs, far lower doses of PPMOs can restore dystrophin sufficiently to reduce disease pathology, increase skeletal and cardiac muscle functions, and prolong survival of animals. The biggest challenge for PPMO is determining safe and effective doses. The toxicity of PPMOs will require caution when moving into the clinic. The first PPMO-based DMD drug is currently in preclinical development for DMD patients who can benefit from skipping exon 50.

post to: CiteULike

Peptide-mediated protein delivery-Which pathways are penetrable?

Publication Date: 2010 Feb 17 PMID: 20170627
Authors: Raagel, H. - Saalik, P. - Pooga, M.
Journal: Biochim Biophys Acta

The growing number of reports on the effective cargo delivery by cell-penetrating peptides (CPPs) has extensively widened our knowledge about the mechanisms involved in CPP-mediated delivery. However, the data available on the internalization mode of CPP-cargo complexes are often conflicting and/or equivocal. Moreover, the intracellular trafficking of CPP-cargo complexes is, to date, relatively unexplored resulting in only minimal information about what is really happening to the complexes inside the cell. This review focuses on defining the endocytic pathways engaged in the transduction of CPP-cargo complexes and seeks to determine the extent of different endocytic routes required for effective uptake. In addition, the intracellular pathways utilized during the trafficking and sorting of CPP-cargo complexes as well as the ultimate fate of the complexes inside cells will be discussed.

post to: CiteULike

The liquid-ordered state comes of age.

Publication Date: 2010 Feb 17 PMID: 20170626
Authors: Mouritsen, O. G.
Journal: Biochim Biophys Acta

Biomembranes are unique states of soft matter that share some of their materials' properties with the mesophases of liquid crystals. Although of genuinely fluid character, membranes can display ordered states under physiological conditions, and it appears that their lateral organization and the related functional properties are intimately coupled to states in-between order and disorder. Hence, the liquid-ordered state of membranes, which owes its existence to the unique ability of cholesterol to mediate between order and disorder, has moved center stage in the characterization of membranes in terms of domains or rafts.

post to: CiteULike

The mechanism of rotating proton pumping ATPases.

Publication Date: 2010 Feb 17 PMID: 20170625
Authors: Nakanishi-Matsui, M. - Sekiya, M. - Nakamoto, R. K. - Futai, M.
Journal: Biochim Biophys Acta

Two proton pumps, the F-ATPase (ATP synthase, F(o)F(1)) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F(1) or V(1)) and a membrane intrinsic (F(o) or V(o)) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60nm, the E. coli F(1) sector was found to rotate at surprisingly high speeds (>400rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the beta subunits, or between the beta and gamma subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F(1)-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.

post to: CiteULike

What are the sources of hydrogen peroxide production by heart mitochondria?

Publication Date: 2010 Feb 17 PMID: 20170624
Authors: Grivennikova, V. G. - Kareyeva, A. V. - Vinogradov, A. D.
Journal: Biochim Biophys Acta

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4-State 3 transition) decreases the succinate- and glutamate plus malate-supported H(2)O(2) production 8- and 1.3-times, respectively. NH(4)(+) strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H(2)O(2) production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H(2)O(2) diffusion by alamethicin treatment are only partially ( approximately 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly ( approximately 6-fold) stimulated by ammonium chloride. NAD(+) inhibits both Complex I-mediated and ammonium-stimulated H(2)O(2) production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).

post to: CiteULike

Marinobufagenin, resibufogenin and preeclampsia.

Publication Date: 2010 Feb 16 PMID: 20167272
Authors: Puschett, J. B. - Agunanne, E. - Uddin, M. N.
Journal: Biochim Biophys Acta

The bufodienolides are cardiac glycosides which have the ability to inhibit the enzyme, Na(+)/K(+) ATPase (sodium potassium adenosine triphosphatase). They are cardiac inotropes, cause vasoconstriction (and, potentially, hypertension) and are natriuretic. Evidence has accrued over time which supports the view that they are mechanistically involved in volume expansion-mediated hypertension. In this communication, the authors summarize data which support the view that the bufodienolides and, in particular, marinobufagenin (MBG) are involved in the pathogenesis of preeclampsia. In a rat model of the syndrome, MBG causes hypertension, proteinuria, intrauterine growth restriction and increased weight gain. All of these phenotypic characteristics are prevented by an antagonist to MBG, resibufogenin (RBG). The "preeclamptic" animals also develop a vascular leak syndrome, resulting in hemoconcentration. Abnormalities in the MAPK (mitogen-activated protein kinase) system play a role in the mechanism by which MBG produces the abnormalities in the pregnant rat. Studies to discover the relevance of these findings to human preeclampsia are currently underway in several laboratories and clinics.

post to: CiteULike

Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure.

Publication Date: 2010 Feb 16 PMID: 20167250
Authors: Mirarefi, A. Y. - Boutet, S. - Ramakrishnan, S. - Kiss, A. J. - Cheng, C. H. - Devries, A. L. - Robinson, I. K. - Zukoski, C. F.
Journal: Biochim Biophys Acta

BACKGROUND: The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (alpha, beta, and gamma). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 degrees C) along with several lenses from Antarctic fish (-2 degrees C) and subtropical bigeye tuna (18 degrees C). METHODS: Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure. RESULTS: We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of alpha-crystallins. CONCLUSIONS: The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller gamma-crystallins in the void space of alpha-crystallins. GENERAL SIGNIFICANCE: Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.

post to: CiteULike

Identification and characterization of proteins that selectively interact with the LHR mRNA binding protein (LRBP) in rat ovaries.

Publication Date: 2010 Feb 16 PMID: 20167237
Authors: Wang, L. - Gulappa, T. - Menon, K. M.
Journal: Biochim Biophys Acta

Luteinizing hormone receptor (LHR) mRNA binding protein (LRBP), identified as mevalonate kinase, has been shown to be a trans factor mediating the post-transcriptional regulation of LHR mRNA expression in ovaries. LRBP binds to the coding region of LHR mRNA and accelerates its degradation. Our previous studies in an in vitro system showed that LRBP represses the translation of LHR mRNA by forming an untranslatable ribonucleoprotein (mRNP) complex, further suggesting that the untranslatable mRNP complex is directed to the mRNA repression/decay machinery for subsequent mRNA turnover. In the present studies, we used yeast two-hybrid system to screen a cDNA library which was constructed from LHR down-regulated ovaries. Two proteins were identified interacting with LRBP: ribosomal protein S20 (RP S20) and ubiquitin conjugating enzyme 2i (UBCE2i). Their interactions with LRBP were confirmed by the mating assay, co-immunoprecipitation analyses and in vitro sumoylation assays. Furthermore, we show that LRBP is a target for modification by SUMO2/3 but not by SUMO1, at K256 and/or K345. Mutation of both lysine residues is sufficient to abrogate the sumoylation of LRBP. These findings suggest that the direct interaction of LRBP with the translation machinery, through RP S20, may be responsible for the transition of LHR mRNA to an untranslatable complex, and that sumoylation of LRBP may play a role in targeting the untranslatable mRNP complex to the mRNA decay machinery in specific cytoplasmic foci.

post to: CiteULike

Gangliosides influence EGFR/ErbB2 heterodimer stability but they do not modify EGF-dependent ErbB2 phosphorylation.

Publication Date: 2010 Feb 12 PMID: 20156584
Authors: Milani, S. - Sottocornola, E. - Zava, S. - Galbiati, M. - Berra, B. - Colombo, I.
Journal: Biochim Biophys Acta

Gangliosides are well-known regulators of cell differentiation through specific interactions with growth factor receptors. Previously, our group provided the first evidence about stable association of ganglioside GM(3) to EGFR/ErbB2 heterodimers in mammary epithelial cells. Goals of the present study were to better define the role of gangliosides in EGFR/ErbB2 heterodimerization and receptor phosphorylation events and to analyze their involvement in mammary cell differentiation. Experiments have been conducted using the ceramide analogue (+/-)-treo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP), which inhibits ceramide glucosyltransferase resulting in the endogenous ganglioside depletion, and the lactogenic hormone mix DIP (dexamethasone, insulin, prolactin), which induces cell differentiation and beta-casein mRNA synthesis. In addition, treatments of ganglioside-depleted cells with exogenous GM(3) have been carried out to ascertain the specific involvement of this ganglioside. Results from co-immunoprecipitation and Western blot experiments have shown that the endogenous ganglioside depletion resulted in the disappearance of SDS-stable EGFR/ErbB2 heterodimers and in the appearance of tyrosine-phosphorylated EGFR also in the absence of EGF stimulation; exogenous GM(3) added in combination with [D]-PDMP reversed both these effects. In contrast, the tyrosine phosphorylation of ErbB2 in ganglioside-depleted cells occurred only after EGF stimulation. Moreover, when ganglioside-depleted cells were treated with DIP in absence of EGF, beta-casein gene expression appeared strongly down-regulated, and beta-casein mRNA levels were partially restored by exogenous GM(3) treatment. Altogether, although the involvement of other ganglioside species cannot be excluded, these findings sustain the ganglioside GM(3) as an essential molecule for EGFR/ErbB2 heterodimer stability and important regulator of EGFR tyrosine phosphorylation, but it is not crucial for tyrosine phosphorylation of the heterodimerization partner ErbB2. Moreover, modulation of EGFR phosphorylation may explain how gangliosides contribute to regulate the lactogenic hormone-induced mammary cell differentiation.

post to: CiteULike

Apolipoprotein A-V associates with intrahepatic lipid droplets and influences triglyceride accumulation.

Publication Date: 2010 Feb 11 PMID: 20153840
Authors: Shu, X. - Nelbach, L. - Ryan, R. O. - Forte, T. M.
Journal: Biochim Biophys Acta

Apolipoprotein A-V (apoA-V), secreted solely by the liver, is a low abundance protein that strongly influences plasma triglyceride (TG) levels. In vitro, in transfected hepatoma cell lines apoA-V is largely retained within the cell in association with cytosolic lipid droplets (LD). To evaluate if this is true in vivo, in the present study the amount of apoA-V in the plasma compartment versus liver tissue was determined in APOA5 transgenic (Tg) mice. The majority of total apoA-V ( approximately 80%) was in the plasma compartment. Injection of APOA5 Tg mice with heparin increased plasma apoA-V protein levels by approximately 25% indicating the existence of a heparin-releasable pool. Intrahepatic apoA-V was associated with LD isolated from livers of wild type (WT) and APOA5 Tg mice. Furthermore, livers from APOA5 Tg mice contained significantly higher amounts of TG than livers from WT or apoa5 knockout mice suggesting that apoA-V influences intrahepatic TG levels.

post to: CiteULike

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation.

Publication Date: 2010 Feb 11 PMID: 20153839
Authors: Mahammad, S. - Dinic, J. - Adler, J. - Parmryd, I.
Journal: Biochim Biophys Acta

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10-20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca(2+). Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more liquid ordered domains forming at the expense of liquid disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD-cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.

post to: CiteULike

Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts.

Publication Date: 2010 Feb 11 PMID: 20153825
Authors: Moran, M. - Rivera, H. - Sanchez-Arago, M. - Blazquez, A. - Merinero, B. - Ugalde, C. - Arenas, J. - Cuezva, J. M. - Martin, M. A.
Journal: Biochim Biophys Acta

Background: Complex I (CI) deficiency is the most frequent cause of OXPHOS disorders. Recent studies have shown increases in reactive oxygen species (ROS) production and mitochondrial network disturbances in patients' fibroblasts harbouring mutations in CI subunits. Objectives: The present work evaluates the impact of mutations in the NDUFA1 and NDUFV1 genes of CI on mitochondrial bioenergetics and dynamics, in fibroblasts from patients suffering isolated CI deficiency. Results: Decreased oxygen consumption rate and slow growth rate were found in patients with severe CI deficiency. Mitochondrial diameter was slightly increased in patients' cells cultured in galactose or treated with 2'-deoxyglucose without evidence of mitochondrial fragmentation. Expression levels of the main proteins involved in mitochondrial dynamics, OPA1, MFN2, and DRP1, were slightly augmented in all patients' cells lines. The study of mitochondrial dynamics showed delayed recovery of the mitochondrial network after treatment with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone (cccp) in patients with severe CI deficiency. Intracellular ROS levels were not increased neither in glucose nor galactose medium in patients' fibroblasts. Conclusion: Our main finding was that severe CI deficiency in patients harbouring mutations in the NDUFA1 and NDUFV1 genes is linked to a delayed mitochondrial network recovery after cccp treatment. However, the CI deficiency is neither associated with massive mitochondrial fragmentation nor with increased ROS levels. The different genetic backgrounds of patients with OXPHOS disorders would explain, at least partially, differences in the pathophysiological manifestations of CI deficiency.

post to: CiteULike

Dopamine and G protein-coupled receptor kinase 4 in the kidney: Role in blood pressure regulation.

Publication Date: 2010 Feb 12 PMID: 20153824
Authors: Jose, P. A. - Soares-da-Silva, P. - Eisner, G. M. - Felder, R. A.
Journal: Biochim Biophys Acta

Complex interactions between genes and environment result in a sodium-induced elevation in blood pressure (salt sensitivity) and/or hypertension that lead to significant morbidity and mortality affecting up to 25% of the middle-aged adult population worldwide. Determining the etiology of genetic and/or environmentally-induced high blood pressure has been difficult because of the many interacting systems involved. Two main pathways have been implicated as principal determinants of blood pressure since they are located in the kidney (the key organ responsible for blood pressure regulation), and have profound effects on sodium balance: the dopaminergic and renin-angiotensin systems. These systems counteract or modulate each other, in concert with a host of intracellular second messenger pathways to regulate sodium and water balance. In particular, the G protein-coupled receptor kinase type 4 (GRK4) appears to play a key role in regulating dopaminergic-mediated natriuresis. Constitutively activated GRK4 gene variants (R65L, A142V, and A486V), by themselves or by their interaction with other genes involved in blood pressure regulation, are associated with essential hypertension and/or salt-sensitive hypertension in several ethnic groups. GRK4gamma 142Vtransgenic mice are hypertensive on normal salt intake while GRK4gamma 486V transgenic mice develop hypertension only with an increase in salt intake. GRK4 gene variants have been shown to hyperphosphorylate, desensitize, and internalize two members of the dopamine receptor family, the D(1) (D(1)R) and D(3) (D(3)R) dopamine receptors, but also increase the expression of a key receptor of the renin-angiotensin system, the angiotensin type 1 receptor (AT(1)R). Knowledge of the numerous blood pressure regulatory pathways involving angiotensin and dopamine may provide new therapeutic approaches to the pharmacological regulation of sodium excretion and ultimately blood pressure control.

post to: CiteULike

ISG15 and immune diseases.

Publication Date: 2010 Feb 12 PMID: 20153823
Authors: Jeon, Y. J. - Yoo, H. M. - Chung, C. H.
Journal: Biochim Biophys Acta

ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.

post to: CiteULike

Functional analysis of H. sapiens DNA polymerase gamma spacer mutation W748S with and without common variant E1143G.

Publication Date: 2010 Feb 12 PMID: 20153822
Authors: Palin, E. J. - Lesonen, A. - Farr, C. L. - Euro, L. - Suomalainen, A. - Kaguni, L. S.
Journal: Biochim Biophys Acta

Mitochondrial DNA polymerase, POLG, is the sole DNA polymerase found in animal mitochondria. In humans, POLGalpha W748S in cis with an E1143G mutation has been linked to a new type of recessive ataxia, MIRAS, which is the most common inherited ataxia in Finland. We investigated the biochemical phenotypes of the W748S amino acid change, using recombinant human POLG. We measured processive and non-processive DNA polymerase activity, DNA binding affinity, enzyme processivity, and subunit interaction with recombinant POLGbeta. In addition, we studied the effects of the W748S and E1143G mutations in primary human cell cultures using retroviral transduction. Here, we examined cell viability, mitochondrial DNA copy number, and products of mitochondrial translation. Our results indicate that the W748S mutant POLGalpha does not exhibit a clear biochemical phenotype, making it indistinguishable from wild type POLGalpha and as such, fail to replicate previously published results. Furthermore, results from the cell models were concurrent with the findings from patients, and support our biochemical findings.

post to: CiteULike

Muscle transfection and permeabilization induced by electrotransfer or pluronic(R) L64 Paired study by optical imaging and MRI.

Publication Date: 2010 Feb 12 PMID: 20153812
Authors: Bureau, M. F. - Juge, L. - Seguin, J. - Rager, M. N. - Scherman, D. - Mignet, N.
Journal: Biochim Biophys Acta

BACKGROUND: Muscle transfection by electrotranfer is an efficient currently used procedure. Recently, the block copolymer pluronic L64 has been reported to improve muscle transfection. Both procedures are known to permeabilize muscle fibres. Relation between muscle transfection and permeabilization by electrotransfer and L64 was investigated herein. METHODS: Muscle transfection was evaluated by optical detection of the luciferase reporter gene activity. Muscle permeabilization was evaluated by the uptake of the T1 contrast agent gadolinium-Dotarem (Gd-DOTA) using Magnetic resonance imaging (MRI). Histological examination of muscle sections was also performed. RESULTS: Electrotransfer and L64 (at a 0.25% concentration) similarly improved muscle transfection, although the interindividual variability was higher for pluronic. On the same animals, the permeabilized volume to the Gd-DOTA was significantly increased after electrotransfer, and L64 from 0.1% to 1%. The concentration of the Gd-DOTA in the permeabilized volume was significantly increased after electrotransfer and L64 at 0.5% and 1%. By histological observation, the inflammation was maximum at day 3 after electrotransfer or L64 injection, and mostly reversed after 7days. The permeabilized volume and the transfection level correlated for the set of all the conditions tested. However, no significant correlation was observed between Gd-DOTA concentration and transfection. GENERAL SIGNIFICANCE: It is possible to use successively on the same animals MRI and optical imaging for paired studies of muscle transfection and permeabilization. Permeabilization is possibly not related to gene transfer but it indicates membrane modification related to transfection by the electrotransfer or co-injection of DNA with the L64.

post to: CiteULike

A conserved function for Inp2 in peroxisome inheritance.

Publication Date: 2010 Feb 11 PMID: 20153784
Authors: Saraya, R. - Cepinska, M. N. - Kiel, J. A. - Veenhuis, M. - der Klei, I. J.
Journal: Biochim Biophys Acta

In budding yeast Saccharomyces cerevisiae, the peroxisomal protein Inp2 is required for inheritance of peroxisomes to the bud, by connecting the organelles to the motor protein Myo2 and the actin cytoskeleton. Recent data suggested that the function of Inp2 may not be conserved in other yeast species. Using in silico analyses we have identified a weakly conserved Inp2-related protein in 18 species of budding yeast and analyzed the role of the identified protein in the methylotrophic yeast Hansenula polymorpha in peroxisome inheritance. Our data show that H. polymorpha Inp2 locates to peroxisomes, interacts with Myo2, and is essential for peroxisome inheritance.

post to: CiteULike

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes.

Publication Date: 2010 Feb 12 PMID: 20153720
Authors: Cui, Z. K. - Bastiat, G. - Jin, C. - Keyvanloo, A. - Lafleur, M.
Journal: Biochim Biophys Acta

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and (2)H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.

post to: CiteULike

Erythrocyte death in vitro induced by starvation in the absence of Ca(2+).

Publication Date: 2010 Feb 12 PMID: 20153719
Authors: Pompeo, G. - Girasole, M. - Cricenti, A. - Boumis, G. - Bellelli, A. - Amiconi, S.
Journal: Biochim Biophys Acta

Human erythrocytes (RBCs), stored at 4 degrees C under nominal absence of external energy sources and calcium ions, show a gradual decrease in membrane roughness (R(rms)) at the end of which the appearance of morphological phenomena (spicules, vesicles and spherocytes) is observed on the cell membrane, phenomena that can mainly be ascribed to the ATP-dependent disconnection of the cortical cytoskeleton from the lipid bilayer. After depletion of the intracellular energy sources obtained under the extreme conditions chosen, treatment with a minimal rejuvenation solution makes the following remarks possible: (i) RBCs are able to regenerate adenosine triphosphate (ATP) and 2,3-bisphosphoglycerate only up to 4days of storage at 4 degrees C, whereas from the eighth day energy stocks cannot be replenished because of a disorder in the transmembrane mechanisms of transport; (ii) the RBCs' roughness may be restored to the initial value (i.e. that observed in fresh RBCs) only in samples stored up to 4-5days, whereas after the eighth day of storage the rejuvenation procedure appears to be inefficient; (iii) membrane physical properties - as measured by R(rms) - are actually controlled by the metabolic production of ATP, necessary to perform the RBCs' basic functions; (iv) once energy stores cannot be replenished, a regulated sequence of the morphological events (represented by local buckles that lead to formation of spicules and vesicles of the lipid bilayer with generation of spherocytes) is reminiscent of the RBCs' apoptotic final stages; (v) the morphological phenomenology of the final apoptotic stages is passive (i.e. determined by simple mechanical forces) and encoded in the mechanical properties of the membrane-skeleton; and (vi) necrotic aspects (e.g. disruption of cell membrane integrity, so that intracellular protein content is easily released) ensue when RBCs are almost totally (>/=90%) depleted in an irreversible way of the energetic stores.

post to: CiteULike

Pore-forming properties of the Bacillus thuringiensis toxin Cry9Ca in Manduca sexta brush border membrane vesicles.

Publication Date: 2010 Feb 11 PMID: 20153718
Authors: Brunet, J. F. - Vachon, V. - Juteau, M. - Van Rie, J. - Larouche, G. - Vincent, C. - Schwartz, J. L. - Laprade, R.
Journal: Biochim Biophys Acta

The toxicity and pore-forming ability of the Bacillus thuringiensis Cry9Ca insecticidal toxin, its single-site mutants, R164A and R164K, and the 55-kDa fragment resulting from its proteolytic cleavage at residue 164 were investigated using Manduca sexta neonate larvae and fifth-instar larval midgut brush border membrane vesicles, respectively. Neither the mutations nor the proteolytic cleavage altered Cry9Ca toxicity. Compared with Cry1Ac, Cry9Ca and its mutants formed large poorly selective pores in the vesicles. Pore formation was highly dependent on pH, however, especially for wild-type Cry9Ca and both mutants. Increasing pH from 6.5 to 10.5 resulted in an irregular step-wise decrease in membrane permeabilization that was not related to a change in the ionic selectivity of the pores. Pore formation was much slower with Cry9Ca and its derivatives, including the 55-kDa fragment, than with Cry1Ac and its rate was not influenced by the presence of protease inhibitors or a reducing agent.

post to: CiteULike

Hypoxia and mitochondrial oxidative metabolism.

Publication Date: 2010 Feb 11 PMID: 20153717
Authors: Solaini, G. - Baracca, A. - Lenaz, G. - Sgarbi, G.
Journal: Biochim Biophys Acta

It is now clear that mitochondrial defects are associated with a large variety of clinical phenotypes. This is the result of the mitochondria's central role in energy production, reactive oxygen species homeostasis, and cell death. These processes are interdependent and may occur under various stressing conditions, among which low oxygen levels (hypoxia) are certainly prominent. Cells exposed to hypoxia respond acutely with endogenous metabolites and proteins promptly regulating metabolic pathways, but if low oxygen levels are prolonged, cells activate adapting mechanisms, the master switch being the hypoxia-inducible factor 1 (HIF-1). Activation of this factor is strictly bound to the mitochondrial function, which in turn is related with the oxygen level. Therefore in hypoxia, mitochondria act as [O(2)] sensors, convey signals to HIF-1directly or indirectly, and contribute to the cell redox potential, ion homeostasis, and energy production. Although over the last two decades cellular responses to low oxygen tension have been studied extensively, mechanisms underlying these functions are still indefinite. Here we review current knowledge of the mitochondrial role in hypoxia, focusing mainly on their role in cellular energy and reactive oxygen species homeostasis in relation with HIF-1 stabilization. In addition, we address the involvement of HIF-1 and the inhibitor protein of F(1)F(0) ATPase in the hypoxia-induced mitochondrial autophagy.

post to: CiteULike

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties, permeabilization and cell death.

Publication Date: 2010 Feb 11 PMID: 20153716
Authors: Montero, J. - Mari, M. - Colell, A. - Morales, A. - Basanez, G. - Garcia-Ruiz, C. - Fernandez-Checa, J. C.
Journal: Biochim Biophys Acta

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.

post to: CiteULike

Sp1 and Sp3 regulate transcription of the chicken GAS41 gene.

Publication Date: 2010 Feb 11 PMID: 20153453
Authors: Hubner, K. - Phi-van, L.
Journal: Biochim Biophys Acta

The 5'-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3' end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene.

post to: CiteULike

DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis.

Publication Date: 2010 Feb 11 PMID: 20153451
Authors: Li, H. - Chang, L. - Howell, J. M. - Turner, R. J.
Journal: Biochim Biophys Acta

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein-protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a "node" in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

post to: CiteULike

Altered lipid A structures and polymyxin hypersensitivity of Rhizobium etli mutants lacking the LpxE and LpxF phosphatases.

Publication Date: 2010 Feb 11 PMID: 20153447
Authors: Ingram, B. O. - Sohlenkamp, C. - Geiger, O. - Raetz, C. R.
Journal: Biochim Biophys Acta

The lipid A of Rhizobium etli, a nitrogen-fixing plant endosymbiont, displays significant structural differences when compared to that of Escherichia coli. An especially striking feature of R. etli lipid A is that it lacks both the 1- and 4'-phosphate groups. The 4'-phosphate moiety of the distal glucosamine unit is replaced with a galacturonic acid residue. The dephosphorylated proximal unit is present as a mixture of the glucosamine hemiacetal and an oxidized 2-aminogluconate derivative. Distinct lipid A phosphatases directed to the 1 or the 4'-positions have been identified previously in extracts of R. etli and Rhizobium leguminosarum. The corresponding structural genes, lpxE and lpxF, respectively, have also been identified. Here, we describe the isolation and characterization of R. etli deletion mutants in each of these phosphatase genes and the construction of a double phosphatase mutant. Mass spectrometry confirmed that the mutant strains completely lacked the wild-type lipid A species and accumulated the expected phosphate-containing derivatives. Moreover, radiochemical analysis revealed that phosphatase activity was absent in membranes prepared from the mutants. Our results indicate that LpxE and LpxF are solely responsible for selectively dephosphorylating the lipid A molecules of R. etli. All the mutant strains showed an increased sensitivity to polymyxin relative to the wild-type. However, despite the presence of altered lipid A species containing one or both phosphate groups, all the phosphatase mutants formed nitrogen-fixing nodules on Phaseolus vulgaris. Therefore, the dephosphorylation of lipid A molecules in R. etli is not required for nodulation but may instead play a role in protecting the bacteria from cationic antimicrobial peptides or other immune responses of plants.

post to: CiteULike

Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods.

Publication Date: 2010 Feb 11 PMID: 20153401
Authors: Tolmachev, V. - Stone-Elander, S.
Journal: Biochim Biophys Acta

BACKGROUND: Dynamic biomedical research is currently yielding a wealth of information about disease-associated molecular alterations on cell surfaces and in the extracellular space. The ability to visualize and quantify these alterations in vivo could provide important diagnostic information and be used to guide individually-optimized therapy. Biotechnology can provide proteinaceous molecular probes with highly specific target recognitions. Suitably labelled, these may be used as tracers for radionuclide-based imaging of molecular disease signatures. If the labels are positron-emitting radionuclides, the superior resolution, sensitivity and quantification capability of positron emission tomography (PET) can be exploited. SCOPE OF REVIEW: This article discusses different approaches to labelling proteins with positron-emitting nuclides with suggestions made depending on the biological features of the tracers. MAJOR CONCLUSIONS: Factors such as matching biological and physical half-lives, availability of the nuclide, labelling yields, and influences of labelling on targeting properties (affinity, charge and lipophilicity, cellular processing and retention of catabolites) should be considered when selecting a labelling strategy for each proteinaceous tracer. GENERAL SIGNIFICANCE: The labelling strategy used can make all the difference between success and failure in a tracer application. This review emphasises chemical, biological and pharmacological considerations in labelling proteins with positron-emitting radionuclides.

post to: CiteULike

Unravelling the molecular basis of the selectivity of the HIV-1 fusion inhibitor sifuvirtide towards phosphatidylcholine-rich rigid membranes.

Publication Date: 2010 Feb 11 PMID: 20153294
Authors: Franquelim, H. G. - Veiga, A. S. - Weissmuller, G. - Santos, N. C. - Castanho, M. A.
Journal: Biochim Biophys Acta

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel HIV-1 fusion inhibitor with improved antiretroviral activity. In this work we evaluated the physical chemistry foundation of the interaction of sifuvirtide with biomembrane model systems. Since this peptide has aromatic residues, fluorescence spectroscopy techniques were mostly used. The interaction was assessed by partition and quenching experiments. Results showed no significant interaction with large unilamellar vesicles composed by sphingomyelin and ceramide. In contrast, sifuvirtide presented selectivity towards vesicles composed by phosphatidylcholines (PC) in the gel phase, in opposition to fluid phase PC vesicles. The interaction of this peptide with gel phase PC membranes (K(p)=1.2x10(2)) is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at an interfacial level. The effects of sifuvirtide on the lipid membranes' structural properties were further evaluated using dipole-potential membrane probes, zeta-potential, dynamic light scattering and atomic force microscopy measurements. The results show that sifuvirtide does not cause a noticeable effect on lipid bilayer structure, except for membranes composed by cationic phospholipids. Altogether, we can conclude that sifuvirtide presents a specific affinity towards rigid PC membranes, and the interaction is mediated by electrostatic factors, not affecting the membrane architecture.

post to: CiteULike

Interaction of the N-terminal segment of HCV protein NS5A with model membranes.

Publication Date: 2010 Feb 11 PMID: 20153293
Authors: Palomares-Jerez, M. F. - Guillen, J. - Villalain, J.
Journal: Biochim Biophys Acta

We have identified a membrane-active region in the HCV NS5A protein by performing an exhaustive study of membrane rupture induced by a NS5A-derived peptide library on model membranes having different phospholipid compositions. We report the identification in NS5A of a highly membranotropic region located at the suggested membrane association domain of the protein. We report the binding and interaction with model membranes of two peptides patterned after this segment, peptides 1A and 1B, derived from the strains 1a_H77 and 1b_HC-4J respectively. We show that they insert into phospholipid membranes, interact with them, and are located in a shallow position in the membrane. The NS5A region where this segment resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the replication complex, and consequently, directly implicated in the HCV life cycle.

post to: CiteULike

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic regulatory domains of ARNO.

Publication Date: 2010 Feb 11 PMID: 20153292
Authors: Merkulova, M. - Bakulina, A. - Thaker, Y. R. - Gruber, G. - Marshansky, V.
Journal: Biochim Biophys Acta

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant K(D)=3.44x10(-)(7)M, similar to the K(D)=3.13x10(-)(7)M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser(392). The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser(392) phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.

post to: CiteULike

Role of FtsH2 in the repair of Photosystem II in mutants of the cyanobacterium Synechocystis PCC 6803 with impaired assembly or stability of the CaMn(4) cluster.

Publication Date: 2010 Feb 11 PMID: 20153291
Authors: Komenda, J. - Knoppova, J. - Krynicka, V. - Nixon, P. J. - Tichy, M.
Journal: Biochim Biophys Acta

The FtsH2 protease, encoded by the slr0228 gene, plays a key role in the selective degradation of photodamaged D1 protein during the repair of Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. To test whether additional proteases might be involved in D1 degradation during high rates of photodamage, we have studied the synthesis and degradation of the D1 protein in DeltaPsbO and DeltaPsbV mutants, in which the CaMn(4) cluster catalyzing oxygen evolution is less stable, and in the D1 processing mutants, D1-S345P and DeltaCtpA, which are unable to assemble a functional cluster. All four mutants exhibited a dramatically increased rate of D1 degradation in high light compared to the wild-type. Additional inactivation of the ftsH2 gene slowed the rate of D1 degradation dramatically and increased the level of PSII complexes. We conclude that FtsH2 plays a major role in the degradation of both precursor and mature forms of D1 following donor-side photoinhibition. However, this conclusion concerned only D1 assembled into larger complexes containing at least D2 and CP47. In the DeltapsbEFLJ deletion mutant blocked at an early stage in PSII assembly, unassembled D1 protein was efficiently degraded in the absence of FtsH2 pointing to the involvement of other protease(s). Significantly, the DeltaPsbO mutant displayed unusually low levels of cellular chlorophyll at extremely low-light intensities. The possibilities that PSII repair may limit the availability of chlorophyll for the biogenesis of other chlorophyll-binding proteins and that PsbO might have a regulatory role in PSII repair are discussed.

post to: CiteULike

Role of calcineurin, hnRNPA2 and Akt in mitochondrial respiratory stress-mediated transcription activation of nuclear gene targets.

Publication Date: 2010 Feb 11 PMID: 20153290
Authors: Guha, M. - Tang, W. - Sondheimer, N. - Avadhani, N. G.
Journal: Biochim Biophys Acta

Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (psim) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca(2+) [Ca(2+)](c) as part of this signaling cascade activates Ca(2+) responsive phosphatase, calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis is a phenotypic hallmark of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFkappaB (cRel:p50) pathway, NFAT, CREB and C/EBPdelta. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFkappaB (cRel:p50), C/EBPdelta, CREB, and NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional coactivator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes.

post to: CiteULike

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site.

Publication Date: 2010 Mar PMID: 20152788
Authors: Fabbro, D. - Manley, P. W. - Jahnke, W. - Liebetanz, J. - Szyttenholm, A. - Fendrich, G. - Strauss, A. - Zhang, J. - Gray, N. S. - Adrian, F. - Warmuth, M. - Pelle, X. - Grotzfeld, R. - Berst, F. - Marzinzik, A. - Cowan-Jacob, S. W. - Furet, P. - Mestan, J.
Journal: Biochim Biophys Acta

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr-Abl, fail to effectively suppress the Bcr-Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr-Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

post to: CiteULike

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL (Agrocybe aegerita lectin).

Publication Date: 2010 Apr PMID: 20122994
Authors: Liang, Y. - Lin, J. C. - Wang, K. - Chen, Y. J. - Liu, H. H. - Luan, R. - Jiang, S. - Che, T. - Zhao, Y. - Li, D. F. - Wang, D. C. - Guo, L. - Sun, H.
Journal: Biochim Biophys Acta

BACKGROUND: We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear. METHODS: We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand. RESULTS: The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment. CONCLUSION: MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL. GENERAL SIGNIFICANCE: These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.

post to: CiteULike

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic, alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7.

Publication Date: 2010 Apr PMID: 20116461
Authors: Subasinghage, A. P. - Conlon, J. M. - Hewage, C. M.
Journal: Biochim Biophys Acta

Peptide XT-7 (GLLGP(5)LLKIA(10)AKVGS(15)NLL.NH(2)) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC(50)=140microM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC(50)>500microM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O mixed solvent system, XT-7 is characterised by a right handed alpha-helical conformation between residues Leu(3) and Leu(17) whereas [G4K]XT-7 adopts a more restricted alpha-helical conformation between residues Leu(6) and Leu(17). A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu(3) and Leu(17). However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H(2)O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly(4)-->Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.

post to: CiteULike

Human CRISP-3 binds serum alpha(1)B-glycoprotein across species.

Publication Date: 2010 Apr PMID: 20116414
Authors: Udby, L. - Johnsen, A. H. - Borregaard, N.
Journal: Biochim Biophys Acta

BACKGROUND: CRISP-3 was previously shown to be bound to alpha(1)B-glycoprotein (A1BG) in human serum/plasma. All mammalian sera are supposed to contain A1BG, although its presence in rodent sera is not well-documented. Since animal sera are often used to supplement buffers in experiments, in particular such that involve cell cultures, binding proteins present in sera might interfere in the experiments. METHODS: We examined sera from five different animal species for CRISP-3 binding proteins using gel filtration and ligand blotting. We developed a rapid method for isolation of proteins that bind to human CRISP-3 and identified the isolated proteins by mass spectrometry and N-terminal sequencing. RESULTS: We identified A1BG as a CRISP-3 binding protein in sera from cow, horse and rabbit. CRISP-3 bound kininogen 1 in mouse serum, whereas rat serum showed no CRISP-3 binding activity. In equine serum, we furthermore detected a possible CRISP, already bound to A1BG. GENERAL SIGNIFICANCE: It seems to be a common mechanism that A1BGs bind CRISPs, also across species. Apart from the possible physiological implications hereof, complex binding of CRISPs by A1BG (and other proteins) may interfere with the detection and function of CRISPs, when these are studied in the presence of animal sera.

post to: CiteULike

PONDR-FIT: A meta-predictor of intrinsically disordered amino acids.

Publication Date: 2010 Apr PMID: 20100603
Authors: Xue, B. - Dunbrack, R. L. - Williams, R. W. - Dunker, A. K. - Uversky, V. N.
Journal: Biochim Biophys Acta

Protein intrinsic disorder is becoming increasingly recognized in proteomics research. While lacking structure, many regions of disorder have been associated with biological function. There are many different experimental methods for characterizing intrinsically disordered proteins and regions; nevertheless, the prediction of intrinsic disorder from amino acid sequence remains a useful strategy especially for many large-scale proteomic investigations. Here we introduced a consensus artificial neural network (ANN) prediction method, which was developed by combining the outputs of several individual disorder predictors. By eight-fold cross-validation, this meta-predictor, called PONDR-FIT, was found to improve the prediction accuracy over a range of 3 to 20% with an average of 11% compared to the single predictors, depending on the datasets being used. Analysis of the errors shows that the worst accuracy still occurs for short disordered regions with less than ten residues, as well as for the residues close to order/disorder boundaries. Increased understanding of the underlying mechanism by which such meta-predictors give improved predictions will likely promote the further development of protein disorder predictors. Access to PONDR-FIT is available at www.disprot.org.

post to: CiteULike

Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans.

Publication Date: 2010 Apr PMID: 20100602
Authors: Sanggaard, K. W. - Hansen, L. - Scavenius, C. - Wisniewski, H. G. - Kristensen, T. - Thogersen, I. B. - Enghild, J. J.
Journal: Biochim Biophys Acta

The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC*TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2*bikunin. In addition, Gg pre-alpha-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein-glycosaminoglycan-protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2*bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-alpha-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC.HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility.

post to: CiteULike

Critical role of interfaces and agitation on the nucleation of Abeta amyloid fibrils at low concentrations of Abeta monomers.

Publication Date: 2010 Apr PMID: 20100601
Authors: Morinaga, A. - Hasegawa, K. - Nomura, R. - Ookoshi, T. - Ozawa, D. - Goto, Y. - Yamada, M. - Naiki, H.
Journal: Biochim Biophys Acta

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid beta-peptide (Abeta) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Abeta amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20muM Abeta in the presence or absence of air-water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Abeta concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Abeta concentrations (10 and 20muM). Importantly, when the 5muM Abeta solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5muM Abeta solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5muM Abeta solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.

post to: CiteULike

Enhanced thermal and ultrasonic stability of a fungal protease encapsulated within biomimetically generated silicate nanospheres.

Publication Date: 2010 Apr PMID: 20100547
Authors: Madadlou, A. - Iacopino, D. - Sheehan, D. - Emam-Djomeh, Z. - Mousavi, M. E.
Journal: Biochim Biophys Acta

BACKGROUND: Dendrimers are highly branched synthetic macromolecules with a globular shape. They have been successfully used for generation of nanospheres at mild conditions via biomimetic silicification. Encapsulation of enzyme molecules within these nanospheres during their synthesis is a promising method for rapid and efficient entrapment of several enzymes. However, encapsulation of proteolytic enzymes has been rarely done via biomimetic silicification. As well, the operational stability of encapsulated enzyme has not been systematically reported. METHODS: A proteolytic enzyme, either alpha-Chymotrypsin or a fungal protease from Aspergilus Oryzea was encapsulated along with iron oxide nanoparticles within particles yielded via biomimetic silicification of different generations of polyamidoamine (PAMAM) dendrimers. Stability of encapsulated enzyme was compared to that of free enzyme during storage at room temperature. As well, their thermal and ultrasonic stabilities were measured. Scanning electron microscopy, transmission electron microscopy and optical microscopy were used to investigate the morphology of nanospheres. RESULTS: Determination of encapsulation efficiency revealed that approximately 85% of fungal protease with concentration 1.4mg mL(-1) stock solution was immobilized within particles yielded by generation 0. Based on microscopic images the generated particles interconnected with each other and had spherical morphologies independent of generation. Kinetic analysis of encapsulated fungal protease demonstrated that Mechaelis-Menten constant (K(m)) slightly increased. CONCLUSION: PAMAM dendrimer generation 0 could be effectively used for rapid encapsulation of a fungal protease from Aspegilus Oryzae. GENERAL SIGNIFICANCE: Encapsulation significantly enhances the thermal and ultrasonic stabilities of enzymes, suggesting a range of diverse applications for them.

post to: CiteULike

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1.

Publication Date: 2010 Mar PMID: 20100457
Authors: Lee, T. H. - Hall, K. N. - Swann, M. J. - Popplewell, J. F. - Unabia, S. - Park, Y. - Hahm, K. S. - Aguilar, M. I.
Journal: Biochim Biophys Acta

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

post to: CiteULike

Energy conservation by Rhodothermus marinus respiratory complex I.

Publication Date: 2010 Apr PMID: 20100453
Authors: Batista, A. P. - Fernandes, A. S. - Louro, R. O. - Steuber, J. - Pereira, M. M.
Journal: Biochim Biophys Acta

A sodium ion efflux, together with a proton influx and an inside-positive DeltaPsi, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by (23)Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.

post to: CiteULike

Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate.

Publication Date: 2010 Apr PMID: 20097313
Authors: Agarwal, N. K. - Mueller, G. A. - Mueller, C. - Streich, J. H. - Asif, A. R. - Dihazi, H.
Journal: Biochim Biophys Acta

Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate-treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p<0.05) in the HL-60 cell proteome after treatment with 2.5microM methotrexate for 72h. We found that three structural alpha4, alpha5, alpha7 proteasome subunits, a non-catalytic beta3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-kappaB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-kappaB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.

post to: CiteULike

Residue 234 is a master switch of the alternative-substrate activity profile of human and rodent theta class glutathione transferase T1-1.

Publication Date: 2010 Apr PMID: 20097269
Authors: Shokeer, A. - Mannervik, B.
Journal: Biochim Biophys Acta

BACKGROUND: The Theta class glutathione transferase GST T1-1 is a ubiquitously occurring detoxication enzyme. The rat and mouse enzymes have high catalytic activities with numerous electrophilic compounds, but the homologous human GST T1-1 has comparatively low activity with the same substrates. A major structural determinant of substrate recognition is the H-site, which binds the electrophile in proximity to the nucleophilic sulfur of the second substrate glutathione. The H-site is formed by several segments of amino acid residues located in separate regions of the primary structure. The C-terminal helix of the protein serves as a lid over the active site, and contributes several residues to the H-site. METHODS: Site-directed mutagenesis of the H-site in GST T1-1 was used to create the mouse Arg234Trp for comparison with the human Trp234Arg mutant and the wild-type rat, mouse, and human enzymes. The kinetic properties were investigated with an array of alternative electrophilic substrates to establish substrate selectivity profiles for the different GST T1-1 variants. RESULTS: The characteristic activity profile of the rat and mouse enzymes is dependent on Arg in position 234, whereas the human enzyme features Trp. Reciprocal mutations of residue 234 between the rodent and human enzymes transform the substrate-selectivity profiles from one to the other. CONCLUSIONS: H-site residue 234 has a key role in governing the activity and substrate selectivity profile of GST T1-1. GENERAL SIGNIFICANCE: The functional divergence between human and rodent Theta class GST demonstrates that a single point mutation can enable or suppress enzyme activities with different substrates.

post to: CiteULike

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments.

Publication Date: 2010 Apr PMID: 20097156
Authors: Yanykin, D. V. - Khorobrykh, A. A. - Khorobrykh, S. A. - Klimov, V. V.
Journal: Biochim Biophys Acta

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O(2) photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O(2) photoreduction on the acceptor side of PSII, there is light-induced O(2) consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O(2) uptake on the donor side of PSII is related to interaction of O(2) with radicals produced by photooxidation of organic molecules. The study of flash-induced O(2) uptake finds that removal of Mn from the WOC leads to O(2) photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O(2) evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O(2) uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10muM, corresponding to 2-4 Mn per RC) of Mn(2+), while at higher concentrations (>100muM) Mn(2+) inhibits the O(2) photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn(2+)) leads to both suppression of flash-induced O(2) uptake and disappearance of the Mn-induced activation of the O(2) photoconsumption. We assume that the light-induced O(2) uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O(2) or its reactive forms in the formation of the inorganic core of the WOC.

post to: CiteULike

Photosystem I light-harvesting complex Lhca4 adopts multiple conformations: Red forms and excited-state quenching are mutually exclusive.

Publication Date: 2010 Apr PMID: 20097154
Authors: Passarini, F. - Wientjes, E. - van Amerongen, H. - Croce, R.
Journal: Biochim Biophys Acta

In this work we have investigated the origin of the multi-exponential fluorescence decay and of the short excited-state lifetime of Lhca4. Lhca4 is the antenna complex of Photosystem I which accommodates the red-most chlorophyll forms and it has been proposed that these chlorophylls can play a role in fluorescence quenching. Here we have compared the fluorescence decay of Lhca4 with that of several Lhca4 mutants that are affected in their red form content. The results show that neither the multi-exponentiality of the decay nor the fluorescence quenching is due to the red forms. The data indicate that Lhca4 exists in multiple conformations. The presence of the red forms, which are very sensitive to changes in the environment, allows to spectrally resolve the different conformations: a "blue" conformation with a short lifetime and a "red" one with a long lifetime. This finding strongly supports the idea that the members of the Lhc family are able to adopt different conformations associated with their light-harvesting and photoprotective roles. The ratio between the conformations is modified by the substitution of lutein by violaxanthin. Finally, it is demonstrated that the red forms cannot be present in the quenched conformation.

post to: CiteULike

Biophysical characterization and mutational analysis of the antibiotic resistance protein NimA from Deinococcus radiodurans.

Publication Date: 2010 Apr PMID: 20096385
Authors: Leiros, H. K. - Brandsdal, B. O. - McSweeney, S. M.
Journal: Biochim Biophys Acta

Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K(a)) were in the range of 5.1-4910(4)M(-1) and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low ( approximately -1kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.

post to: CiteULike

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases.

Publication Date: 2010 Apr PMID: 20096384
Authors: Pollet, A. - Lagaert, S. - Eneyskaya, E. - Kulminskaya, A. - Delcour, J. A. - Courtin, C. M.
Journal: Biochim Biophys Acta

Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the +1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate-protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the +2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the +3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the +3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord - a long loop flanking the aglycon subsites of GH11 xylanases - proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.

post to: CiteULike

Specific interaction between E2F1 and Sp1 regulates the expression of murine CTP:phosphocholine cytidylyltransferase alpha during the S phase.

Publication Date: 2010 Apr PMID: 20096375
Authors: Elena, C. - Banchio, C.
Journal: Biochim Biophys Acta

CTP:phosphocholine cytidylyltransferase alpha (CCTalpha) is a key enzyme for phosphatidylcholine biosynthesis in mammalian cells. This enzyme plays an essential role in all processes that require membrane biosynthesis such as cell proliferation and viability. Thus, CCTalpha activity and expression fluctuate during the cell cycle to achieve PtdCho requirements. We demonstrated, for the first time, that CCTalpha is localized in the nucleus in cells transiting the S phase, whereas it is localized in the cytoplasm of G(0)-arrested cells, suggesting a specific role of nuclear CCTalpha during the S phase. We also investigated how E2F1 influences the regulation of the CCTalpha-promoter during the S phase; we demonstrated that E2F1 is necessary, but not sufficient, to activate CCTalpha expression when this factor is over-expressed. However, when E2F1 and Sp1 were over-expressed, the transcription from the CCTalpha-promoter reporter construct was super-activated. Transient transfection studies demonstrated that E2F1 could super-activate Sp1-dependent transcription in a promoter containing only the Sp1 binding sites "B" or "C", and that Sp1 could activate Sp1-dependent transcription in a promoter containing the E2F site, thus, further demonstrating a functional interaction of these factors. In conclusion, the present results allowed us to portray the clearest picture of the CCTalpha-gene expression in proliferating cells, and understand the mechanism by which cells coordinate cell cycle progression with the requirement for phosphatidylcholine.

post to: CiteULike

Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives.

Publication Date: 2010 Mar PMID: 20093105
Authors: Acosta, E. J. - Policova, Z. - Lee, S. - Dang, A. - Hair, M. L. - Neumann, A. W.
Journal: Biochim Biophys Acta

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113kDa and 213kDa. The other two additives were cationic peptides, polylysine 50kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive-surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50mul/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive-surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500mul/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant).

post to: CiteULike

Mitochondria isolated in nearly isotonic KCl buffer: Focus on cardiolipin and organelle morphology.

Publication Date: 2010 Mar PMID: 20093104
Authors: Corcelli, A. - Saponetti, M. S. - Zaccagnino, P. - Lopalco, P. - Mastrodonato, M. - Liquori, G. E. - Lorusso, M.
Journal: Biochim Biophys Acta

Rat liver mitochondria were isolated in parallel in two different isolation buffers: a standard buffer containing mannitol/sucrose and a nearly physiological KCl based solution. The two different organelle preparations were comparatively characterized by respiratory activity, heme content, microsomal and Golgi contamination, electron microscopy and lipid analyses. The substitution of saccharides with KCl in the isolation buffer does not induce the formation of mitoplasts or disruption of mitochondria. Mitochondria isolated in KCl buffer are coupled and able to maintain a stable transmembrane charge separation. A number of biochemical and functional differences between the two organelle preparations are described; in particular KCl mitochondria exhibit lower cardiolipin content and smaller intracristal compartments in comparison with the standard mitochondrial preparation.

post to: CiteULike

Hydroimidazolone modification of human alphaA-crystallin: Effect on the chaperone function and protein refolding ability.

Publication Date: 2010 Apr PMID: 20085807
Authors: Gangadhariah, M. H. - Wang, B. - Linetsky, M. - Henning, C. - Spanneberg, R. - Glomb, M. A. - Nagaraj, R. H.
Journal: Biochim Biophys Acta

AlphaA-crystallin is a molecular chaperone; it prevents aggregation of denaturing proteins. We have previously demonstrated that upon modification by a metabolic alpha-dicarbonyl compound, methylglyoxal (MGO), alphaA-crystallin becomes a better chaperone. AlphaA-crystallin also assists in refolding of denatured proteins. Here, we have investigated the effect of mild modification of alphaA-crystallin by MGO (with 20-500microM) on the chaperone function and its ability to refold denatured proteins. Under the conditions used, mildly modified protein contained mostly hydroimidazolone modifications. The modified protein exhibited an increase in chaperone function against thermal aggregation of beta(L)- and gamma-crystallins, citrate synthase (CS), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and chemical aggregation of insulin. The ability of the protein to assist in refolding of chemically denatured beta(L)- and gamma-crystallins, MDH and LDH, and to prevent thermal inactivation of CS were unchanged after mild modification by MGO. Prior binding of catalytically inactive, thermally denatured MDH or the hydrophobic probe, 2-p-toluidonaphthalene-6-sulfonate (TNS) abolished the ability of alphaA-crystallin to assist in the refolding of denatured MDH. However, MGO modification of chaperone-null TNS-bound alphaA-crystallin resulted in partial regain of the chaperone function. Taken together, these results demonstrate that: 1) hydroimidazolone modifications are sufficient to enhance the chaperone function of alphaA-crystallin but such modifications do not change its ability to assist in refolding of denatured proteins, 2) the sites on the alphaA-crystallin responsible for the chaperone function and refolding are the same in the native alphaA-crystallin and 3) additional hydrophobic sites exposed upon MGO modification, which are responsible for the enhanced chaperone function, do not enhance alphaA-crystallin's ability to refold denatured proteins.

post to: CiteULike

Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process.

Publication Date: 2010 Mar PMID: 20085747
Authors: Galdiero, S. - Falanga, A. - Vitiello, G. - Vitiello, M. - Pedone, C. - D'Errico, G. - Galdiero, M.
Journal: Biochim Biophys Acta

The entry of enveloped viruses involves attachment followed by close apposition of the viral and plasma membranes. Then, either on the cell surface or in an endocytotic vesicle, the two membranes fuse by an energetically unfavourable process requiring the destabilisation of membrane microenvironment in order to release the viral nucleocapsid into the cytoplasm. The core fusion machinery, conserved throughout the herpesvirus family, involves glycoprotein B (gB) and the non-covalently associated complex of glycoproteins H and L (gH/gL). Both gB and gH possess several hydrophobic domains necessary for efficient induction of fusion, and synthetic peptides corresponding to these regions are able to associate to membranes and induce fusion of artificial liposomes. Here, we describe the first application of surface plasmon resonance (SPR) to the study of the interaction of viral membranotropic peptides with model membranes in order to enhance our molecular understanding of the mechanism of membrane fusion. SPR spectroscopy data are supported by tryptophan fluorescence, circular dichroism and electron spin resonance spectroscopy (ESR). We selected peptides from gB and gH and also analysed the behaviour of HIV gp41 fusion peptide and the cationic antimicrobial peptide melittin. The combined results of SPR and ESR showed a marked difference between the mode of action of the HSV peptides and the HIV fusion peptide compared to melittin, suggesting that viral-derived membrane interacting peptides all act via a similar mechanism, which is substantially different from that of the non-cell selective lytic peptide melittin.

post to: CiteULike

Intestinal lipid alterations occur prior to antibody-induced prostaglandin E2 production in a mouse model of ischemia/reperfusion.

Publication Date: 2010 Apr PMID: 20083230
Authors: Sparkes, B. L. - Slone, E. E. - Roth, M. - Welti, R. - Fleming, S. D.
Journal: Biochim Biophys Acta

Ischemia/reperfusion (IR) induced injury results in significant tissue damage in wild-type, but not antibody-deficient, Rag-1(-/-) mice. However, Rag-1(-/-) mice sustain intestinal damage after administration of wild-type antibodies or naturally occurring, specific anti-phospholipid related monoclonal antibodies, suggesting involvement of a lipid antigen. We hypothesized that IR initiates metabolism of cellular lipids, resulting in production of an antigen recognized by anti-phospholipid antibodies. At multiple time points after Sham or IR treatment, lipids extracted from mouse jejunal sections were analyzed by electrospray ionization triple quadrupole mass spectrometry. Within 15min of reperfusion, IR induced significantly more lysophosphatidylcholine (lysoPC), lysophosphatidylglycerol (lysoPG) and free arachidonic acid (AA) production than Sham treatment. While lysoPC, lysoPG, and free AA levels were similar in C57Bl/6 (wild-type) and Rag-1(-/-) mice, IR led to Cox-2 activation and prostaglandin E(2) (PGE(2)) production in wild-type, but not in the antibody-deficient, Rag-1(-/-) mice. Administration of wild-type antibodies to Rag-1(-/-) mice restored PGE(2) production and intestinal damage. These data indicate that IR-induced intestinal damage requires antibodies for Cox-2 stimulated PGE(2) production but not for production of lysoPC and free AA.

post to: CiteULike

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2.

Publication Date: 2010 Apr PMID: 20083229
Authors: Amara, S. - Barouh, N. - Lecomte, J. - Lafont, D. - Robert, S. - Villeneuve, P. - De Caro, A. - Carriere, F.
Journal: Biochim Biophys Acta

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.

post to: CiteULike

Conversion of the g=4.1 EPR signal to the multiline conformation during the S(2) to S(3) transition of the oxygen evolving complex of Photosystem II.

Publication Date: 2010 Apr PMID: 20083085
Authors: Chrysina, M. - Zahariou, G. - Ioannidis, N. - Petrouleas, V.
Journal: Biochim Biophys Acta

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S(0)-S(1),...,S(3)-(S(4))S(0) during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)(4)Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S(2) shows a notable heterogeneity with two spectral forms; a g=2 (S=1/2) multiline, and a g=4.1 (S=5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S(3) they exhibit the same characteristic g=10 (S=3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g=4.1 conformation during advancement to S(3) is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S(2) to S(3) transition (ca 230K). We observed that, above about 220K the g=4.1 signal converts to a multiline form prior to advancement to S(3). We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S(2) to S(3) transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)(4)Ca with a low-spin ground state advances to S(3).

post to: CiteULike

Biofilm-induced modifications in the proteome of Pseudomonas aeruginosa planktonic cells.

Publication Date: 2010 Apr PMID: 20080211
Authors: Nigaud, Y. - Cosette, P. - Collet, A. - Song, P. C. - Vaudry, D. - Vaudry, H. - Junter, G. A. - Jouenne, T.
Journal: Biochim Biophys Acta

While recent studies focused on Quorum Sensing (QS) role in the cell-to-cell communication in free or biofilm cultures, no work has been devoted up to now to investigate the communication between sessile and planktonic bacteria. In this aim, we elaborated an original two-chambered bioreactor and used a proteomic approach to study the alterations induced by Pseudomonas aeruginosa biofilm cells on protein expression in planktonic counterparts (named SIPs for Surface-Influenced Planktonics). Proteomic analyses revealed the existence of 31 proteins whose amount varied in SIPs, among which five corresponded to hypothetic proteins and two (the Fur and BCP proteins) are involved in bacterial response to oxidative stress. An increase in the concentration of C(4)-HSL (rhlR-rhlI-dependent QS) and 3-oxo-C(12)-HSL (lasR-lasI-dependent QS) autoinducer molecules was shown in the planktonic compartment. Interestingly, among proteins that were accumulated by SIPs was 3-oxoacyl-[acyl-carrier-protein] reductase, a protein involved in the production of the autoinducer 3-oxo-C(12)-HSL. These results demonstrate that planktonic organisms are able to detect the presence of a biofilm in their close environment and to modify their gene expression in consequence.

post to: CiteULike

X-ray absorption studies of Zn(2+)-binding sites in Escherichia coli transhydrogenase and its betaH91K mutant.

Publication Date: 2010 Apr PMID: 20080075
Authors: Veronesi, G. - Whitehead, S. J. - Francia, F. - Giachini, L. - Boscherini, F. - Venturoli, G. - Cotton, N. P. - Jackson, J. B.
Journal: Biochim Biophys Acta

Transhydrogenase couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. The binding of Zn(2+) to the enzyme was shown previously to inhibit steps associated with proton transfer. Using Zn K-edge X-ray absorption fine structure (XAFS), we report here on the local structure of Zn(2+) bound to Escherichia coli transhydrogenase. Experiments were performed on wild-type enzyme and a mutant in which betaHis91 was replaced by Lys (betaH91K). This well-conserved His residue, located in the membrane-spanning domain of the protein, has been suggested to function in proton transfer, and to act as a ligand of the inhibitory Zn(2+). The XAFS analysis has identified a Zn(2+)-binding cluster formed by one Cys, two His, and one Asp/Glu residue, arranged in a tetrahedral geometry. The structure of the site is consistent with the notion that Zn(2+) inhibits proton translocation by competing with H(+) binding to the His residues. The same cluster of residues with very similar bond lengths best fits the spectra of wild-type transhydrogenase and betaH91K. Evidently, betaHis91 is not directly involved in Zn(2+) binding. The locus of betaHis91 and that of the Zn-binding site, although both on (or close to) the proton-transfer pathway of transhydrogenase, are spatially separate.

post to: CiteULike

Mitochondrial metabolism during fasting-induced daily torpor in mice.

Publication Date: 2010 Apr PMID: 20080074
Authors: Brown, J. C. - Staples, J. F.
Journal: Biochim Biophys Acta

During fasting, mice (Mus musculus) undergo daily bouts of torpor, considerably reducing body temperature (T(b)) and metabolic rate (MR). We examined females of different laboratory strains (Balb/c, C57/6N, and CD1) to determine whether liver mitochondrial metabolism is actively reduced during torpor. In all strains, we found that state 3 (phosphorylating) respiration rate measured at 37 degrees C was reduced up to 35% during torpor for at least one of the substrates (glutamate and succinate) used to fuel respiration. The extent of this suppression varied and was correlated with T(b) at sampling. This suggests that, at the biochemical level, the transition to and from a hypometabolic torpid state is gradual. In fasted non-torpid animals, T(b) and MR still fluctuated greatly: T(b) dropped by as much as 4 degrees C and MR was reduced up to 25% compared to fed controls. Changes in T(b) and MR in fasted, non-torpid animals were correlated with changes in mitochondrial state 3 respiration rate measured at 37 degrees C. This suggests that fasting mice may conserve energy even when not torpid by occasionally reducing T(b) and mitochondrial oxidative capacity to reduce MR. Furthermore, proton conductance was higher in torpid compared to non-torpid animals when measured at 15 degrees C (the lower limit of torpid T(b)). This pattern is similar to that reported previously for daily torpor in Phodopus sungorus.

post to: CiteULike

Effects of congenital cataract mutation R116H on alphaA-crystallin structure, function and stability.

Publication Date: 2010 Apr PMID: 20079887
Authors: Pang, M. - Su, J. T. - Feng, S. - Tang, Z. W. - Gu, F. - Zhang, M. - Ma, X. - Yan, Y. B.
Journal: Biochim Biophys Acta

alpha-crystallin is a molecular chaperone that maintains the optical properties of the lens and delays the onset scattering caused by aging-related protein aggregation. In this research, we found that the missense mutation R116H resulted in an altered size distribution, impaired packing of the secondary structures and modified quaternary structure with great hydrophobic exposure. The mutant exhibited a substrate-dependent chaperone (aggregation-inhibition) or anti-chaperone (aggregation-promotion) effect. Equilibrium unfolding experiments indicated that the mutation stabilized an aggregation-prone intermediate which was not populated during the unfolding of the wild-type protein. The accumulation of this intermediate greatly promoted the formation of non-native large oligomers or aggregates during unfolding. These results suggested that both the aggregation of the mutant upon stress and co-deposition with the target proteins were likely to be responsible for the onset of cataract.

post to: CiteULike

Abscisic acid pretreatment enhances salt tolerance of rice seedlings: Proteomic evidence.

Publication Date: 2010 Apr PMID: 20079886
Authors: Li, X. J. - Yang, M. F. - Chen, H. - Qu, L. Q. - Chen, F. - Shen, S. H.
Journal: Biochim Biophys Acta

Enhanced salt tolerance of rice seedlings by abscisic acid (ABA) pretreatment was observed from phenotypic and physiological analyses. Total proteins from rice roots treated with ABA plus subsequent salt stress were analyzed by using proteomics method. Results showed that, 40 protein spots were uniquely upregulated in the seedlings under the condition of ABA pretreatment plus subsequent salt stress, whereas only 16 under the condition of salt treatment. About 78% (31 spots) of the 40 protein spots were only upregulated in the presence of the subsequent salt stress, indicating that plants might have an economical strategy to prevent energy loss under a false alarm. The results also showed that more enzymes involved in energy metabolism, defense, primary metabolism, etc. were upregulated uniquely in ABA-pretreated rice seedlings, suggesting more abundant energy supply, more active anabolism (nitrogen, nucleotide acid, carbohydrate, etc), and more comprehensive defense systems in ABA-pretreated seedlings than in salt stressed ones.

post to: CiteULike

Interaction of l-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

Publication Date: 2010 Apr PMID: 20079884
Authors: Olivieri, A. - O'Sullivan, J. - Fortuny, L. R. - Vives, I. L. - Tipton, K. F.
Journal: Biochim Biophys Acta

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of l-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since d-lysine, l-lysine ethyl ester and epsilon-acetyl-l-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.

post to: CiteULike

beta-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E(2) synthesis in rat hepatocytes-Kupffer cells.

Publication Date: 2010 Apr PMID: 20079880
Authors: Du, Z. Y. - Ma, T. - Winterthun, S. - Kristiansen, K. - Froyland, L. - Madsen, L.
Journal: Biochim Biophys Acta

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E(2) (PGE(2)) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE(2) synthesis in a rat hepatocyte-Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) beta-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE(2) synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased beta-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE(2) synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.

post to: CiteULike

The glycosylation and characterization of the candidate Gc macrophage activating factor.

Publication Date: 2010 Apr PMID: 20079467
Authors: Ravnsborg, T. - Olsen, D. T. - Thysen, A. H. - Christiansen, M. - Houen, G. - Hojrup, P.
Journal: Biochim Biophys Acta

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.

post to: CiteULike

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod.

Publication Date: 2010 Apr PMID: 20079466
Authors: Matusovsky, O. S. - Shelud'ko, N. S. - Permyakova, T. V. - Zukowska, M. - Sobieszek, A.
Journal: Biochim Biophys Acta

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca(2+); and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin-myosin-myorod complex increased the actin-activated Mg(2+)-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.

post to: CiteULike

Pathological roles of MAPK signaling pathways in human diseases.

Publication Date: 2010 Apr PMID: 20079433
Authors: Kim, E. K. - Choi, E. J.
Journal: Biochim Biophys Acta

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH(2)-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.

post to: CiteULike

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins.

Publication Date: 2010 Apr PMID: 20079404
Authors: De Vos, W. H. - Houben, F. - Hoebe, R. A. - Hennekam, R. - van Engelen, B. - Manders, E. M. - Ramaekers, F. C. - Broers, J. L. - Van Oostveldt, P.
Journal: Biochim Biophys Acta

BACKGROUND: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.

post to: CiteULike

Templating membrane assembly, structure, and dynamics using engineered interfaces.

Publication Date: 2010 Apr PMID: 20079336
Authors: Oliver, A. E. - Parikh, A. N.
Journal: Biochim Biophys Acta

The physical and chemical properties of biological membranes are intimately linked to their bounding aqueous interfaces. Supported phospholipid bilayers, obtained by surface-assisted rupture, fusion, and spreading of vesicular microphases, offer a unique opportunity, because engineering the substrate allows manipulation of one of the two bilayer interfaces as well. Here, we review a collection of recent efforts, which illustrates deliberate substrate-membrane coupling using structured surfaces exhibiting chemical and topographic patterns. Vesicle fusion on chemically patterned substrates results in co-existing lipid phases, which reflect the underlying pattern of surface energy and wettability. These co-existing bilayer/monolayer morphologies are useful both for fundamental biophysical studies (e.g., studies of membrane asymmetry) as well as for applied work, such as synthesizing large-scale arrays of bilayers or living cells. The use of patterned, static surfaces provides new models to design complex membrane topographies and curvatures. Dynamic switchable-topography surfaces and sacrificial trehalose based-substrates reveal abilities to dynamically introduce membrane curvature and change the nature of the membrane-substrate interface. Taken together, these studies illustrate the importance of controlling interfaces in devising model membrane platforms for fundamental biophysical studies and bioanalytical devices.

post to: CiteULike

Palmitoylation of R-Ras by human DHHC19, a palmitoyl transferase with a CaaX box.

Publication Date: 2010 Mar PMID: 20074548
Authors: Baumgart, F. - Corral-Escariz, M. - Perez-Gil, J. - Rodriguez-Crespo, I.
Journal: Biochim Biophys Acta

Mammalian proteins that contain an aspartate-histidine-histidine-cysteine-(DHHC) motif have been recently identified as a group of membrane-associated palmitoyl acyltransferases (PATs). Among the several protein substrates known to become palmitoylated by DHHC PATs are small GTPases prenylated at their carboxy-terminal end, such as H-Ras or N-Ras, eNOS, kinases myristoylated at their N-terminal end, such as Lck, and many transmembrane proteins and channels. We have focused our studies on the product of the human gene DHHC19, a putative palmitoyl transferase that, interestingly, displays a conserved CaaX box at its carboxy-terminal end. We show herein that the amino acid sequence present at the carboxy-terminus of DHHC19 is able to exclude a green fluorescent protein (GFP) reporter from the nucleus and direct it towards perinuclear regions. Transfection of full-length DHHC19 in COS7 cells reveals a perinuclear distribution, in analogy to other palmitoyl transferases, with a strong colocalization with the trans-Golgi markers Gal-T and TGN38. We have tested several small GTPases that are known to be palmitoylated as possible substrates of DHHC19. Although DHHC19 failed to increase the palmitoylation of H-Ras, N-Ras, K-Ras4A, RhoB or Rap2 it increased the palmitoylation of R-Ras approximately two-fold. The increased palmitoylation of R-Ras cotransfected with DHHC19 is accompanied by an augmented association with membranes as well as with rafts/caveolae. Finally, using both wild-type and an activated GTP bound form of R-Ras (G38V), we also show that the increased palmitoylation of R-Ras due to DHHC19 coexpression is accompanied by an enhanced viability of the transfected cells.

post to: CiteULike

Identification of a slowly inducible zeaxanthin-dependent component of non-photochemical quenching of chlorophyll fluorescence generated under steady-state conditions in Arabidopsis.

Publication Date: 2010 Apr PMID: 20067757
Authors: Nilkens, M. - Kress, E. - Lambrev, P. - Miloslavina, Y. - Muller, M. - Holzwarth, A. R. - Jahns, P.
Journal: Biochim Biophys Acta

The induction and relaxation of non-photochemical quenching (NPQ) under steady-state conditions, i.e. during up to 90min of illumination at saturating light intensities, was studied in Arabidopsis thaliana. Besides the well-characterized fast qE and the very slow qI component of NPQ, the analysis of the NPQ dynamics identified a zeaxanthin (Zx) dependent component which we term qZ. The formation (rise time 10-15min) and relaxation (lifetime 10-15min) of qZ correlated with the synthesis and epoxidation of Zx, respectively. Comparative analysis of different NPQ mutants from Arabidopsis showed that qZ was clearly not related to qE, qT or qI and thus represents a separate, Zx-dependent NPQ component.

post to: CiteULike

Peroxisomes, lipid metabolism and lipotoxicity.

Publication Date: 2010 Mar PMID: 20064629
Authors: Wanders, R. J. - Ferdinandusse, S. - Brites, P. - Kemp, S.
Journal: Biochim Biophys Acta

Peroxisomes play an essential role in cellular lipid metabolism as exemplified by the existence of a number of genetic diseases in humans caused by the impaired function of one of the peroxisomal enzymes involved in lipid metabolism. Key pathways in which peroxisomes are involved include: (1.) fatty acid beta-oxidation; (2.) etherphospholipid biosynthesis, and (3.) fatty acid alpha-oxidation. In this paper we will describe these different pathways in some detail and will provide an overview of peroxisomal disorders of metabolism and in addition discuss the toxicity of the intermediates of peroxisomal metabolism as they accumulate in the different peroxisomal deficiencies.

post to: CiteULike

Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway.

Publication Date: 2010 Apr PMID: 20064588
Authors: Wu, X. Z. - Chang, W. Q. - Cheng, A. X. - Sun, L. M. - Lou, H. X.
Journal: Biochim Biophys Acta

BACKGROUND: Plagiochin E (PLE) is an antifungal active macrocyclic bis(bibenzyl) isolated from liverwort Marchantia polymorpha L. To elucidate the mechanism of action, previous studies revealed that the antifungal effect of PLE was associated with the accumulation of ROS, an important regulator of apoptosis in Candida albicans. The present study was designed to find whether PLE caused apoptosis in C. albicans. METHODS: We assayed the cell cycle by flow cytometry using PI staining, observed the ultrastructure by transmission electron microscopy, studied the nuclear fragmentation by DAPI staining, and investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V staining. The effect of PLE on expression of CDC28, CLB2, and CLB4 was determined by RT-PCR. Besides, the activity of metacaspase was detected by FITC-VAD-FMK staining, and the release of cytochrome c from mitochondria was also determined. Furthermore, the effect of antioxidant L-cysteine on PLE-induced apoptosis in C. albicans was also investigated. RESULTS: Cells treated with PLE showed typical markers of apoptosis: G(2)/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. The expression of CDC28, CLB2, and CLB4 was down-regulated by PLE, which may contribute to PLE-induced G(2)/M cell cycle arrest. Besides, PLE promoted the cytochrome c release and activated the metacaspase, which resulted in the yeast apoptosis. The addition of L-cysteine prevented PLE-induced nuclear fragmentation, phosphatidylserine exposure, and metacaspase activation, indicating the ROS was an important mediator of PLE-induced apoptosis. CONCLUSIONS: PLE induced apoptosis in C. albicans through a metacaspase-dependent apoptotic pathway. GENERAL SIGNIFICANCE: In this study, we reported for the first time that PLE induced apoptosis in C. albicans through activating the metacaspase. These results would conduce to elucidate its underlying antifungal mechanism.

post to: CiteULike

The molecular structure of the IsiA-Photosystem I supercomplex, modelled from high-resolution, crystal structures of Photosystem I and the CP43 protein.

Publication Date: 2010 Apr PMID: 20064486
Authors: Zhang, Y. - Chen, M. - Church, W. B. - Lau, K. W. - Larkum, A. W. - Jermiin, L. S.
Journal: Biochim Biophys Acta

We present the molecular structure of the IsiA-Photosystem I (PSI) supercomplex, inferred from high-resolution, crystal structures of PSI and the CP43 protein. The structure of iron-stress-induced A protein (IsiA) is similar to that of CP43, albeit with the difference that IsiA is associated with 15 chlorophylls (Chls), one more than previously assumed. The membrane-spanning helices of IsiA contain hydrophilic residues many of which bind Chl. The optimal structure of the IsiA-PSI supercomplex was inferred by systematically rearranging the IsiA monomers and PSI trimer in relation to each other. For each of the 6,969,600 structural configurations considered, we counted the number of optimal Chl-Chl connections (i.e., cases where Chl-bound Mg atoms are
post to: CiteULike

Prolyl-peptidyl isomerase, Pin1, phosphorylation is compromised in association with the expression of the HFE polymorphic allele, H63D.

Publication Date: 2010 Apr PMID: 20060900
Authors: Hall, E. C. 2nd - Lee, S. Y. - Simmons, Z. - Neely, E. B. - Nandar, W. - Connor, J. R.
Journal: Biochim Biophys Acta

There is substantial interest in HFE gene variants as putative risk factors in neurodegenerative diseases such as Alzheimer disease (AD). Previous studies in cell models have shown the H63D HFE variant to result in increased cellular iron, oxidative stress, glutamate dyshomeostasis, and an increase in tau phosphorylation; all processes thought to contribute to AD pathology. Pin1 is a prolyl-peptidyl cis/trans isomerase that can regulate the dephosphorylation of the amyloid and tau proteins. Hyperphosphorylation of these later proteins is implicated in the pathogenesis of AD and Pin1 levels are reportedly decreased in AD brains. Because of the relationship between Pin1 loss of function by oxidative stress and the increase in oxidative stress in cells with the H63D polymorphism it was logical to interrogate a relationship between Pin1 and HFE status. To test our hypothesis that H63D HFE would be associated with less Pin1 activity, we utilized stably transfected human neuroblastoma SH-SY5Y cell lines expressing the different HFE polymorphisms. Under resting conditions, total Pin1 levels were unchanged between the wild type and H63D HFE cells, yet there was a significant increase in phosphorylation of Pin1 at its serine 16 residue suggesting a loss of Pin1 activity in H63D variant cells. To evaluate whether cellular iron status could influence Pin1, we treated the WT HFE cells with exogenous iron and found that Pin1 phosphorylation increased with increasing levels of iron. Iron exposure to H63D variant cells did not impact Pin1 phosphorylation beyond that already seen suggesting a ceiling effect. Because HFE H63D cells have been shown to have more oxidative stress, the cells were treated with the antioxidant Trolox which resulted in a decrease in Pin1 phosphorylation in H63D cells with no change in WT HFE cells. In a mouse model carrying the mouse equivalent of the H63D allele, there was an increase in the phosphorylation status of Pin1 providing in vivo evidence for our findings in the cell culture model. Thus, we have shown another cellular mechanism that HFE polymorphisms influence; further supporting their role as neurodegenerative disease modifiers.

post to: CiteULike

Lipid raft disruption protects mature neurons against amyloid oligomer toxicity.

Publication Date: 2010 Apr PMID: 20060899
Authors: Malchiodi-Albedi, F. - Contrusciere, V. - Raggi, C. - Fecchi, K. - Rainaldi, G. - Paradisi, S. - Matteucci, A. - Santini, M. T. - Sargiacomo, M. - Frank, C. - Gaudiano, M. C. - Diociaiuti, M.
Journal: Biochim Biophys Acta

A specific neuronal vulnerability to amyloid protein toxicity may account for brain susceptibility to protein misfolding diseases. To investigate this issue, we compared the effects induced by oligomers from salmon calcitonin (sCTOs), a neurotoxic amyloid protein, on cells of different histogenesis: mature and immature primary hippocampal neurons, primary astrocytes, MG63 osteoblasts and NIH-3T3 fibroblasts. In mature neurons, sCTOs increased apoptosis and induced neuritic and synaptic damages similar to those caused by amyloid beta oligomers. Immature neurons and the other cell types showed no cytotoxicity. sCTOs caused cytosolic Ca(2+) rise in mature, but not in immature neurons and the other cell types. Comparison of plasma membrane lipid composition showed that mature neurons had the highest content in lipid rafts, suggesting a key role for them in neuronal vulnerability to sCTOs. Consistently, depletion in gangliosides protected against sCTO toxicity. We hypothesize that the high content in lipid rafts makes mature neurons especially vulnerable to amyloid proteins, as compared to other cell types; this may help explain why the brain is a target organ for amyloid-related diseases.

post to: CiteULike

In-vivo visualization of key molecular processes involved in Alzheimer's disease pathogenesis: Insights from neuroimaging research in humans and rodent models.

Publication Date: 2010 Apr PMID: 20060898
Authors: Higuchi, M. - Maeda, J. - Ji, B. - Maruyama, M. - Okauchi, T. - Tokunaga, M. - Ono, M. - Suhara, T.
Journal: Biochim Biophys Acta

Diverse age-associated neurodegenerative disorders are featured at a molecular level by depositions of self-aggregating molecules, as represented by amyloid beta peptides (Abeta) and tau proteins in Alzheimer's disease, and cascade-type chain reactions are supposedly commenced with biochemical aberrancies of these amyloidogenic components. Mutagenesis and multiplication of the genes encoding Abeta, tau and other pathogenic initiators may accelerate the incipient process at the cascade top, rationalizing generations of transgenic and knock-in animal models of these illnesses. Meanwhile, these genetic manipulations do not necessarily compress the timelines of crucial intermediate events linking amyloidogenesis and neuronal lethality, resulting in an incomplete recapitulation of the diseases. Requirements for modeling the entire cascade can be illustrated by a side-by-side comparison of humans and animal models with the aid of imaging-based biomarkers commonly applicable to different species. Notably, key components in a highly reactive state are assayable by probe-assisted neuroimaging techniques exemplified by positron emission tomography (PET), providing critical information on the in-vivo accessibility of these target molecules. In fact, multispecies PET studies in conjunction with biochemical, electrophysiological and neuropathological tests have revealed putative neurotoxic subspecies of Abeta assemblies, translocator proteins accumulating in aggressive but not neuroprotective microglia, and functionally active neuroreceptors available to endogenous neurotransmitters and exogenous agonistic ligands. Bidirectional translational studies between human cases and model strains based on this experimental paradigm are presently aimed at clarifying the tau pathogenesis, and would be expanded to analyses of disrupted calcium homeostasis and mitochondrial impairments. Since reciprocal causalities among the key processes have indicated an architectural interchangeability between cascade and network connections as an etiological representation, longitudinal imaging assays with manifold probes covering the cascade from top to bottom virtually delineate the network dynamics continuously altering in the course of the disease and its treatment, and therefore expedite the evaluation and optimization of therapeutic strategies intended for suppressing the neurodegenerative pathway over its full length.

post to: CiteULike

The role of the autonomic nervous liver innervation in the control of energy metabolism.

Publication Date: 2010 Apr PMID: 20060897
Authors: Yi, C. X. - la Fleur, S. E. - Fliers, E. - Kalsbeek, A.
Journal: Biochim Biophys Acta

Despite a longstanding research interest ever since the early work by Claude Bernard, the functional significance of autonomic liver innervation, either sympathetic or parasympathetic, is still ill defined. This scarcity of information not only holds for the brain control of hepatic metabolism, but also for the metabolic sensing function of the liver and the way in which this metabolic information from the liver affects the brain. Clinical information from the bedside suggests that successful human liver transplantation (implying a complete autonomic liver denervation) causes no life threatening metabolic derangements, at least in the absence of severe metabolic challenges such as hypoglycemia. However, from the benchside, data are accumulating that interference with the neuronal brain-liver connection does cause pronounced changes in liver metabolism. This review provides an extensive overview on how metabolic information is sensed by the liver, and how this information is processed via neuronal pathways to the brain. With this information the brain controls liver metabolism and that of other organs and tissues. We will pay special attention to the hypothalamic pathways involved in these liver-brain-liver circuits. At this stage, we still do not know the final destination and processing of the metabolic information that is transferred from the liver to the brain. On the other hand, in recent years, there has been a considerable increase in the understanding which brain areas are involved in the control of liver metabolism via its autonomic innervation. However, in view of the ever rising prevalence of type 2 diabetes, this potentially highly relevant knowledge is still by far too limited. Thus the autonomic innervation of the liver and its role in the control of metabolism needs our continued and devoted attention.

post to: CiteULike

The bile acid sensor FXR regulates insulin transcription and secretion.

Publication Date: 2010 Mar PMID: 20060466
Authors: Renga, B. - Mencarelli, A. - Vavassori, P. - Brancaleone, V. - Fiorucci, S.
Journal: Biochim Biophys Acta

Farnesoid X Receptor plays an important role in maintaining bile acid, cholesterol homeostasis and glucose metabolism. Here we investigated whether FXR is expressed by pancreatic beta-cells and regulates insulin signaling in pancreatic beta-cell line and human islets. We found that FXR activation induces positive regulatory effects on glucose-induced insulin transcription and secretion by genomic and non-genomic activities. Genomic effects of FXR activation relay on the induction of the glucose regulated transcription factor KLF11. Indeed, results from silencing experiments of KLF11 demonstrate that this transcription factor is essential for FXR activity on glucose-induced insulin gene transcription. In addition FXR regulates insulin secretion by non-genomic effects. Thus, activation of FXR in betaTC6 cells increases Akt phosphorylation and translocation of the glucose transporter GLUT2 at plasma membrane, increasing the glucose uptake by these cells. In vivo experiments on Non Obese Diabetic (NOD) mice demonstrated that FXR activation delays development of signs of diabetes, hyperglycemia and glycosuria, by enhancing insulin secretion and by stimulating glucose uptake by the liver. These data established that an FXR-KLF11 regulated pathway has an essential role in the regulation of insulin transcription and secretion induced by glucose.

post to: CiteULike

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids.

Publication Date: 2010 Mar PMID: 20060379
Authors: Pande, A. H. - Kar, S. - Tripathy, R. K.
Journal: Biochim Biophys Acta

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.

post to: CiteULike

Liquid-ordered phases induced by cholesterol: A compendium of binary phase diagrams.

Publication Date: 2010 Mar PMID: 20060378
Authors: Marsh, D.
Journal: Biochim Biophys Acta

Mixtures of phospholipids with cholesterol are able to form liquid-ordered phases that are characterised by short-range orientational order and long-range translational disorder. These L(o)-phases are distinct from the liquid-disordered, fluid L(alpha)-phases and the solid-ordered, gel L(beta)-phases that are assumed by the phospholipids alone. The liquid-ordered phase can produce spatially separated in-plane fluid domains, which, in the form of lipid rafts, are thought to act as platforms for signalling and membrane sorting in cells. The areas of domain formation are defined by the regions of phase coexistence in the phase diagrams for the binary mixtures of lipid with cholesterol. In this paper, the available binary phase diagrams of lipid-cholesterol mixtures are all collected together. It is found that there is not complete agreement between different determinations of the phase diagrams for the same binary mixture. This can be attributed to the indirect methods largely used to establish the phase boundaries. Intercomparison of the various data sets allows critical assessment of which phase boundaries are rigorously established from direct evidence for phase coexistence.

post to: CiteULike

Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin.

Publication Date: 2010 Apr PMID: 20056172
Authors: Frasconi, M. - Favero, G. - Boer, H. - Koivula, A. - Mazzei, F.
Journal: Biochim Biophys Acta

The electrochemical studies of laccase-mediator systems are aimed at understanding the mechanism of their redox transformation and their efficiency in both homogeneous and heterogeneous reactions; this topic has paramount application spanning from bleaching of paper pulp and the enzymatic degradation of lignin to the biosensors and biofuel cell development. In this paper four different laccases from Trametes hirsuta (ThL), Trametes versicolor (TvL), Melanocarpus albomyces (r-MaL) and Rhus vernicifera (RvL) were characterized from both biochemical and electrochemical points of view. Two of them (TvL and ThL) are high redox potential and two (RvL and r-MaL) are low redox potential laccases. The outline of this work is focused on the determination of catalytic and bioelectrochemical properties of these four enzymes in homogenous solution as well as immobilized onto electrode surface in the presence of a set of different redox mediators. The results measured in the homogenous reaction system correlated well with those measured with the immobilized enzymes. In addition, they are in good agreement with those reported with reference techniques, suggesting that the electrochemical methods employed in this work can be applied well in place of the traditional techniques commonly used for the kinetic characterization of laccases. These results are also discussed in terms of the known amino acid sequences and three-dimensional (3D) structures of the laccases.

post to: CiteULike

Analysis of proton exchange kinetics with time-dependent exchange rate.

Publication Date: 2010 Apr PMID: 20056171
Authors: Rutkowska-Wlodarczyk, I. - Kierdaszuk, B. - Wlodarczyk, J.
Journal: Biochim Biophys Acta

Mass spectrometry is used to probe the kinetics of hydrogen-deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen-deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.

post to: CiteULike

Adipose tissue expandability, lipotoxicity and the Metabolic Syndrome - An allostatic perspective.

Publication Date: 2010 Mar PMID: 20056169
Authors: Virtue, S. - Vidal-Puig, A.
Journal: Biochim Biophys Acta

While the link between obesity and type 2 diabetes is clear on an epidemiological level, the underlying mechanism linking these two common disorders is not as clearly understood. One hypothesis linking obesity to type 2 diabetes is the adipose tissue expandability hypothesis. The adipose tissue expandability hypothesis states that a failure in the capacity for adipose tissue expansion, rather than obesity per se is the key factor linking positive energy balance and type 2 diabetes. All individuals possess a maximum capacity for adipose expansion which is determined by both genetic and environmental factors. Once the adipose tissue expansion limit is reached, adipose tissue ceases to store energy efficiently and lipids begin to accumulate in other tissues. Ectopic lipid accumulation in non-adipocyte cells causes lipotoxic insults including insulin resistance, apoptosis and inflammation. This article discusses the links between adipokines, inflammation, adipose tissue expandability and lipotoxicity. Finally, we will discuss how considering the concept of allostasis may enable a better understanding of how diabetes develops and allow the rational design of new anti diabetic treatments.

post to: CiteULike

Modulation of plasma TG lipolysis by Angiopoietin-like proteins and GPIHBP1.

Publication Date: 2010 Apr PMID: 20056168
Authors: Lichtenstein, L. - Kersten, S.
Journal: Biochim Biophys Acta

There is evidence that elevated plasma triglycerides (TG) serve as an independent risk factor for coronary heart disease. Plasma TG levels are determined by the balance between the rate of production of chylomicrons and VLDL in intestine and liver, respectively, and their rate of clearance in peripheral tissues. Lipolytic processing of TG-rich lipoproteins is mediated by the enzyme lipoprotein lipase (LPL), which is tethered to the capillary endothelium via heparin sulphate proteoglycans. In recent years the Angiopoietin-like proteins ANGPTL3 and ANGPTL4 have emerged as novel modulators of LPL activity. Studies in transgenic animals supported by in vitro experiments have demonstrated that ANGPTL3 and ANGPTL4 impair plasma TG clearance by inhibiting LPL activity. In humans, genetic variation within the ANGPTL3 and ANGPTL4 genes contributes to variation in plasma TG and HDL levels, thereby validating the importance of ANGPTLs in the regulation of lipoprotein metabolism in humans. Combined with the discovery of GPIHBP1 as a likely LPL anchor, these findings have led to a readjustment of the mechanism of LPL function. This review provides an overview of our current understanding of the role and regulation of ANGPTL3, ANGPTL4 and GPIHBP1, and places the newly acquired knowledge in the context of the established function and mechanism of LPL-mediated lipolysis.

post to: CiteULike

Obese and anorexic yeasts: Experimental models to understand the metabolic syndrome and lipotoxicity.

Publication Date: 2010 Mar PMID: 20056167
Authors: Kohlwein, S. D.
Journal: Biochim Biophys Acta

Lipotoxicity is the pathological consequence of lipid overflow in non-adipose tissue, mediated through reactive lipid moieties which may even lead to lipid-induced cell death (lipoapoptosis). This derailment of cellular and organismal fat homeostasis is the consequence of obesity due to continued over-feeding, and contributes substantially to the pathogenesis of insulin resistance, type 2 diabetes mellitus and cardiovascular disease, which are all components of the metabolic syndrome. Now, does yeast, a single-celled eukaryote, ever suffer from the metabolic syndrome and what can we potentially learn from studies in this organism about the underlying molecular mechanism that lead to lipid-associated pathologies in human cells? In this review I will summarize the remarkably conserved metabolic and regulatory processes relevant to establishing cellular energy and lipid homeostasis, as well as recent findings that provide detailed insights into the molecular mechanisms underlying fat-induced cellular malfunction and cell death, with potential implications also for mammalian cells.

post to: CiteULike

Insulin alters the expression of components of the Wnt signaling pathway including TCF-4 in the intestinal cells.

Publication Date: 2010 Mar PMID: 20056134
Authors: Sun, J. - Wang, D. - Jin, T.
Journal: Biochim Biophys Acta

BACKGROUND: Epidemiological and experimental evidence that support the correlation between Type 2 diabetes mellitus (T2D) and increased risks of colorectal cancer formation have led us to hypothesize the existence of molecular crosstalk between insulin and canonical Wnt signaling pathways. Insulin was shown to stimulate Wnt target gene expression, utilizing the effector of the Wnt signaling pathway. Whether insulin affects expression of components of Wnt pathway has not been extensively examined. METHODS: cDNA microarray was utilized to assess the effect of insulin on gene expression profile in the rat intestinal non-cancer IEC-6 cell line, followed by real-time RT-PCR, Western blotting and reporter gene analyses in intestinal cancer and non-cancer cells. RESULTS: Insulin was shown to alter the expression of a dozen of Wnt pathway related genes including TCF-4 (=TCF7L2) and frizzled- (Fzd-4). The stimulatory effect of insulin on TCF-4 expression was then confirmed by real-time RT-PCR, Western blotting and luciferase reporter analyses, while the activation on Fzd-4 was confirmed by real-time PCR. GENERAL SIGNIFICANCE: Our observations suggest that insulin may crosstalk with the Wnt signaling pathway in a multi-level fashion, involving insulin regulation of the expression of Wnt target genes, a Wnt receptor, as well as mediators of the Wnt signaling pathway.

post to: CiteULike

Differential mechanism of light-induced and oxygen-dependent restoration of the high-potential form of cytochrome b(559) in Tris-treated Photosystem II membranes.

Publication Date: 2010 Apr PMID: 20056104
Authors: Pospisil, P. - Tiwari, A.
Journal: Biochim Biophys Acta

The effect of illumination and molecular oxygen on the redox and the redox potential changes of cytochrome b(559) (cyt b(559)) has been studied in Tris-treated spinach photosystem II (PSII) membranes. It has been demonstrated that the illumination of Tris-treated PSII membranes induced the conversion of the intermediate-potential (IP) to the reduced high-potential (HP(Fe2+)) form of cyt b(559), whereas the removal of molecular oxygen resulted in the conversion of the IP form to the oxidized high-potential (HP(Fe3+)) form of cyt b(559). Light-induced conversion of cyt b(559) from the IP to the HP form was completely inhibited above pH 8 or by the modification of histidine ligand that prevents its protonation. Interestingly, no effect of high pH or histidine modification was observed during the conversion of the IP to the HP form of cyt b(559) after the removal of molecular oxygen. These results indicate that conversion from the IP to the HP form of cyt b(559) proceeds via different mechanisms. Under illumination, conversion of the IP to the HP form of cyt b(559) depends primarily on the protonation of the histidine residue, whereas under anaerobic conditions, the conversion of the IP to the HP form of cyt b(559) is driven by higher hydrophobicity of the environment around the heme iron resulting from the absence of molecular oxygen.

post to: CiteULike

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei.

Publication Date: 2010 Apr PMID: 20056101
Authors: Kido, Y. - Sakamoto, K. - Nakamura, K. - Harada, M. - Suzuki, T. - Yabu, Y. - Saimoto, H. - Yamakura, F. - Ohmori, D. - Moore, A. - Harada, S. - Kita, K.
Journal: Biochim Biophys Acta

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338muM and V(max) of 601mumol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

post to: CiteULike

Reaction mechanism of 5,8-linoleate diol synthase, 10R-dioxygenase, and 8,11-hydroperoxide isomerase of Aspergillus clavatus.

Publication Date: 2010 Apr PMID: 20045744
Authors: Jerneren, F. - Garscha, U. - Hoffmann, I. - Hamberg, M. - Oliw, E. H.
Journal: Biochim Biophys Acta

Aspergilli express fusion proteins of an animal haem peroxidase domain with fatty acid dioxygenase (DOX) activity ( approximately 600 amino acids) and a functional or non-functional hydroperoxide isomerase/cytochrome P450 domain ( approximately 500 amino acids with EXXR and GPHXCLG motifs). 5,8-Linoleate diol synthases (LDS; ppoA) and 10R-DOX (ppoC) of Aspergillusnidulans and A. fumigatus belong to this group. Our objective was to determine the oxylipins formed from linoleic acid by A. clavatus and their mechanism of biosynthesis. A. clavatus oxidized linoleic acid to (8R)-hydroperoxylinoleic acid (8R-HPODE), (10R)-hydroperoxy-8(E),12(Z)-octadecadienoic acid (10R-HPODE), and to (5S,8R)-dihydroxy- and (8R,11S)-dihydroxylinoleic acids (DiHODE) as major products. This occurred by abstraction of the pro-S hydrogen at C-8 and antarafacial dioxygenation at C-8 or at C-10 with double bond migration. 8R-HPODE was then isomerized to 5S,8R-DiHODE and to 8R,11S-DiHODE by abstraction of the pro-S hydrogens at C-5 and C-11 of 8R-HPODE, respectively, followed by suprafacial oxygenation. The genome of A. clavatus codes for two enzymes, which can be aligned with >65% amino acid identity to 10R-DOX and 5,8-LDS, respectively. The 5,8-LDS homologue likely forms and isomerizes 8R-HPODE to 5S,8R-DiHODE. A third gene (ppoB) codes for a protein which carries a serine residue at the cysteine position of the P450 motif. This Cys to Ser replacement is known to abolish P450 2B4 catalysis and the hydroperoxide isomerase activity of 5,8-LDS, suggesting that ppoB of A. clavatus may not be involved in the biosynthesis of 8R,11S-DiHODE.

post to: CiteULike

Obesity as a clinical and public health problem: Is there a need for a new definition based on lipotoxicity effects?

Publication Date: 2010 Mar PMID: 20045743
Authors: Sorensen, T. I. - Virtue, S. - Vidal-Puig, A.
Journal: Biochim Biophys Acta

The risk functions for obesity (defined as the quantitative relation between degree of obesity throughout its range and the risk of health problems) have been used to define 'obesity' as an excess storage of fat in the body to such an extent that it causes health problems leading to increased mortality. The lipotoxicity theory implies that the fat stored in droplets of triglycerides in the cells are biologically inert and that the metabolic dysfunctions are primarily due to the increased exposure of the cells to fatty acids. If this is true, it has profound implications for the interpretations of the multiple epidemiological studies of the risk functions. It is obvious from all these studies that the sizes of the fat depots are risk indicators of health effects in various ways. Paradoxically, the sizes of the fat stores are also indicators of the preceding implementation of the ability of the body to protect itself against the toxic effects of the free fatty acids. The current risk of metabolic dysfunctions appears to be determined by the balance between the rate of loading of the body with fatty acids and the rate of eliminating the fatty acids by either triglyceride storage or oxidation. The progress in the development of the dysfunction then depends on the persistence of the imbalance leading to future cumulative exposure of the cells to the toxic effects of the fatty acids rather than on the current size of the fat depots. This may be considered as a reason for changing the definition of obesity to one based on better estimates of future risks of health problems derived from later metabolic dysfunctions rather than on the past coping with the exposure to the fatty acids by storage as triglycerides. Implementation of such definition would require a test that measures this residual capacity to avoid excess exposure of the cells to the fatty acids before the metabolic dysfunctions have emerged. In analogy with the glucose tolerance test, a fatty acid tolerance test may be needed to identify individuals who are at a level of risk for developing lipotoxicity induced metabolic dysfunctions such that they require intervention. This test would ideally be a single biomarker that would determine residual capacity for adipose expansion, fatty acid oxidation and safe ectopic lipid deposition.

post to: CiteULike

Regulatory role for phosphatidylcholine transfer protein/StarD2 in the metabolic response to peroxisome proliferator activated receptor alpha (PPARalpha).

Publication Date: 2010 Apr PMID: 20045742
Authors: Kang, H. W. - Kanno, K. - Scapa, E. F. - Cohen, D. E.
Journal: Biochim Biophys Acta

Phosphatidylcholine transfer protein (PC-TP, a.k.a. StarD2) is abundantly expressed in liver and is regulated by PPARalpha. When fed the synthetic PPARalpha ligand fenofibrate, Pctp(-/-) mice exhibited altered lipid and glucose metabolism. Microarray profiling of livers from fenofibrate fed wild type and Pctp(-/-) mice revealed differential expression of a broad array of metabolic genes, as well as their regulatory transcription factors. PC-TP expression in cell culture controlled the activities of both PPARalpha and HNF4alpha, suggesting that the mechanism by which it modulates hepatic metabolism is at least in part via activation of transcription factors that govern nutrient homeostasis.

post to: CiteULike

Low-density lipoprotein and oxysterols suppress the transcription of CTP:Phosphoethanolamine cytidylyltransferase in vitro.

Publication Date: 2010 Apr PMID: 20045741
Authors: Ando, H. - Horibata, Y. - Yamashita, S. - Oyama, T. - Sugimoto, H.
Journal: Biochim Biophys Acta

The rate-limiting step in phosphatidylethanolamine (PE) synthesis by the CDP-ethanolamine pathway is the second step, which is catalyzed by CTP:phosphoethanolamine cytidylyltransferase (ET). The rate-limiting step for phosphatidylcholine biosynthesis by the CDP-choline pathway is also the second step, which is catalyzed by CTP:phosphocholine cytidylyltransferase (CT). The transcription of the most active form of CT, CTalpha, in serum-starved cells was stimulated by fetal bovine serum (FBS). Therefore, we were interested in the effects of FBS on the transcription of ET. Unexpectedly, the ET mRNA levels were significantly increased after NIH3T3 cells were cultured in serum-starved medium (0.5% FBS) longer than 8h, and the increase was suppressed by the addition of FBS. Actinomycin-D inhibited the increased ET mRNA levels in serum-starved cells. ET enzyme activities and protein amounts were also increased after serum starvation. These results suggest that FBS contains substances that inhibit the transcription of ET. To identify these substances, cells were incubated with several fractions of FBS separated by molecular sizes. As expected from the results, low-density lipoprotein, 25-hydroxycholesterol (25-OHC), 24-OHC, 27-OHC, 24(S),25-epoxycholesterol and mevalonolactate suppressed the ET mRNA levels in serum-starved cells, similar to 3-hydroxy-3-methylglutaryl-CoA reductase but not CTalpha. These results suggest that oxysterols are important regulating lipids for the suppression of ET transcription and may help maintain the contents of PE and cholesterol at the same ratio in the cellular membrane.

post to: CiteULike

MeuTXKbeta1, a scorpion venom-derived two-domain potassium channel toxin-like peptide with cytolytic activity.

Publication Date: 2010 Apr PMID: 20045493
Authors: Zhu, S. - Gao, B. - Aumelas, A. - Del Carmen Rodriguez, M. - Lanz-Mendoza, H. - Peigneur, S. - Diego-Garcia, E. - Martin-Eauclaire, M. F. - Tytgat, J. - Possani, L. D.
Journal: Biochim Biophys Acta

Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report systematic characterization of a new two-domain peptide (named MeuTXKbeta1) belonging to the TsTXKbeta molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1-21 and 22-72 fragments (named N(1-21) and C(22-72), respectively) was produced in Escherichia coli by an on-column refolding approach. Recombinant peptide (rMeuTXKbeta1) exhibited a low affinity for K(+) channels and cytolytic effects against bacteria and several eukaryotic cells. N(1-21) was found to preserve anti-Plasmodium activity in contrast to haemolytic activity, whereas C(22-72) retains these two activities. Circular dichroism analysis demonstrates that rMeuTXKbeta1 presents a typical scorpion toxin scaffold in water and its alpha-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1-21) and an ab initio structure model of MeuTXKbeta1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKbeta-related peptides and antiparasitic scorpines which both comprise the betaSPN (beta-KTxs and scorpines) family.

post to: CiteULike

Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements.

Publication Date: 2010 Mar PMID: 20045393
Authors: Koynova, R. - Tihova, M.
Journal: Biochim Biophys Acta

Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20-30wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.

post to: CiteULike

Membrane charge dependent states of the beta-amyloid fragment Abeta (16-35) with differently charged micelle aggregates.

Publication Date: 2010 Mar PMID: 20045392
Authors: Grimaldi, M. - Scrima, M. - Esposito, C. - Vitiello, G. - Ramunno, A. - Limongelli, V. - D'Errico, G. - Novellino, E. - D'Ursi, A. M.
Journal: Biochim Biophys Acta

Abeta (16-35) is the hydrophobic central core of beta-amyloid peptide, the main component of plaques found in the brain tissue of Alzheimer's disease patients. Depending on the conditions present, beta-amyloid peptides undergo a conformational transition from random coil or alpha-helical monomers, to highly toxic beta-sheet oligomers and aggregate fibrils. The behavior of beta-amyloid peptide at plasma membrane level has been extensively investigated, and membrane charge has been proved to be a key factor modulating its conformational properties. In the present work we probed the conformational behavior of Abeta (16-35) in response to negative charge modifications of the micelle surface. CD and NMR conformational analyses were performed in negatively charged pure SDS micelles and in zwitterionic DPC micelles "doped" with small amounts of SDS. To analyze the tendency of Abeta (16-35) to interact with these micellar systems, we performed EPR experiments on three spin-labeled analogues of Abeta (16-35), bearing the methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl) methanethiolsulfonate spin label at the N-terminus, in the middle of the sequence and at the C-terminus, respectively. Our conformational data show that, by varying the negative charge of the membrane, Abeta (16-35) undergoes a conformational transition from a soluble helical-kink-helical structure, to a U-turn shaped conformation that resembles protofibril models.

post to: CiteULike

Coupling effects of distal loops on structural stability and enzymatic activity of Escherichia coli dihydrofolate reductase revealed by deletion mutants.

Publication Date: 2010 Apr PMID: 20045086
Authors: Horiuchi, Y. - Ohmae, E. - Tate, S. I. - Gekko, K.
Journal: Biochim Biophys Acta

Residues distal from the active site in dihydrofolate reductase (DHFR) have regulatory roles in catalytic reaction and also folding stability. The couplings of the distal residues to the ones in the active site have been analyzed using site-directed mutants. To expand our understanding of the structural and functional influences of distal residue mutation, we explored the structural stability and enzymatic activity of deletion mutants. Deletion has greater structural and dynamical impacts on the corresponding part than site-directed mutation does. Thus, deletion amplifies the effects caused by distal mutations, which should make the mutual couplings among the distant residues more apparent. We focused on residues 52, 67, 121, and 145 in the four distinct loops of DHFR. All the single-residue deletion mutants showed marked reduction in stability, except for Delta52 in an alphaC-betaC loop. Double deletion mutants showed that the loop alphaC-betaC has nonadditive couplings with the betaF-betaG and betaG-betaH loops regarding stability. Single deletion to the loops alphaC-betaC or betaC-betaD resulted in considerable activity reduction, demonstrating that the loops couple to the residues near the active site. The four loops were shown to be functionally interdependent from the double deletion experiments.

post to: CiteULike

Chaperonin-encapsulation of proteins for NMR.

Publication Date: 2010 Apr PMID: 20045085
Authors: Tanaka, S. - Kawata, Y. - Otting, G. - Dixon, N. E. - Matsuzaki, K. - Hoshino, M.
Journal: Biochim Biophys Acta

A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR.

post to: CiteULike

Special issue on lipotoxicity.

Publication Date: 2010 Mar PMID: 20045080
Authors: Vidal-Puig, A. - Unger, R. H.
Journal: Biochim Biophys Acta

post to: CiteULike

Functional characterization of lysophosphatidic acid phosphatase from Arabidopsis thaliana.

Publication Date: 2010 Apr PMID: 20045079
Authors: Reddy, V. S. - Rao, D. K. - Rajasekharan, R.
Journal: Biochim Biophys Acta

Lysophosphatidic acid (LPA) acts as a signaling molecule that regulates diverse cellular processes and it can rapidly be metabolized by phosphatase and acyltransferase. LPA phosphatase gene has not been identified and characterized in plants so far. The BLAST search revealed that the At3g03520 is similar to phospholipase family, and distantly related to bacterial phosphatases. The conserved motif, (J)4XXXNXSFD, was identified in both At3g03520 like phospholipases and acid phosphatases. In silico expression analysis of At3g03520 revealed a high expression during phosphate starvation and abiotic stresses. This gene was overexpressed in Escherichia coli and shown to posses LPA specific phosphatase activity. These results suggest that this gene possibly plays a role in signal transduction and storage lipid synthesis.

post to: CiteULike

Hepcidin induction limits mobilisation of splenic iron in a mouse model of secondary iron overload.

Publication Date: 2010 Mar PMID: 20045050
Authors: Camberlein, E. - Abgueguen, E. - Fatih, N. - Canonne-Hergaux, F. - Leroyer, P. - Turlin, B. - Ropert, M. - Brissot, P. - Loreal, O.
Journal: Biochim Biophys Acta

Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.

post to: CiteULike

(15)N Solid-state NMR spectroscopic studies on phospholamban at its phosphorylated form at Ser-16 in aligned phospholipid bilayers.

Publication Date: 2010 Mar PMID: 20044975
Authors: Chu, S. - Abu-Baker, S. - Lu, J. - Lorigan, G. A.
Journal: Biochim Biophys Acta

Wild-type phospholamban (WT-PLB) is a pentameric transmembrane protein that regulates the cardiac cycle (contraction and relaxation). From a physiological prospective, unphosphorylated WT-PLB inhibits sarcoplasmic reticulum ATPase activity; whereas, its phosphorylated form relieves the inhibition in a mechanism that is not completely understood. In this study, site-specifically (15)N-Ala-11- and (15)N-Leu-7-labeled WT-PLB and the corresponding phosphorylated forms (P-PLB) were incorporated into 1,2-dioleoyl-sn-glycero-3-phosphocholine/2-dioleoyl-sn-glycero-3-phosphoet hanolamine (DOPC/DOPE) mechanically oriented lipid bilayers. The aligned (15)N-labeled Ala-11 and Leu-7 WT-PLB samples show (15)N resonance peaks at approximately 71ppm and 75ppm, respectively, while the corresponding phosphorylated forms P-PLB show (15)N peaks at 92ppm and 99ppm, respectively. These (15)N chemical shift changes upon phosphorylation are significant and in agreement with previous reports, which indicate that phosphorylation of WT-PLB at Ser-16 alters the structural properties of the cytoplasmic domain with respect to the lipid bilayers.

post to: CiteULike

Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective.

Publication Date: 2010 Apr PMID: 20044974
Authors: Garcia-Manyes, S. - Sanz, F.
Journal: Biochim Biophys Acta

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.

post to: CiteULike

Structural and functional changes in a synthetic S5 segment of KvLQT1 channel as a result of a conserved amino acid substitution that occurs in LQT1 syndrome of human.

Publication Date: 2010 Mar PMID: 20044973
Authors: Verma, R. - Ghosh, J. K.
Journal: Biochim Biophys Acta

Mutations in various voltage gated cardiac ion channels are the cause of different forms of long QT syndrome (LQTS), which is an inherited arrhythmic disorder marked as a prolonged QT interval on electrocardiogram. Of these LQTS1 is associated with mutations in the gene encoding KCNQ1 (KvLQT1) channel. One responsible mutation, G269S, in the S5 segment of KvLQT1, that affects the proper expression and function of channel protein leads to LQTS1. Our objective was to study how G269S mutation interferes with the structure and function of a synthetic S5 segment of KvLQT1 channel. One wild type 22-residue peptide and another mutant peptide of the same length with G269S mutation, derived from the S5 segment were synthesized and labeled with fluorescent probes. The mutant peptide exhibited lower affinity towards phospholipid vesicles as compared to the wild type peptide and showed impaired assembly and localization onto the lipid vesicles as evidenced by membrane-binding, energy transfer and proteolytic cleavage experiments. Loss in the helical content of S5 mutant peptide in membrane-mimetic environments was observed. Furthermore, it was observed that G269S mutation significantly inhibited the ability of S5 peptide to permeabilize the lipid vesicles. The present studies show the basis of change in function of the selected S5 segment as a result of G269S mutation which is associated with LQT1 syndrome. We speculate that the structural and functional changes related to the glycine to serine amino acid substitution in the S5 segment may also influence the activity of the whole KvLQT1 channel.

post to: CiteULike

Stimulation of F(1)-ATPase activity by sodium dodecyl sulfate.

Publication Date: 2010 Apr PMID: 20044971
Authors: Hossain, M. D. - Furuike, S. - Onoue, Y. - Adachi, K. - Yoshida, M. - Kinosita, K. Jr
Journal: Biochim Biophys Acta

F(1)-ATPase is a rotary molecular motor in which the gamma subunit rotates inside the cylinder made of alpha(3)beta(3) subunits. We have studied the effects of sodium dodecyl sulfate (SDS) on the rotational and ATP hydrolysis activities of F(1)-ATPase. Bulk hydrolysis activity at various SDS concentrations was examined at 2mM ATP. Maximal stimulation was obtained at 0.003% (w/v) SDS, the initial (least inhibited) activity being about 1.4 times and the steady-state activity 3-4 times the values in the absence of SDS. Rotation rates observed with a 40-nm gold bead or a 0.29-mum bead duplex as well as the torque were unaffected by the presence of 0.003% SDS. The fraction of beads that rotated, in contrast, tended to increase in the presence of SDS. SDS seems to bring inactive F(1) molecules into an active form but it does not alter or enhance the function of already active F(1) molecules significantly.

post to: CiteULike

Inhibitors of protein kinases.

Publication Date: 2010 Mar PMID: 20044042
Authors: Shugar, D.
Journal: Biochim Biophys Acta

post to: CiteULike

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response.

Publication Date: 2010 Apr PMID: 20044041
Authors: Gomes-Alves, P. - Couto, F. - Pesquita, C. - Coelho, A. V. - Penque, D.
Journal: Biochim Biophys Acta

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca(2+)-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be 'restored', i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.

post to: CiteULike

Hyperforin induces Ca(2+)-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A(2) activation through select phospholipid interactions.

Publication Date: 2010 Apr PMID: 20044029
Authors: Hoffmann, M. - Lopez, J. J. - Pergola, C. - Feisst, C. - Pawelczik, S. - Jakobsson, P. J. - Sorg, B. L. - Glaubitz, C. - Steinhilber, D. - Werz, O.
Journal: Biochim Biophys Acta

Here, we investigated the modulation of cytosolic phospholipase A(2) (cPLA(2))-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10microM) versus unstimulated cells, which was blocked by cPLA(2)alpha-inhibition, and induced translocation of cPLA(2) to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca(2+) by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA(2) at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA(2) phosphorylation. However, neither AA release nor translocation of cPLA(2) was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA(2) in the presence of Ca(2+). However, when Ca(2+) was omitted, hyperforin caused a prominent increase in cPLA(2) activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional (1)H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA(2) activation by enabling its access towards select lipid membranes independent of Ca(2+) ions. Such Ca(2+)- and phosphorylation-independent mechanism of cPLA(2) activation may apply also to other membrane-interfering molecules.

post to: CiteULike

alpha(1)-Fetoprotein transcription factor (FTF)/liver receptor homolog-1 (LRH-1) is an essential lipogenic regulator.

Publication Date: 2010 Apr PMID: 20044028
Authors: Xu, Z. - Ouyang, L. - Del Castillo-Olivares, A. - Pandak, W. M. - Gil, G.
Journal: Biochim Biophys Acta

alpha(1)-Fetoprotein transcription factor (FTF), also known as liver receptor homolog 1 (LRH-1) is highly expressed in the liver and intestine, where it is implicated in the regulation of cholesterol, bile acid and steroid hormone homeostasis. FTF is an important regulator of bile acid metabolism. We show here that FTF plays a key regulatory role in lipid homeostasis including triglyceride and cholesterol homeostasis. FTF deficient mice developed lower levels of serum triglyceride and cholesterol as a result of lower expression of several hepatic FTF target genes. Chenodeoxycholic acid repressed FTF expression resulting in a decrease in serum triglyceride in wild-type mice. The absence of chenodeoxycholic acid-mediated repression in FTF(+/-) mice demonstrated the essential role of FTF in triglyceride metabolism. Taken together, our results identify the nuclear receptor FTF as a central regulator of lipid metabolism.

post to: CiteULike

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae.

Publication Date: 2010 Apr PMID: 20044027
Authors: Schuiki, I. - Schnabl, M. - Czabany, T. - Hrastnik, C. - Daum, G.
Journal: Biochim Biophys Acta

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[(3)H(G)]serine and [(14)C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of "correct" PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.

post to: CiteULike

Role of loop 9 on the function of neuronal nicotinic receptors.

Publication Date: 2010 Mar PMID: 20043866
Authors: Criado, M. - Castillo, M. - Mulet, J. - Sala, F. - Sala, S.
Journal: Biochim Biophys Acta

We have studied the role of loop 9 in the function of neuronal nicotinic receptors. By systematically mutating the residues in the loop we have determined that the most important amino acids determining the coupling of binding to gating are the ones closer to the transmembrane region. Single mutations at location E173 in homomeric alpha7 receptors destroyed their function by completely abolishing the current while preserving the expression at the membrane. In contrast, heteromeric receptor alpha3beta4 with the same mutations retained some function. We conclude that loop 9 has a different role in the function of homomeric and heteromeric receptors.

post to: CiteULike

Structure and function of yeast glutaredoxin 2 depend on postranslational processing and are related to subcellular distribution.

Publication Date: 2010 Apr PMID: 20036764
Authors: Porras, P. - McDonagh, B. - Pedrajas, J. R. - Barcena, J. A. - Padilla, C. A.
Journal: Biochim Biophys Acta

We have previously shown that glutaredoxin 2 (Grx2) from Saccharomyces cerevisiae localizes at 3 different subcellular compartments, cytosol, mitochondrial matrix and outer membrane, as the result of different postranslational processing of one single gene. Having set the mechanism responsible for this remarkable phenomenon, we have now aimed at defining whether this diversity of subcellular localizations correlates with differences in structure and function of the Grx2 isoforms. We have determined the N-terminal sequence of the soluble mitochondrial matrix Grx2 by mass spectrometry and have determined the exact cleavage site by Mitochondrial Processing Peptidase (MPP). As a consequence of this cleavage, the mitochondrial matrix Grx2 isoform possesses a basic tetrapeptide extension at the N-terminus compared to the cytosolic form. A functional relationship to this structural difference is that mitochondrial Grx2 displays a markedly higher activity in the catalysis of GSSG reduction by the mitochondrial dithiol dihydrolipoamide. We have prepared Grx2 mutants affected on key residues inside the presequence to direct the protein to one single cellular compartment; either the cytosol, the mitochondrial membrane or the matrix and have analyzed their functional phenotypes. Strains expressing Grx2 only in the cytosol are equally sensitive to H(2)O(2) as strains lacking the gene, whereas those expressing Grx2 exclusively in the mitochondrial matrix are more resistant. Mutations on key basic residues drastically affect the cellular fate of the protein, showing that evolutionary diversification of Grx2 structural and functional properties are strictly dependent on the sequence of the targeting signal peptide.

post to: CiteULike

Cloning and molecular characterization of Dashurin encoded by C20orf116, a PCI-domain containing protein.

Publication Date: 2010 Apr PMID: 20036718
Authors: Neziri, D. - Ilhan, A. - Maj, M. - Majdic, O. - Baumgartner-Parzer, S. - Cohen, G. - Base, W. - Wagner, L.
Journal: Biochim Biophys Acta

BACKGROUND: Characterization of gene products originating from undefined open reading frames and delineation of biological functions has become the task after the human genome has been decoded. METHODS: We cloned the human C20orf 116 and defined its transcript in liver, kidney and various brain regions by Northern analysis. Antibodies against recombinant protein used for immunofluorescence and immunoblots confirmed its expression in these tissues. With the focus on kidney, its tubular expression and presence in glomerula were shown. RESULTS: A 28 aa long signal peptide predicted by in silico analysis is reflected by visualization of size variants of approximately 3kDa difference suggesting a signal peptidase cleavage of the proform. Cell compartment separation confirmed the presence of Dashurin in peroxisomes/mitochondria, microsomes, cytosol and nucleus. This is in line with green fluorescent protein (GFP)-Dashurin fusion protein shuttling between cytosol and nucleus. Luciferase reporter studies revealed a 2-3 fold increase of promoter activities upon over-expression. Bioinformatic analysis identified a PCI-domain at the C-terminus providing protein-protein interaction capabilities. CONCLUSION: Our present findings suggest the involvement of Dashurin in gene transcription or mRNA translation. GENERAL SIGNIFICANCE: Dashurin shares the PCI-domain with three multisubunit protein complexes (26S proteasome, COP9 signalosome and eIF3 translation initiation factor).

post to: CiteULike

Strong oligomerization behavior of PDGFbeta receptor transmembrane domain and its regulation by the juxtamembrane regions.

Publication Date: 2010 Mar PMID: 20036637
Authors: Oates, J. - King, G. - Dixon, A. M.
Journal: Biochim Biophys Acta

The platelet-derived growth factor beta-receptor (PDGFbetaR) represents an important subclass of receptor tyrosine kinase (RTK) thought to be activated by ligand-induced dimerization. Interestingly, the receptor is also activated by the bovine papillomavirus E5 oncoprotein, an interaction involving the transmembrane domains of both proteins and resulting in constitutive downstream signalling. This unique mode of activation along with emerging data for other RTKs raises important questions about the role of the PDGFbetaR transmembrane domain in signalling. To address this, we have investigated the murine PDGFbetaR transmembrane and juxtamembrane domains. We show for the first time the strong oligomerization behavior of PDGFbetaR transmembrane domain, forming dimers and trimers in natural membranes and detergents; and that these self-interactions are mediated by a leucine-zipper-like motif. The juxtamembrane regions are found to regulate these helix-helix interactions and select specifically for dimer formation. These data provide evidence that PDGFbetaR is able to form ligand-independent dimers, supporting similar observations in a number of other RTK's. A point mutant in the PDGFbetaR juxtamembrane domain previously shown to cause receptor activation was studied and yielded no change in oligomerization or folding, suggesting (in-line with observations of the c-Kit receptor) that it may moderate interactions with other regions of PDGFbetaR.

post to: CiteULike

Multimeric forms of the small multidrug resistance protein EmrE in anionic detergent.

Publication Date: 2010 Mar PMID: 20036636
Authors: Bay, D. C. - Budiman, R. A. - Nieh, M. P. - Turner, R. J.
Journal: Biochim Biophys Acta

Escherichia coli multidrug resistance protein E (EmrE) is a four transmembrane alpha-helix protein, and a member of the small multidrug resistance protein family that confers resistance to a broad range of quaternary cation compounds (QCC) via proton motive force. The multimeric states of EmrE protein during transport or ligand binding are variable and specific to the conditions of study. To explore EmrE multimerization further, EmrE extracted from E. coli membranes was solubilized in anionic detergent, sodium dodecyl sulphate (SDS), at varying protein concentrations. At low concentrations (/=100nm sizes) altered in the presence of TPP. Circular dichroism spectropolarimetry displayed no differences in secondary structure under the conditions studied. Fluorescence spectroscopy of SDS-EmrE protein demonstrated that aromatic residues, Trp and Tyr, are more susceptible to SDS concentration than TPP addition, but both residues exhibit enhanced quenching at high ligand concentrations. Hence, EmrE forms various multimers in SDS that are influenced by detergent concentration and TPP substrate addition.

post to: CiteULike

Light harvesting, energy transfer and electron cycling of a native photosynthetic membrane adsorbed onto a gold surface.

Publication Date: 2010 Mar PMID: 20036635
Authors: Magis, G. J. - den Hollander, M. J. - Onderwaater, W. G. - Olsen, J. D. - Hunter, C. N. - Aartsma, T. J. - Frese, R. N.
Journal: Biochim Biophys Acta

Photosynthetic membranes comprise a network of light harvesting and reaction center pigment-protein complexes responsible for the primary photoconversion reactions: light absorption, energy transfer and electron cycling. The structural organization of membranes of the purple bacterial species Rb. sphaeroides has been elucidated in most detail by means of polarized light spectroscopy and atomic force microscopy. Here we report a functional characterization of native and untreated membranes of the same species adsorbed onto a gold surface. Employing fluorescence confocal spectroscopy and light-induced electrochemistry we show that adsorbed membranes maintain their energy and electron transferring functionality. Gold-adsorbed membranes are shown to generate a steady high photocurrent of 10muA/cm(2) for several minutes and to maintain activity for up to three days while continuously illuminated. The surface-adsorbed membranes exhibit a remarkable functionality under aerobic conditions, even when exposed to light intensities well above that of direct solar irradiation. The component at the interface of light harvesting and electron cycling, the LH1 complex, displays exceptional stability, likely contributing to the robustness of the membranes. Peripheral light harvesting LH2 complexes show a light intensity dependent decoupling from photoconversion. LH2 can act as a reversible switch at low-light, an increased emitter at medium light and photobleaches at high light.

post to: CiteULike

Residual Factor VIII-like cofactor activity of thioredoxin and related oxidoreductases.

Publication Date: 2010 Mar PMID: 20036319
Authors: Bayele, H. K. - Murdock, P. J. - Pasi, K. J.
Journal: Biochim Biophys Acta

BACKGROUND: Factor VIII is the cofactor for Factor X activation by Factor IXa. Activated Factor X, Factor Xa, in turn activates prothrombin in a sequence that leads to fibrin clot formation at the site of vascular injury. Although the biochemistry of the cascade has been well studied, the molecular mechanism underlying the cofactor role of Factor VIII is not understood. METHODS: We screened a bacterial peptide display library with Factor IXa and Factor X co-immobilized on tosylactivated Dynabeads which were then used as platelet surrogates. Validation of peptide selection procedure and comparison of Factor VIII-like cofactor activity of oxidoreductases was performed using COATEST assays. Determination of Factor VIII as a folding catalyst with potential disulphide isomerase activity was determined using the RNase A renaturation assay. RESULTS: We set out to identify the cofactor requirements of the Factor IXa/Factor X procoagulant complex by random peptide display, and isolated a peptide with the active-site sequence, CGPC, of thioredoxin. This peptide was able to activate Factor X in a Factor IXa-dependent manner. Redox catalysts or oxidoreductases with homologous active-site vicinal cysteines such as PDI and DsbA also mimicked Factor VIII in their requirement of Factor IXa in Factor X activation. However, the cofactor activity of these peptides was up to a 1000-fold lower than that of Factor VIII and they were therefore unable to catalyse blood coagulation. Factor X activation by PDI and by Factor VIII was abolished by oxidation in an isolated system, which implies a possible role for thiol-disulphide exchange in the activity of the tenase complex. Using scrambled RNase A as a surrogate substrate, we also found that Factor VIII could renature this enzyme. CONCLUSION: Our findings suggest that Factor VIII may be a specialized folding catalyst with disulphide isomerase activity. We suggest that it is this activity that may underlie its cofactor function in Factor X activation, and that this function is interchangeable with classical oxidoreductases. GENERAL SIGNIFICANCE: The possible involvement of thiol-disulphide interchange as a mechanism underlying Factor VIII cofactor activity may provide some insight into the biochemistry of the intrinsic tenase complex.

post to: CiteULike

RNA and DNA interactions with zwitterionic and charged lipid membranes - A DSC and QCM-D study.

Publication Date: 2010 Apr PMID: 20036213
Authors: Michanek, A. - Kristen, N. - Hook, F. - Nylander, T. - Sparr, E.
Journal: Biochim Biophys Acta

The aim of the present study is to establish under which conditions tRNA associates with phospholipid bilayers, and to explore how this interaction influences the lipid bilayer. For this purpose we have studied the association of tRNA or DNA of different sizes and degrees of base pairing with a set of model membrane systems with varying charge densities, composed of zwitterionic phosphatidylcholines (PC) in mixtures with anionic phosphatidylserine (PS) or cationic dioctadecyl-dimethyl-ammoniumbromide (DODAB), and with fluid or solid acyl-chains (oleoyl, myristoyl and palmitoyl). To prove and quantify the attractive interaction between tRNA and model-lipid membrane we used quartz crystal microbalance with dissipation (QCM-D) monitoring to study the tRNA adsorption to deposit phospholipid bilayers from solutions containing monovalent (Na(+)) or divalent (Ca(2+)) cations. The influence of the adsorbed polynucleic acids on the lipid phase transitions and lipid segregation was studied by means of differential scanning calorimetry (DSC). The basic findings are: i) tRNA adsorbs to zwitterionic liquid-crystalline and gel-phase phospholipid bilayers. The interaction is weak and reversible, and cannot be explained only on the basis of electrostatic attraction. ii) The adsorbed amount of tRNA is higher for liquid-crystalline bilayers compared to gel-phase bilayers, while the presence of divalent cations show no significant effect on the tRNA adsorption. iii) The adsorption of tRNA can lead to segregation in the mixed 1,2-dimyristoyl-sn-glycerol-3-phosphatidylcholine (DMPC)-1,2-dimyristoyl-sn-glycero-3-phosphatidylserine (DMPS) and DMPC-DODAB bilayers, where tRNA is likely excluded from the anionic DMPS-rich domains in the first system, and associated with the cationic DODAB-rich domains in the second system. iv) The addition of shorter polynucleic acids influence the chain melting transition and induce segregation in a mixed DMPC-DMPS system, while larger polynucleic acids do not influence the melting transition in these system. The results in this study on tRNA-phospholipid interactions can have implications for understanding its biological function in, e.g., the cell nuclei, as well as in applications in biotechnology and medicine.

post to: CiteULike

Excitation energy transfer to Photosystem I in filaments and heterocysts of Nostoc punctiforme.

Publication Date: 2010 Mar PMID: 20036211
Authors: Cardona, T. - Magnuson, A.
Journal: Biochim Biophys Acta

Cyanobacteria adapt to varying light conditions by controlling the amount of excitation energy to the photosystems. On the minute time scale this leads to redirection of the excitation energy, usually referred to as state transitions, which involves movement of the phycobilisomes. We have studied short-term light adaptation in isolated heterocysts and intact filaments from the cyanobacterium Nostoc punctiforme ATCC 29133. In N.punctiforme vegetative cells differentiate into heterocysts where nitrogen fixation takes place. Photosystem II is inactivated in the heterocysts, and the abundancy of Photosystem I is increased relative to the vegetative cells. To study light-induced changes in energy transfer to Photosystem I, pre-illumination was made to dark adapted isolated heterocysts. Illumination wavelengths were chosen to excite Photosystem I (708nm) or phycobilisomes (560nm) specifically. In heterocysts that were pre-illuminated at 708nm, fluorescence from the phycobilisome terminal emitter was observed in the 77K emission spectrum. However, illumination with 560nm light caused quenching of the emission from the terminal emitter, with a simultaneous increase in the emission at 750nm, indicating that the 560nm pre-illumination caused trimerization of Photosystem I. Excitation spectra showed that 560nm pre-illumination led to an increase in excitation transfer from the phycobilisomes to trimeric Photosystem I. Illumination at 708nm did not lead to increased energy transfer from the phycobilisome to Photosystem I compared to dark adapted samples. The measurements were repeated using intact filaments containing vegetative cells, and found to give very similar results as the heterocysts. This demonstrates that molecular events leading to increased excitation energy transfer to Photosystem I, including trimerization, are independent of Photosystem II activity.

post to: CiteULike

De novo design of a non-natural fold for an iron-sulfur protein: Alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core.

Publication Date: 2010 Mar PMID: 20035711
Authors: Grzyb, J. - Xu, F. - Weiner, L. - Reijerse, E. J. - Lubitz, W. - Nanda, V. - Noy, D.
Journal: Biochim Biophys Acta

Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe(4)S(4)](+) (S=1/2), with g values of g(z,y)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe(4)S(4)](+) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.

post to: CiteULike

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII).

Publication Date: 2010 Mar PMID: 20035710
Authors: Schaller, S. - Latowski, D. - Jemiola-Rzeminska, M. - Wilhelm, C. - Strzalka, K. - Goss, R.
Journal: Biochim Biophys Acta

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.

post to: CiteULike

Modulation of advanced glycation endproduct synthesis by kynurenines in human lens proteins.

Publication Date: 2010 Apr PMID: 20026434
Authors: Nagaraj, R. H. - Padmanabha, S. - Mailankot, M. - Staniszewska, M. - Mun, L. J. - Glomb, M. A. - Linetsky, M. D.
Journal: Biochim Biophys Acta

Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5mM ribose or 5mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200-500microM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10-200microM) promoted pentosidine synthesis, but high concentrations (200-500microM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by approximately 90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100-500muM) spontaneously produced copious amounts of H(2)O(2) (10-25muM), but externally added H(2)O(2) had only a mild stimulating effect on pentosidine but had no effect on N(epsilon)-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100-500muM. Argpyrimidine synthesis from 5mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100-500muM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.

post to: CiteULike

Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer.

Publication Date: 2010 Mar PMID: 20026433
Authors: Eck, M. J. - Yun, C. H.
Journal: Biochim Biophys Acta

EGFR and other ErbB-family tyrosine kinases are overexpressed in many human tumors, and their aberrant expression and mutational activation is associated with the development, progression and aggressiveness of a number of malignancies. Thus the EGFR kinase has long been recognized as a potential drug target in oncology, and small-molecule inhibitors have been under development for more than two decades. As a result of their effectiveness in treating non-small cell lung cancers (NSCLCs) driven by somatic mutations in the EGFR kinase, gefitinib and erlotinib were the first EGFR tyrosine kinase inhibitors (TKIs) approved for clinical use. Ironically, these drugs found their target against mutant forms of the EGFR kinase, which have altered enzyme active sites, and not against the wild type (WT) kinase against which their potency and selectivity was carefully honed. Here we review recent structural and enzymological studies that explore the exquisite sensitivity of a subset of these lung cancer mutants to gefitinib and erlotinib. We discuss available structural evidence for the mechanisms of activation of the EGFR kinase by these mutants, and compare it to physiologic activation of the kinase by ligand-induced dimerization. Finally, we consider the mechanisms by which the secondary T790M "gatekeeper" mutation confers resistance to gefitinib and erlotinib.

post to: CiteULike

Divorcing folding from function: How acylation affects the membrane-perturbing properties of an antimicrobial peptide.

Publication Date: 2010 Apr PMID: 20026432
Authors: Vad, B. - Thomsen, L. A. - Bertelsen, K. - Franzmann, M. - Pedersen, J. M. - Nielsen, S. B. - Vosegaard, T. - Valnickova, Z. - Skrydstrup, T. - Enghild, J. J. - Wimmer, R. - Nielsen, N. C. - Otzen, D. E.
Journal: Biochim Biophys Acta

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an alpha-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8-C16 alkyl chains and the inclusion of anionic lipids both increase Nc's ability to form alpha-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of alpha-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of alpha-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing "plugging" effect.

post to: CiteULike

Overcoming rapid inactivation of lung surfactant: Analogies between competitive adsorption and colloid stability.

Publication Date: 2010 Apr PMID: 20026298
Authors: Zasadzinski, J. A. - Stenger, P. C. - Shieh, I. - Dhar, P.
Journal: Biochim Biophys Acta

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air-liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air-water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. added to LS, or polyelectrolytes such as chitosan, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer.

post to: CiteULike

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants.

Publication Date: 2010 Apr PMID: 20026288
Authors: Vlasits, J. - Furtmuller, P. G. - Jakopitsch, C. - Zamocky, M. - Obinger, C.
Journal: Biochim Biophys Acta

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H(2)O(2), is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 degrees C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2x10(4)M(-1)s(-1), 30M(-1)s(-1), 3.4x10(3)M(-1)s(-1), and 8.6x10(3)M(-1)s(-1), respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.

post to: CiteULike

Fatty acid 2-Hydroxylation in mammalian sphingolipid biology.

Publication Date: 2010 Apr PMID: 20026285
Authors: Hama, H.
Journal: Biochim Biophys Acta

2-Hydroxy fatty acids (hFA) are important components of a subset of mammalian sphingolipids. The presence of hFA in sphingolipids is best described in the nervous system, epidermis, and kidney. However, the literature also indicates that various hFA-sphingolipids are present in additional tissues and cell types, as well as in tumors. Biosynthesis of hFA-sphingolipids requires fatty acid 2-hydroyxlase, and degradation of hFA-sphingolipids depends, at least in part, on lysosomal acid ceramidase and the peroxisomal fatty acid alpha-oxidation pathway. Mutations in the fatty acid 2-hydroxylase gene, FA2H, have been associated with leukodystrophy and spastic paraparesis in humans, underscoring the importance of hFA-sphingolipids in the nervous system. In the epidermis, hFA-ceramides are essential for the permeability barrier function. Physiological function of hFA-sphingolipids in other organs remains largely unknown. Recent evidence indicates that hFA-sphingolipids have specific roles in cell signaling.

post to: CiteULike

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse.

Publication Date: 2010 Apr PMID: 20026284
Authors: Wu, G. - Sher, R. B. - Cox, G. A. - Vance, D. E.
Journal: Biochim Biophys Acta

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase beta, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase beta is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase alpha is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb(-/-) mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A(2) in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb(-/-) mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb(-/-) mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb(-/-) mice is due to abundant choline kinase alpha and the stable homeostasis of phosphatidylcholine.

post to: CiteULike

Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion.

Publication Date: 2010 Mar PMID: 20026269
Authors: Pedrozo, Z. - Sanchez, G. - Torrealba, N. - Valenzuela, R. - Fernandez, C. - Hidalgo, C. - Lavandero, S. - Donoso, P.
Journal: Biochim Biophys Acta

Type-2 ryanodine receptors (RyR2) - the calcium release channels of cardiac sarcoplasmic reticulum - have a central role in cardiac excitation-contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems - calpains, the proteasome and autophagy - on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8h of ischemia followed by 16h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia.

post to: CiteULike

Dipeptidyl peptidase 9 (DPP9) from bovine testes: Identification and characterization as the short form by mass spectrometry.

Publication Date: 2010 Apr PMID: 20026260
Authors: Dubois, V. - Lambeir, A. M. - Vandamme, S. - Matheeussen, V. - Guisez, Y. - Scharpe, S. - De Meester, I.
Journal: Biochim Biophys Acta

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

post to: CiteULike

Methionine oxidation stabilizes non-toxic oligomers of alpha-synuclein through strengthening the auto-inhibitory intra-molecular long-range interactions.

Publication Date: 2010 Mar PMID: 20026206
Authors: Zhou, W. - Long, C. - Reaney, S. H. - Di Monte, D. A. - Fink, A. L. - Uversky, V. N.
Journal: Biochim Biophys Acta

Oxidative stress and aggregation of the presynaptic protein alpha-synuclein (alpha-Syn) are implied in the pathogenesis of Parkinson's disease and several other neurodegenerative diseases. Various posttranslational modifications, such as oxidation, nitration and truncation, have significant effects on the kinetics of alpha-Syn fibrillation in vitro. alpha-Syn is a typical natively unfolded protein, which possesses some residual structure. The existence of long-range intra-molecular interactions between the C-terminal tail (residues 120-140) and the central part of alpha-Syn (residues 30-100) was recently established (Bertoncini et al. (2005) Proc Natl Acad Sci U S A 102, 1430-1435). Since alpha-Syn has four methionines, two of which (Met 1 and 5) are at the N-terminus and the other two (Met 116 and 127) are in the hydrophobic cluster at the C-terminus of protein, the perturbation of these residues via their oxidation represents a good model for studying the effect of long-range interaction on alpha-Syn fibril formation. In this paper we show that Met 1, 116, and 127 are more protected from the oxidation than Met 5 likely due to the residual structure in the natively unfolded alpha-Syn. In addition to the hydrophobic interactions between the C-terminal hydrophobic cluster and hydrophobic central region of alpha-Syn, there are some long-range electrostatic interactions in this protein. Both of these interactions likely serve as auto-inhibitors of alpha-Syn fibrillation. Methionine oxidation affects both electrostatic and hydrophobic long-range interactions in alpha-Syn. Finally, oxidation of methionines by H(2)O(2) greatly inhibited alpha-Syn fibrillation in vitro, leading to the formation of relatively stable oligomers, which are not toxic to dopaminergic and GABAergic neurons.

post to: CiteULike

3beta-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes.

Publication Date: 2010 Mar PMID: 20026188
Authors: Sangeetha, K. N. - Sujatha, S. - Muthusamy, V. S. - Anand, S. - Nithya, N. - Velmurugan, D. - Balakrishnan, A. - Lakshmi, B. S.
Journal: Biochim Biophys Acta

BACKGROUND: The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage. METHODS: Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process. RESULTS: The study reports (i) the isolation of a bioactive compound 3beta-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3beta. GENERAL SIGNIFICANCE: This study demonstrates the dual activity of 3beta-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3beta-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.

post to: CiteULike

Selective localization of phosphatidylcholine-derived signaling in detergent-resistant membranes from synaptic endings.

Publication Date: 2010 Mar PMID: 20026046
Authors: Mateos, M. V. - Salvador, G. A. - Giusto, N. M.
Journal: Biochim Biophys Acta

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.

post to: CiteULike

NBD-cholesterol probes to track cholesterol distribution in model membranes.

Publication Date: 2010 Mar PMID: 20026044
Authors: Ramirez, D. M. - Ogilvie, W. W. - Johnston, L. J.
Journal: Biochim Biophys Acta

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L(o)) and liquid-disordered (L(o)) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L(o) domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L(o) domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.

post to: CiteULike

Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: The effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium.

Publication Date: 2010 Mar PMID: 20026040
Authors: Davydov, D. R. - Sineva, E. V. - Sistla, S. - Davydova, N. Y. - Frank, D. J. - Sligar, S. G. - Halpert, J. R.
Journal: Biochim Biophys Acta

We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55+/-6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high-spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein.

post to: CiteULike

Redox potential of the Rieske iron-sulfur protein Quantum-chemical and electrostatic study.

Publication Date: 2010 Mar PMID: 20026009
Authors: Kuznetsov, A. M. - Zueva, E. M. - Masliy, A. N. - Krishtalik, L. I.
Journal: Biochim Biophys Acta

Quantum-chemical study of structures, energies, and effective partial charge distribution for several models of the Rieske protein redox center is performed in terms of the B3LYP density functional method in combination with the broken symmetry approach using three different atomic basis sets. The structure of the redox complex optimized in vacuum differs markedly from that inside the protein. This means that the protein matrix imposes some stress on the active site resulting in distortion of its structure. The redox potentials calculated for the real active site structure are in a substantially better agreement with the experiment than those calculated for the idealized structure. This shows an important role of the active site distortion in tuning its redox potential. The reference absolute electrode potential of the standard hydrogen electrode is used that accounts for the correction caused by the water surface potential. Electrostatic calculations are performed in the framework of the polarizable solute model. Two dielectric permittivities of the protein are employed: the optical permittivity for calculation of the intraprotein electric field, and the static permittivity for calculation of the dielectric response energy. Only this approach results in a reasonable agreement of the calculated and experimental redox potentials.

post to: CiteULike

Chloroplastic oxidative burst induced by tenuazonic acid, a natural photosynthesis inhibitor, triggers cell necrosis in Eupatorium adenophorum Spreng.

Publication Date: 2010 Mar PMID: 20026008
Authors: Chen, S. - Yin, C. - Qiang, S. - Zhou, F. - Dai, X.
Journal: Biochim Biophys Acta

Tenuazonic acid (TeA), a nonhost-specific phytotoxin produced by Alternaria alternata, was determined to be a novel natural photosynthesis inhibitor owning several action sites in chloroplasts. To further elucidate the mode of its action, studies were conducted to assess the production and involvement of reactive oxygen species (ROS) in the toxic activity of TeA. A series of experiments indicated that TeA treatment can induce chloroplast-derived ROS generation including not only (1)O(2) but also O(2)(-), H(2)O(2) and ()OH in Eupatorium adenophorum mesophyll cells, resulting from electron leakage and charge recombination in PSII as well as thylakoid overenergization due to inhibition of the PSII electron transport beyond Q(A) and the reduction of end acceptors on the PSI acceptor side and chloroplast ATPase activity. The initial production of TeA-induced ROS was restricted to chloroplasts and accompanied with a certain degree of chloroplast damage. Subsequently, abundant ROS were quickly dispersed throughout whole cell and cellular compartments, causing a series of irreversible cellular harm such as chlorophyll breakdown, lipid peroxidation, plasma membrane rupture, chromatin condensation, DNA cleavage, and organelle disintegration, and finally resulting in rapid cell destruction and leaf necrosis. These results show that TeA causing cell necrosis of host-plants is a result of direct oxidative damage from chloroplast-mediated ROS eruption.

post to: CiteULike

Hofmeister effects on activity and stability of alkaline phosphatase.

Publication Date: 2010 Apr PMID: 20025997
Authors: Yang, Z. - Liu, X. J. - Chen, C. - Halling, P. J.
Journal: Biochim Biophys Acta

We have studied the effects on alkaline phosphatase of adding high concentrations (normally 1.0 M) of simple salts. It is necessary to allow for significant effects of salts on the extinction coefficient of the reaction product, and on the apparent pH of the buffer. Both activity and stability of the enzyme correlate well with the Hofmeister series in terms of the salt's kosmotropic/chaotropic properties, which are assessed by the Jones-Dole viscosity B coefficients (B(+) for cations and B(-) for anions). The catalytic activity or V(max)/K(m) of the enzyme showed a bell-shaped relationship with the (B(-)-B(+)) values of the salts present, being optimal with salts (such as NaCl, KCl, and KNO(3)) where the anion and cation have similar kosmotropic/chaotropic properties. This effect is believed to be enzyme-specific and relates to the impact of both cations and anions on the enzyme's surface pH, active site, and catalytic mechanism. Anions play a more predominant role than cations in affecting enzyme stability. The rate of irreversible thermal inactivation is strongly reduced by addition of kosmotropic anions like SO(4)(2-) (half-life increased from 8 to 580 min at 60 degrees C). This effect is general and the mechanism probably involves the ability of the ions to affect the water solvation layer around the enzyme molecule and to interact with both the surface and internal structure of the enzyme.

post to: CiteULike

A novel function of the human CLS1 in phosphatidylglycerol synthesis and remodeling.

Publication Date: 2010 Apr PMID: 20025994
Authors: Nie, J. - Hao, X. - Chen, D. - Han, X. - Chang, Z. - Shi, Y.
Journal: Biochim Biophys Acta

Phosphatidylglycerol (PG) is a precursor for the biosynthesis of cardiolipin and a signaling molecule required for various cellular functions. PG is subjected to remodeling subsequent to its de novo biosynthesis in mitochondria to incorporate appropriate acyl content for its biological functions and to prevent the harmful effect of lysophosphatidylglycerol (LPG) accumulation. Yet, a gene encoding a mitochondrial LPG acyltransferase has not been identified. In this report, we identified a novel function of the human cardiolipin synthase (hCLS1) in regulating PG remodeling. In addition to the reported cardiolipin synthase activity, the recombinant hCLS1 protein expressed in COS-7 cells and Sf-9 insect cells exhibited a strong acyl-CoA-dependent LPG acyltransferase activity, which was further confirmed by purified hCLS1 protein overexpressed in Sf-9 cells. The recombinant hCLS1 displayed an acyl selectivity profile in the order of in the order of C18:1>C18:2>C18:0>C16:0, which is similar to that of hCLS1 toward PGs in cardiolipin synthesis, suggesting that the PG remodeling by hCLS1 is an intrinsic property of the enzyme. In contrast, no significant acyltransferase activity was detected from the recombinant hCLS1 enzyme toward lysocardiolipin which shares a similar structure with LPG. In support of a key function of hCLS1 in PG remodeling, overexpression of hCLS1 in COS-7 cells significantly increased PG biosynthesis concurrent with elevated levels of cardiolipin without any significant effects on the biosynthesis of other phospholipids. These results demonstrate for the first time that hCLS1 catalyzes two consecutive steps in cardiolipin biosynthesis by acylating LPG to PG and then converting PG to cardiolipin.

post to: CiteULike

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc(1) complex.

Publication Date: 2010 Mar PMID: 20025846
Authors: Berry, E. A. - Huang, L. S. - Lee, D. W. - Daldal, F. - Nagai, K. - Minagawa, N.
Journal: Biochim Biophys Acta

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc(1) complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q(i) site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q(o) site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A(3) in a fashion similar to the Q(o) site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q(i) and the Q(o) sites of the fungal cytochrome bc(1) complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc(1) complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q(o) and Q(i) sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc(1) complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q(i) and Q(o) sites. Moreover, crystal structure of chicken cytochrome bc(1) complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q(i) and Q(o) sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc(1) inhibitor that acts at both of the active sites of this enzyme.

post to: CiteULike

(d)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha.

Publication Date: 2010 Mar PMID: 20018259
Authors: Nickl, C. K. - Raidas, S. K. - Zhao, H. - Sausbier, M. - Ruth, P. - Tegge, W. - Brayden, J. E. - Dostmann, W. R.
Journal: Biochim Biophys Acta

The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.

post to: CiteULike

Host proteins involved in HIV infection: New therapeutic targets.

Publication Date: 2010 Mar PMID: 20018238
Authors: Arhel, N. - Kirchhoff, F.
Journal: Biochim Biophys Acta

Current treatment of HIV/AIDS consists of a combination of three to five agents targeting different viral proteins, i.e. the reverse transcriptase, protease, integrase and envelope, and aims to suppress viral replication below detectable levels. This "highly active antiretroviral therapy" (HAART) has brought an enormous benefit for life expectancy and quality in HIV-1-infected individuals, at least in industrialized countries. However, significant limitations with regard to efficiency, drug resistance, side effect and costs still exist. Recent data suggest that cellular factors also represent useful targets for therapy. Here, we summarize findings from several genome-wide screens that identified a large number of cellular factors exploited by HIV-1 at each step of its life cycle. Furthermore, we discuss the evidence that humans are equipped with powerful intrinsic defense mechanisms against retroviruses but that HIV-1 has evolved elaborate ways to counteract or evade them. Preventing the use of host cell proteins obligatory for viral replication or strengthening the cellular defense mechanisms may help to reduce viral replication to harmless levels. A better understanding of the host factors that promote or restrict HIV-1 replication may thus lead to the development of novel therapeutics against HIV/AIDS.

post to: CiteULike

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies.

Publication Date: 2010 Mar PMID: 20018171
Authors: Kulma, M. - Herec, M. - Grudzinski, W. - Anderluh, G. - Gruszecki, W. I. - Kwiatkowska, K. - Sobota, A.
Journal: Biochim Biophys Acta

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

post to: CiteULike

Thermal aggregation of glycated bovine serum albumin.

Publication Date: 2010 Apr PMID: 20006741
Authors: Rondeau, P. - Navarra, G. - Cacciabaudo, F. - Leone, M. - Bourdon, E. - Militello, V.
Journal: Biochim Biophys Acta

Aggregation and glycation processes in proteins have a particular interest in medicine fields and in food technology. Serum albumins are model proteins which are able to self-assembly in aggregates and also sensitive to a non-enzymatic glycation in cases of diabetes. In this work, we firstly reported a study on the glycation and oxidation effects on the structure of bovine serum albumin (BSA). The experimental approach is based on the study of conformational changes of BSA at secondary and tertiary structures by FTIR absorption and fluorescence spectroscopy, respectively. Secondly, we analysed the thermal aggregation process on BSA glycated with different glucose concentrations. Additional information on the aggregation kinetics are obtained by light scattering measurements. The results show that glycation process affects the native structure of BSA. Then, the partial unfolding of the tertiary structure which accompanies the aggregation process is similar both in native and glycated BSA. In particular, the formation of aggregates is progressively inhibited with growing concentration of glucose incubated with BSA. These results bring new insights on how aggregation process is affected by modification of BSA induced by glycation.

post to: CiteULike

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions.

Publication Date: 2010 Mar PMID: 20006572
Authors: Unitt, D. C. - Hollis, V. S. - Palacios-Callender, M. - Frakich, N. - Moncada, S.
Journal: Biochim Biophys Acta

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O(2)) conditions. The system measures the concentrations of O(2) and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O(2) concentration and electron turnover of the enzyme. At a high O(2) concentration (70 muM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O(2). At low O(2) (15 muM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O(2) consumption. At both high and low O(2) concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.

post to: CiteULike

Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2.

Publication Date: 2010 Apr PMID: 20005974
Authors: Pedrosa, A. M. - Faine, L. A. - Grosso, D. M. - de Las Heras, B. - Bosca, L. - Abdalla, D. S.
Journal: Biochim Biophys Acta

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264.7 macrophages with LDL(-) for 24 h resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-); incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells. CD95 (Fas), CD95 ligand (FasL), CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells. However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL. Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression. These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages.

post to: CiteULike

Cross-linking with bifunctional reagents and its application to the study of the molecular symmetry and the arrangement of subunits in hexameric protein oligomers.

Publication Date: 2010 Apr PMID: 20005307
Authors: Azem, A. - Tsfadia, Y. - Hajouj, O. - Shaked, I. - Daniel, E.
Journal: Biochim Biophys Acta

Cross-linking with a bifunctional reagent and subsequent SDS gel electrophoresis is a simple but effective method to study the symmetry and arrangement of subunits in oligomeric proteins. In this study, theoretical expressions for the description of cross-linking patterns were derived for protein homohexamers through extension of the method used for tetramers by Hajdu et al. (1976). The derived equations were used for the analysis of cross-linking by glutardialdehyde of four protein hexamers: beef liver glutamate dehydrogenase (GDH), jack bean urease, hemocyanin from the spiny lobster Panulirus pencillatus (PpHc), Escherichia coli glutamate decarboxylase (GDC) and for analysis of published data on the cross-linking of hexameric E. coli rho by dimethyl suberimidate. Best fit models showed that the subunits in the first four proteins are arranged according to D(3) symmetry in two layers, each subunit able to cross-link to three neighboring subunits for GDH and urease, or to four for PpHc and GDC. The findings indicate a dimer-of-trimers eclipsed arrangement of subunits for GDH and urease and a trimer-of-dimers staggered one for PpHc and GDC. In rho, the subunits are arranged according to D(3) symmetry in a trimer-of-dimers ring. The conclusions from cross-linking of GDH and GDC, PpHc and rho are consistent with results from X-ray crystal structure, those for urease with findings from electron microscopy.

post to: CiteULike

Dissecting the role of mTOR: Lessons from mTOR inhibitors.

Publication Date: 2010 Mar PMID: 20005306
Authors: Dowling, R. J. - Topisirovic, I. - Fonseca, B. D. - Sonenberg, N.
Journal: Biochim Biophys Acta

Recent years have observed significant advances in our understanding of how the serine/threonine kinase target of rapamycin (TOR) controls key cellular processes such as cell survival, growth and proliferation. Consistent with its role in cell proliferation, the mTOR pathway is frequently hyperactivated in a number of human malignancies and is thus considered to be an attractive target for anti-cancer therapy. Rapamycin and its analogs (rapalogs) function as allosteric inhibitors of mTORC1 and are currently used in the treatment of advanced renal cell carcinoma. Rapamycin and its derivatives bind to the small immunophilin FKBP12 to inhibit mTORC1 signalling through a poorly understood mechanism. Rapamycin/FKBP12 efficiently inhibit some, but not all, functions of mTOR and hence much interest has been placed in the development of drugs that target the kinase activity of mTOR directly. Several novel active-site inhibitors of mTOR, which inhibit both mTORC1 and mTORC2, were developed in the last year. In this manuscript, we provide a brief outline of our current understanding of the mTOR signalling pathway and review the molecular underpinnings of the action of rapamycin and novel active-site mTOR inhibitors as well as potential advantages and caveats associated with the use of these drugs in the treatment of cancer.

post to: CiteULike

Structure-guided expansion of kinase fragment libraries driven by support vector machine models.

Publication Date: 2010 Mar PMID: 20005305
Authors: Erickson, J. A. - Mader, M. M. - Watson, I. A. - Webster, Y. W. - Higgs, R. E. - Bell, M. A. - Vieth, M.
Journal: Biochim Biophys Acta

This work outlines a new de novo design process for the creation of novel kinase inhibitor libraries. It relies on a profiling paradigm that generates a substantial amount of kinase inhibitor data from which highly predictive QSAR models can be constructed. In addition, a broad diversity of X-ray structure information is needed for binding mode prediction. This is important for scaffold and substituent site selection. Borrowing from FBDD, the process involves fragmentation of known actives, proposition of binding mode hypotheses for the fragments, and model-driven recombination using a pharmacophore derived from known kinase inhibitor structures. The support vector machine method, using Merck atom pair derived fingerprint descriptors, was used to build models from activity from 6 kinase assays. These models were qualified prospectively by selecting and testing compounds from the internal compound collection. Overall hit and enrichment rates of 82% and 2.5%, respectively, qualified the models for use in library design. Using the process, 7 novel libraries were designed, synthesized and tested against these same 6 kinases. The results showed excellent results, yielding a 92% hit rate for the 179 compounds that made up the 7 libraries. The results of one library designed to include known literature compounds, as well as an analysis of overall substituent frequency, are discussed.

post to: CiteULike

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

Publication Date: 2010 Mar PMID: 20004641
Authors: Nervi, P. - Li-Blatter, X. - Aanismaa, P. - Seelig, A.
Journal: Biochim Biophys Acta

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.

post to: CiteULike

Infrared reflection-absorption spectroscopy: Principles and applications to lipid-protein interaction in Langmuir films.

Publication Date: 2010 Apr PMID: 20004639
Authors: Mendelsohn, R. - Mao, G. - Flach, C. R.
Journal: Biochim Biophys Acta

Infrared reflection-absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.

post to: CiteULike

Low-temperature electron transfer suggests two types of Q(A) in intact photosystem II.

Publication Date: 2010 Mar PMID: 20004638
Authors: Bao, H. - Zhang, C. - Ren, Y. - Zhao, J.
Journal: Biochim Biophys Acta

The correlation between the reduction of Q(A) and the oxidation of Tyr(Z) or Car/Chl(Z)/Cyt(b559) in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g=1.95-1.86 Q(A)(-*)EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q(A)(-*), Car(+*)/Chl(+*) and oxidized Cyt(b559), while enhancing the amplitude of the S(1)Tyr(Z)* EPR signal in the intact PSII sample. The quantification and stability of the g=1.95-1.86 EPR signal and signals arising from the oxidized Tyr(Z) and the side-path electron donors, respectively, indicate that the EPR-detectable g=1.95-1.86 Q(A)(-*) signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl(Z)/Cyt(b559)), but not of Tyr(Z). These results imply that two types of Q(A)(-*) probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q(A) are discussed.

post to: CiteULike

A single-point mutation in FGFR2 affects cell cycle and Tgfbeta signalling in osteoblasts.

Publication Date: 2010 Mar PMID: 20004243
Authors: Lee, K. M. - Santos-Ruiz, L. - Ferretti, P.
Journal: Biochim Biophys Acta

Fgf and Tgfbeta are key regulators of bone development. It is not known, however, whether there is a relationship between defective Fgf signalling, resulting in a premature cranial suture fusion, and Tgfbeta signalling. We used mouse calvaria osteoblasts carrying a mutation (hFGFR2-C278F) associated with Crouzon and Pfeiffer syndromes to investigate effects of this mutation on cell growth and possible mechanisms underlying it. Mutated osteoblasts displayed reduced S-phase, increased apoptosis and increased differentiation. As Tgfbeta signalling appeared to be required in an autocrine/paracrine manner for osteoblast proliferation, we tested the hypothesis that reduced growth might be due, at least in part, to an altered balance between FGF and Tgfbeta signalling. Tgfbeta expression was indeed decreased in mutated osteoblasts, as compared to osteoblasts carrying the wild type hFGFR2. Treatment with Tgfbeta, however, neither increased proliferation in mutated osteoblasts, unlike in controls, nor rescued proliferation in control osteoblasts treated with an Erk1/2 inhibitor. Significantly, Erk2, that is important for proliferation, was reduced relatively to Erk1 in mutated cells. Altogether this study suggests that the hFGFR2-C278F mutation affects the osteoblast ability to respond to Tgfbeta stimulation via the Erk pathway and that the overall effect of the mutation is a loss of function.

post to: CiteULike

Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.

Publication Date: 2010 Mar PMID: 20004233
Authors: Clarke, M. J. - Artero, J. B. - Moulin, M. - Callow, P. - Carver, J. A. - Griffiths, P. C. - Haertlein, M. - Harding, J. J. - Meek, K. M. - Timmins, P. - Regini, J. W.
Journal: Biochim Biophys Acta

alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2) O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.

post to: CiteULike

Interaction with membrane mimics of transmembrane fragments 16 and 17 from the human multidrug resistance ABC transporter 1 (hMRP1/ABCC1) and two of their tryptophan variants.

Publication Date: 2010 Mar PMID: 20004175
Authors: de Foresta, B. - Vincent, M. - Gallay, J. - Garrigos, M.
Journal: Biochim Biophys Acta

The human multidrug resistance-associated protein 1 (hMRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily. Together with P-glycoprotein (ABCB1) and the breast cancer resistance protein (BCRP/ABCG2), hMRP1 confers resistance to a large number of structurally diverse drugs. The current topological model of hMRP1 includes two cytosolic nucleotide-binding domains and 17 putative transmembrane (TM) helices forming three membrane-spanning domains. Mutagenesis and labeling studies have shown TM16 and TM17 to be important for function. We characterized the insertion of the TM16 fragment into dodecylphosphocholine (DPC) or n-dodecyl-beta-d-maltoside (DM) micelles as membrane mimics and extended our previous work on TM17 (Vincent et al., 2007, Biochim. Biophys. Acta 1768, 538). We synthesized TM16 and TM17, with the Trp residues, W1198 in TM16 and W1246 in TM17, acting as an intrinsic fluorescent probe, and TM16 and TM17 Trp variants, to probe different positions in the peptide sequence. We assessed the interaction of peptides with membrane mimics by evaluating the increase in fluorescence intensity resulting from such interactions. In all micelle-bound peptides, the tryptophan residue appeared to be located, on average, in the head group micelle region, as shown by its fluorescence spectrum. Each tryptophan residue was partially accessible to both acrylamide and the brominated acyl chains of two DM analogs, as shown by fluorescence quenching. Tryptophan fluorescence lifetimes were found to depend on the position of the tryptophan residue in the various peptides, probably reflecting differences in local structures. Far UV CD spectra showed that TM16 contained significant beta-strand structures. Together with the high Trp correlation times, the presence of these structures suggests that TM16 self-association may occur at the interface. In conclusion, this experimental study suggests an interfacial location for both TM16 and TM17 in membrane mimics. In terms of overall hMRP1 structure, the experimentally demonstrated amphipathic properties of these TM are consistent with a role in the lining of an at least partly hydrophilic transport pore, as suggested by the currently accepted structural model, the final structure being modified by interaction with other TM helices.

post to: CiteULike

Design of peptide-targeted liposomes containing nucleic acids.

Publication Date: 2010 Mar PMID: 20004174
Authors: Santos, A. O. - da Silva, L. C. - Bimbo, L. M. - de Lima, M. C. - Simoes, S. - Moreira, J. N.
Journal: Biochim Biophys Acta

Anticancer systemic gene silencing therapy has been so far limited by the inexistence of adequate carrier systems that ultimately provide an efficient intracellular delivery into target tumor cells. In this respect, one promising strategy involves the covalent attachment of internalizing-targeting ligands at the extremity of PEG chains grafted onto liposomes. Therefore, the present work aims at designing targeted liposomes containing nucleic acids, with small size, high encapsulation efficiency and able to be actively internalized by SCLC cells, using a hexapeptide (antagonist G) as a targeting ligand. For this purpose, the effect of the liposomal preparation method, loading material (ODN versus siRNA) and peptide-coupling procedure (direct coupling versus post-insertion) on each of the above-mentioned parameters was assessed. Post-insertion of DSPE-PEG-antagonist G conjugates into preformed liposomes herein named as stabilized lipid particles, resulted in targeted vesicles with a mean size of about 130 nm, encapsulation efficiency close to 100%, and a loading capacity of approximately 5 nmol siRNA/mumol of total lipid. In addition, the developed targeted vesicles showed increased internalization in SCLC cells, as well as in other tumor cells and HMEC-1 microvascular endothelial cells. The improved cellular association, however, did not correlate with enhanced downregulation of the target protein (Bcl-2) in SCLC cells. These results indicate that additional improvements need to be performed in the future, namely by ameliorating the access of the nucleic acids to the cytoplasm of the tumor cells following receptor-mediated endocytosis.

post to: CiteULike

Differential effects of conjugated linoleic acid isomers on the biophysical and biochemical properties of model membranes.

Publication Date: 2010 Mar PMID: 20004173
Authors: Subbaiah, P. V. - Sircar, D. - Aizezi, B. - Mintzer, E.
Journal: Biochim Biophys Acta

Conjugated linoleic acids (CLA) are known to exert several isomer-specific biological effects, but their mechanisms of action are unclear. In order to determine whether the physicochemical effects of CLA on membranes play a role in their isomer-specific effects, we synthesized phosphatidylcholines (PCs) with 16:0 at sn-1 position and one of four CLA isomers (trans 10 cis 12 (A), trans 9 trans 11 (B), cis 9 trans 11 (C), and cis 9 cis 11 (D)) at sn-2, and determined their biophysical properties in monolayers and bilayers. The surface areas of the PCs with the two natural CLA (A and C) were similar at all pressures, but they differed significantly in the presence of cholesterol, with PC-A condensing more than PC-C. Liposomes of PC-A similarly showed increased binding of cholesterol compared to PC-C liposomes. PC-A liposomes were less permeable to carboxyfluorescein compared to PC-C liposomes. The PC with two trans double bonds (B) showed the highest affinity to cholesterol and lowest permeability. The two natural CLA-PCs (A and C) stimulated lecithin-cholesterol acyltransferase activity by 2-fold, whereas the unnatural CLA-PCs (B and D) were inhibitory. These results suggest that the differences in the biophysical properties of CLA isomers A and C may partly contribute to the known differences in their biological effects.

post to: CiteULike

Cell membrane extensions, generated by mechanical constraint, are associated with a sustained lipid raft patching and an increased cell signaling.

Publication Date: 2010 Mar PMID: 19962956
Authors: Larive, R. M. - Baisamy, L. - Urbach, S. - Coopman, P. - Bettache, N.
Journal: Biochim Biophys Acta

Platelet activation triggers an imbalance in plasma membrane phospholipids by a specific aminophospholipid outflux, resulting in filopodia formation. Similarly, the addition of a phospholipid excess in the outer leaflet of the plasma membrane induces cellular extensions and actin polymerization. The implication of membrane microdomains in sustaining these mechanical constraints remains, however, unknown and was investigated in human platelets and mouse fibroblasts. The disruption of lipid rafts by cholesterol depletion prevents actin polymerization and formation of cellular extensions. Phospholipid excess triggers raft patching underneath the cell extensions, recruitment of protein raft markers and increase of tyrosine phosphorylation of raft proteins. Using a mass spectrometric analysis of isolated platelet rafts, we identified tyrosine kinases and proteins implicated in the formation of cell membrane extensions, cell adhesion and motility. They are recruited to rafts in response to a mechanical constraint. Taken together, our results demonstrate that exogenous phospholipid addition causes a modulation of the lateral plasma membrane organization and an activation of the cell signaling triggering actin remodeling and the formation of cellular protrusions. Raft disruption abolishes these processes, demonstrating that their integrity is crucial for cell shape changes in response to a mechanical constraint on plasma membrane.

post to: CiteULike

Mechanism of strong quenching of photosystem II chlorophyll fluorescence under drought stress in a lichen, Physciella melanchla, studied by subpicosecond fluorescence spectroscopy.

Publication Date: 2010 Mar PMID: 19962955
Authors: Komura, M. - Yamagishi, A. - Shibata, Y. - Iwasaki, I. - Itoh, S.
Journal: Biochim Biophys Acta

The mechanism of the severe quenching of chlorophyll (Chl) fluorescence under drought stress was studied in a lichen Physciella melanchla, which contains a photobiont green alga, Trebouxia sp., using a streak camera and a reflection-mode fluorescence up-conversion system. We detected a large 0.31 ps rise of fluorescence at 715 and 740 nm in the dry lichen suggesting the rapid energy influx to the 715-740 nm bands from the shorter-wavelength Chls with a small contribution from the internal conversion from Soret bands. The fluorescence, then, decayed with time constants of 23 and 112 ps, suggesting the rapid dissipation into heat through the quencher. The result confirms the accelerated 40 ps decay of fluorescence reported in another lichen (Veerman et al., 2007 [36]) and gives a direct evidence for the rapid energy transfer from bulk Chls to the longer-wavelength quencher. We simulated the entire PS II fluorescence kinetics by a global analysis and estimated the 20.2 ns(-1) or 55.0 ns(-1) energy transfer rate to the quencher that is connected either to the LHC II or to the PS II core antenna. The strong quenching with the 3-12 times higher rate compared to the reported NPQ rate, suggests the operation of a new type of quenching, such as the extreme case of Chl-aggregation in LHCII or a new type of quenching in PS II core antenna in dry lichens.

post to: CiteULike

Photochemical processes observed during the reaction of superoxide reductase from Desulfoarculus baarsii with superoxide Re-evaluation of the reaction mechanism.

Publication Date: 2010 Apr PMID: 19962458
Authors: Bonnot, F. - Houee-Levin, C. - Favaudon, V. - Niviere, V.
Journal: Biochim Biophys Acta

Superoxide reductase SOR is an enzyme involved in superoxide detoxification in some microorganisms. Its active site consists of a non-heme ferrous center in an unusual [Fe(NHis)(4) (SCys)(1)] square pyramidal pentacoordination that efficiently reduces superoxide into hydrogen peroxide. In previous works, the reaction mechanism of the SOR from Desulfoarculus baarsii enzyme, studied by pulse radiolysis, was shown to involve the formation of two reaction intermediates T1 and T2. However, the absorption spectrum of T2 was reported with an unusual sharp band at 625 nm, very different from that reported for other SORs. In this work, we show that the sharp band at 625 nm observed by pulse radiolysis reflects the presence of photochemical processes that occurs at the level of the transient species formed during the reaction of SOR with superoxide. These processes do not change the stoichiometry of the global reaction. These data highlight remarkable photochemical properties for these reaction intermediates, not previously suspected for iron-peroxide species formed in the SOR active site. We have reinvestigated the reaction mechanism of the SOR from D. baarsii by pulse radiolysis in the absence of these photochemical processes. The T1 and T2 intermediates now appear to have absorption spectra similar to those reported for the Archaeoglobus fulgidus SOR enzymes. Although for some enzymes of the family only one transient was reported, on the whole, the reaction mechanisms of the different SORs studied so far seem very similar, which is in agreement with the strong sequence and structure homologies of their active sites.

post to: CiteULike

Somatic mutations in PI3Kalpha: Structural basis for enzyme activation and drug design.

Publication Date: 2010 Mar PMID: 19962457
Authors: Gabelli, S. B. - Mandelker, D. - Schmidt-Kittler, O. - Vogelstein, B. - Amzel, L. M.
Journal: Biochim Biophys Acta

The PI3K pathway is a communication hub coordinating critical cell functions including cell survival, cell growth, proliferation, motility and metabolism. Because PI3Kalpha harbors recurrent somatic mutations resulting in gains of function in human cancers, it has emerged as an important drug target for many types of solid tumors. Various PI3K isoforms are also being evaluated as potential therapeutic targets for inflammation, heart disease, and hematological malignancies. Structural biology is providing insights into the flexibility of the PI3Ks, and providing basis for understanding the effects of mutations, drug resistance and specificity.

post to: CiteULike

ADD1/SREBP1c activates the PGC1-alpha promoter in brown adipocytes.

Publication Date: 2010 Apr PMID: 19962449
Authors: Hao, Q. - Hansen, J. B. - Petersen, R. K. - Hallenborg, P. - Jorgensen, C. - Cinti, S. - Larsen, P. J. - Steffensen, K. R. - Wang, H. - Collins, S. - Wang, J. - Gustafsson, J. A. - Madsen, L. - Kristiansen, K.
Journal: Biochim Biophys Acta

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that beta-adrenergic stimulation induced expression of LXRalpha, ADD1/SREBP1c and PGC1alpha in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1alpha expression via a conserved E box in the proximal promoter and that beta-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1alpha promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1alpha expression in brown adipose tissue.

post to: CiteULike

Effect of fumagillin on adipocyte differentiation and adipogenesis.

Publication Date: 2010 Apr PMID: 19961899
Authors: Scroyen, I. - Christiaens, V. - Lijnen, H. R.
Journal: Biochim Biophys Acta

BACKGROUND: Inhibition of angiogenesis may impair adipose tissue development. METHODS: The effect of fumagillin (a methionine aminopeptidase-2 inhibitor) on adipocyte differentiation and de novo adipogenesis was investigated in murine model systems. RESULTS: During in vitro differentiation of murine 3T3-F442A preadipocytes, administration of fumagillin (>/=1 muM) resulted in reduced expression of methionine aminopeptidase-2, and in enhanced differentiation rate. In vivo, de novo development of adipose tissue following injection of preadipocytes in nude mice kept on high fat diet was somewhat, but not significantly (p=0.06), reduced by administration of fumagillin (1 mg/kg/day during 4 weeks by oral gavage). This was not associated with effects on blood vessel size or density, whereas blood vessel density normalized to adipocyte density was enhanced upon fumagillin treatment. In vivo BrdU incorporation experiments did not reveal effects of fumagillin on cell proliferation in adipose tissues, and cellular apoptosis was also not affected. Treatment with fumagillin enhances in vitro differentiation of preadipocytes, but has only a minor effect on in vivo adipogenesis. GENERAL SIGNIFICANCE: These studies on in vitro and in vivo preadipcoyte differentiation thus do not support an anti-obesity effect of fumagillin as a result of effects on adipocyte differentiation.

post to: CiteULike

Dynamic and energetic mechanisms for the distinct permeation rate in AQP1 and AQP0.

Publication Date: 2010 Mar PMID: 19961829
Authors: Qiu, H. - Ma, S. - Shen, R. - Guo, W.
Journal: Biochim Biophys Acta

Despite sharing overall sequence and structural similarities, water channel aquaporin 0 (AQP0) transports water more slowly than other aquaporins. Using molecular dynamics simulations of AQP0 and AQP1, we find that there is a sudden decrease in the distribution profile of water density along the pore of AQP0 in the region of residue Tyr23, which significantly disrupts the single file water chain by forming hydrogen bond with permeating water molecules. Comparisons of free-energy and interaction-energy profiles for water conduction between AQP0 and AQP1 indicate that this interruption of the water chain causes a huge energy barrier opposing water translocation through AQP0. We further show that a mutation of Tyr23 to phenylalanine leads to a 2- to 4-fold enhancement in water permeability of AQP0, from (0.5+/-0.2)x10(-14) cm(3)s(-1) to (1.9+/-0.6)x10(-14) cm(3)s(-1). Therefore, Tyr23 is a dominate factor leading to the low water permeability in AQP0.

post to: CiteULike