Biochimica et Biophysica Acta

Charge distribution and imperfect amphipathicity affect pore formation by antimicrobial peptides.

Publication Date: 2012 Jan 25 PMID: 22290189
Authors: Mihajlovic, M. - Lazaridis, T.
Journal: Biochim Biophys Acta

Antimicrobial peptides often permeabilize biological membranes via a pore mechanism. Two pore types have been proposed: toroidal, where the pore is partly lined by lipid, and barrel-stave, where a cylindrical pore is completely lined by peptides. What drives the preference of antimicrobial peptides for a certain pore type is not yet fully understood. According to neutron scattering and oriented circular dichroism, melittin and MG-H2 induce toroidal pores whereas alamethicin forms barrel-stave pores. In previous work we found that indeed melittin seems to favor toroidal pores whereas alamethicin favors cylindrical pores. Here we designed mutants of these two peptides and the magainin analog MG-H2, aimed to probe how the distribution of charges along the helix and its imperfectly amphipathic structure influence pore formation. Molecular dynamics (MD) simulations of the peptides in a pre-formed cylindrical pore have been performed. The duration of the simulations was 136ns to 216ns. We found that a melittin mutant with lysine 7 neutralized favors cylindrical pores whereas a MG-H2 mutant with lysines in the N-terminal half of these peptides neutralized and an alamethicin mutant with a positive charge at the position 7 form semitoroidal pores. These results suggest that charged residues within the N-terminal half are important for the toroidal pore formation. Toroidal pores produced by MG-H2 are more disordered than the melittin pores, likely because of the charged residues located in the middle of the MG-H2 helix (K11 and K14). Imperfect amphipathicity of melittin seems to play a role in its preference for toroidal pores since the substitutions of charged residues located within the nonpolar face by hydrophobic residues suppress evolution of a toroidal pore. The mutations change the position of lysine 7 near the N-terminus, relative to the lower leaflet headgroups. The MD simulations also show that the melittin P14A mutant forms a toroidal pore, but its configuration diverges from that of melittin and it is probably metastable.

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Na,K-ATPase activity modulates Src activation: A role for ATP/ADP ratio.

Publication Date: 2012 Jan 24 PMID: 22290188
Authors: Weigand, K. M. - Swarts, H. G. - Fedosova, N. U. - Russel, F. G. - Koenderink, J. B.
Journal: Biochim Biophys Acta

Digitalis-like compounds (DLCs), specific inhibitors of Na,K-ATPase, are implicated in cellular signaling. Exposure of cell cultures to ouabain, a well-known DLC, leads to up- or down regulation of various processes and involves activation of Src kinase. Since Na,K-ATPase is the only known target for DLC binding an in vitro experimental setup using highly purified Na,K-ATPase from pig kidney and commercially available recombinant Src was used to investigate the mechanism of coupling between the Na,K-ATPase and Src. Digoxin was used as a representative DLC for inhibition of Na,K-ATPase. The activation of Src kinase was measured as the degree of its autophosphorylation. It was observed that in addition to digoxin, Src activation was dependent on concentrations of other specific ligands of Na,K-ATPase: Na(+), K(+), vanadate, ATP and ADP. The magnitude of the steady-state ATPase activity therefore seemed to affect Src activation. Further experiments with an ATP regenerating system showed that the ATP/ADP ratio determined the extent of Src activation. Thus, our model system which represents the proposed very proximal part of the Na,K-ATPase-Src signaling cascade, shows that Src kinase activity is regulated by both ATP and ADP concentrations and provides no evidence for a direct interaction between Na,K-ATPase and Src.

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Photosynthetic cytochrome c550.

Publication Date: 2012 Jan 24 PMID: 22289879
Authors: Roncel, M. - Kirilovsky, D. - Guerrero, F. - Serrano, A. - Ortega, J. M.
Journal: Biochim Biophys Acta

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl(-) and Ca(2+) to the oxygen evolving complex and protects the Mn4Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (Em) of the purified soluble form (from -250 to -314mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an Em value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22A) between cyt c550 and the nearest redox cofactor (Mn4Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial Photosynthesis.

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Oncogenic role and therapeutic target of leptin signaling in breast cancer and cancer stem cells.

Publication Date: 2012 Jan 24 PMID: 22289780
Authors: Guo, S. - Liu, M. - Wang, G. - Torroella-Kouri, M. - Gonzalez-Perez, R. R.
Journal: Biochim Biophys Acta

Significant correlations between obesity and incidence of various cancers have been reported. Obesity, considered a mild inflammatory process, is characterized by a high level of secretion of several cytokines from adipose tissue. These molecules have disparate effects, which could be relevant to cancer development. Among the inflammatory molecules, leptin, mainly produced by adipose tissue and overexpressed with its receptor (Ob-R) in cancer cells is the most studied adipokine. Mutations of leptin or Ob-R genes associated with obesity or cancer are rarely found. However, leptin is an anti-apoptotic molecule in many cell types, and its central roles in obesity-related cancers are based on its pro-angiogenic, pro-inflammatory and mitogenic actions. Notably, these leptin actions are commonly reinforced through entangled crosstalk with multiple oncogenes, cytokines and growth factors. Leptin-induced signals comprise several pathways commonly triggered by many cytokines (i.e., canonical: JAK2/STAT; MAPK/ERK1/2 and PI-3K/AKT1 and, non-canonical signaling pathways: PKC, JNK and p38 MAP kinase). Each of these leptin-induced signals is essential to its biological effects on food intake, energy balance, adiposity, immune and endocrine systems, as well as oncogenesis. This review is mainly focused on the current knowledge of the oncogenic role of leptin in breast cancer. Additionally, leptin pro-angiogenic molecular mechanisms and its potential role in breast cancer stem cells will be reviewed. Strict biunivocal binding-affinity and activation of leptin/Ob-R complex makes it a unique molecular target for prevention and treatment of breast cancer, particularly in obesity contexts.

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Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase.

Publication Date: 2012 Jan 25 PMID: 22289630
Authors: Mehrotra, S. - B Ningappa, M. - Raman, J. - Anand, R. P. - Balaram, H.
Journal: Biochim Biophys Acta

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.

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Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia.

Publication Date: 2012 Jan 27 PMID: 22289388
Authors: Khatchadourian, A. - Bourque, S. D. - Richard, V. R. - Titorenko, V. I. - Maysinger, D.
Journal: Biochim Biophys Acta

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 alpha/beta (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A(2) (cPLA(2)-alpha), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 alpha/beta in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.

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The taste of heavy metals: Gene regulation by MTF-1.

Publication Date: 2012 Jan 20 PMID: 22289350
Authors: Gunther, V. - Lindert, U. - Schaffner, W.
Journal: Biochim Biophys Acta

The metal-responsive transcription factor-1 (MTF-1, also termed MRE-binding transcription factor-1 or metal regulatory transcription factor-1) is a pluripotent transcriptional regulator involved in cellular adaptation to various stress conditions, primarily exposure to heavy metals but also to hypoxia or oxidative stress. MTF-1 is evolutionarily conserved from insects to humans and is the main activator of metallothionein genes, which encode small cysteine-rich proteins that can scavenge toxic heavy metals and free radicals. MTF-1 has been suggested to act as an intracellular metal sensor but evidence for direct metal sensing was scarce. Here we review recent advances in our understanding of MTF-1 regulation with a focus on the mechanism underlying heavy metal responsiveness and transcriptional activation mediated by mammalian or Drosophila MTF-1. This article is part of a Special Issue entitled: Cell Biology of Metals.

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Knock-down of Pdcd4 stimulates angiogenesis via up-regulation of angiopoietin-2.

Publication Date: 2012 Jan 24 PMID: 22289349
Authors: Krug, S. - Huth, J. - Goke, F. - Buchholz, M. - Gress, T. M. - Goke, R. - Lankat-Buttgereit, B.
Journal: Biochim Biophys Acta

The tumor suppressor Pdcd4 is involved in multiple pathways. Considering its biological action conflicting data in the literature exist and, consequently, our own studies point to a cell type specific action of Pdcd4. In the present study, using several Pdcd4 knock down cell lines we succeeded to identify angiopoietin-2 (Ang-2) as a gene up-regulated on the mRNA and protein level. The subsequent enhanced peptide secretion forced wild type Bon-1 cells in a neoplastic direction demonstrated by increased proliferation and colony formation while cell adhesion was decreased. Most likely, the stimulation of Ang-2 is in part mediated by increased activation of AP-1 but different signal transduction pathways may also be involved since we found opposite activation of PI3K/Akt/mTOR and MAPK7ERK pathways (both known to regulate in Ang-2 expression). Ang-2 is a modulator of vascular remodeling. Therefore, we analyzed the effect of supernatants from Pdcd4 knock-down cell lines on endothelial cells. Again, we detected reduced cell adhesion and increased colony formation. Probably, the most impressive effect was described on tube formation in a model for angiogenesis. Tube length and junctions of endothelial cells treated with conditioned medium from Pdcd4 knock-down cells were considerably increased. Both, up-regulation of Ang-2 and down-regulation of Pdcd4 are described for many tumors. However, this is the first study showing a direct impact of Pdcd4 on Ang-2 levels and, thereby, angiogenesis. Our data suggest a completely new mechanism for Pdcd4 to act as a tumor suppressor rendering Pdcd4 an attractive target for new therapeutic strategies in cancer treatment.

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Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast.

Publication Date: 2012 Jan 20 PMID: 22285845
Authors: Traxlmayr, M. W. - Faissner, M. - Stadlmayr, G. - Hasenhindl, C. - Antes, B. - Ruker, F. - Obinger, C.
Journal: Biochim Biophys Acta

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85 degrees C was developed. Besides library construction by error prone PCR, strong heat stress at 79 degrees C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.

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Interleukin 17 inhibits myogenic and promotes osteogenic differentiation of C2C12 myoblasts by activating ERK1,2.

Publication Date: 2012 Jan 17 PMID: 22285818
Authors: Kocic, J. - Santibanez, J. F. - Krstic, A. - Mojsilovic, S. - Dordevic, I. O. - Trivanovic, D. - Ilic, V. - Bugarski, D.
Journal: Biochim Biophys Acta

The present study evaluated the role of interleukin (IL) 17 in multilineage commitment of C2C12 myoblastic cells and investigated associated signaling pathways. The results concerning the effects on cell function showed that IL-17 inhibits the migration of C2C12 cells, while not affecting their proliferation. The data regarding the influence on differentiation demonstrated that IL-17 inhibits myogenic differentiation of C2C12 cells by down-regulating the myogenin mRNA level, myosin heavy chain expression and myotube formation, but promotes their osteogenic differentiation by up-regulating the Runt-related transcription factor 2 mRNA level, cyclooxygenase-2 expression and alkaline phosphatase activity. IL-17 exerted these effects by activating ERK1,2 mitogen activated protein kinase signaling pathway, which in turn regulated the expression of relevant genes and proteins to inhibit myogenic differentiation and induce osteogenic differentiation. Additional analysis showed that the induction of osteogenic differentiation by IL-17 is independent of BMP signaling. The results obtained demonstrate the potential of IL-17 not only to inhibit the myogenic differentiation of C2C12 myoblasts but also to convert their differentiation pathway into that of osteoblast lineage providing new insight into the capacities of IL-17 to modulate the differentiation commitment.

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The CCAAT box-binding transcription factor NF-Y regulates basal expression of human proteasome genes.

Publication Date: 2012 Jan 20 PMID: 22285817
Authors: Xu, H. - Fu, J. - Ha, S. W. - Ju, D. - Zheng, J. - Li, L. - Xie, Y.
Journal: Biochim Biophys Acta

Protein degradation by the proteasome plays an important role in all major cellular pathways. Aberrant proteasome activity is associated with numerous human diseases including cancer and neurological disorders, but the underlying mechanism is virtually unclear. At least part of the reason for this is due to lack of understanding of the regulation of human proteasome genes. In this study, we found that a large set of human proteasome genes carry the CCAAT box in their promoters. We further demonstrated that the basal expression of these CCAAT box-containing proteasome genes is regulated by the transcription factor NF-Y. Knockdown of NF-YA, an essential subunit of NF-Y, reduced proteasome gene expression and compromised the cellular proteasome activity. In addition, we showed that knockdown of NF-YA sensitized breast cancer cells to the proteasome inhibitor MG132. This study unveils a new role for NF-Y in the regulation of human proteasome genes and suggests that NF-Y may be a potential target for cancer therapy.

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Cellular and mitochondrial iron homeostasis in vertebrates.

Publication Date: 2012 Jan 18 PMID: 22285816
Authors: Chen, C. - Paw, B. H.
Journal: Biochim Biophys Acta

Iron plays an essential role in cellular metabolism and biological processes. However, due to its intrinsic redox activity, free iron is a potentially toxic molecule in cellular biochemistry. Thus, organisms have developed sophisticated ways to import, sequester, and utilize iron. The transferrin cycle is a well-studied iron uptake pathway that is important for most vertebrate cells. Circulating iron can also be imported into cells by mechanisms that are independent of transferrin. Once imported into erythroid cells, iron is predominantly consumed by the mitochondria for the biosynthesis of heme and iron sulfur clusters. This review focuses on canonical transferrin-mediated and the newly discovered, non-transferrin mediated iron uptake pathways, as well as, mitochondrial iron homeostasis in higher eukaryotes. This article is part of a Special Issue entitled: Cell Biology of Metals.

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Structural determinants of the specificity of a membrane binding domain of the scaffold protein Ste5 of budding yeast: Implications in signaling by the scaffold protein in MAPK pathway.

Publication Date: 2012 Jan 20 PMID: 22285780
Authors: Bhunia, A. - Mohanram, H. - Bhattacharjya, S.
Journal: Biochim Biophys Acta

In the mitogen activated protein kinase (MAPK) cascades of budding yeast, the scaffold protein Ste5 is recruited to the plasma membrane to transmit pheromone induced signal. A region or domain of Ste5 i.e. residues P44-R67, referred here as Ste5PM24, has been known to be involved in direct interactions with the membrane. In order to gain structural insights into membrane interactions of Ste5, here, we have investigated structures and interactions of two synthetic peptide fragments of Ste5, Ste5PM24, and a hyperactive mutant, Ste5PM24LM, by NMR, ITC, and fluorescence spectroscopy, with lipid membranes. We observed that Ste5PM24 predominantly interacted only with the anionic lipid vesicles. By contrast, Ste5PM24LM exhibited binding with negatively charged as well as zwitterionic or mixed lipid vesicles. Binding of Ste5 peptides with the negatively charged lipid vesicles were primarily driven by hydrophobic interactions. NMR studies revealed that Ste5PM24 assumes dynamic or transient conformations in zwitterionic dodecylphosphocholine (DPC) micelles. By contrast, NMR structure, obtained in anionic sodium dodecyl sulphate (SDS), demonstrated amphipathic helical conformations for the central segment of Ste5PM24. The hydrophobic surface of the helix was found to be buried inside the micelles. Taken together, these results provide important insights toward the structure and specificity determinants of the scaffold protein interactions with the plasma membrane.

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The amphipathic helix of an enzyme that regulates phosphatidylcholine synthesis remodels membranes into highly curved nanotubules.

Publication Date: 2012 Jan 18 PMID: 22285779
Authors: Taneva, S. G. - Lee, J. M. - Cornell, R. B.
Journal: Biochim Biophys Acta

CTP:phosphocholine cytidylyltransferase (CCT) is an amphitropic protein regulating phosphatidylcholine synthesis. Lipid-induced folding of its amphipathic helical (AH) membrane-binding domain activates the enzyme. In this study we examined the membrane deforming property of CCT in vitro by monitoring conversion of vesicles to tubules, using transmission electron microscopy. Vesicle tubulation was proportional to the membrane density of CCT and proceeded either as growth from a pre-formed surface bud, or as a global transformation of roughly spherical vesicles into progressively thinner tubules. The tubulation pathway depended on the lipid compositional heterogeneity of the vesicles, with heterogeneous mixtures supporting the bud-extension pathway. Co-existence of vesicles alongside thick and thin tubules suggested that CCT can discriminate between flat membrane surfaces and those with emerging curvature, binding preferentially to the latter. Thin tubules had a limiting diameter of ~12nm, likely representing bilayer cylinders with a very high density of 1 CCT/50 lipids. The AH segment was necessary and sufficient for tubulation. AH regions from diverse CCT sources, including C. elegans, had tubulation activity that correlated with alpha-helical length. The AH motifs in CCT and the Parkinson's-related protein, alpha-synuclein, have similar features, however the CCT AH was more effective in its membrane remodeling function. That CCT can deform vesicles of physiologically relevant composition suggests that CCT binding to membranes may initiate deformations required for organelle morphogenesis and at the same time stimulate synthesis of the PC required for the development of these regions.

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Different fractions of human serum glycoproteins bind galectin-1 or galectin-8, and their ratio may provide a refined biomarker for pathophysiological conditions in cancer and inflammatory disease.

Publication Date: 2012 Jan 17 PMID: 22285770
Authors: Carlsson, M. C. - Balog, C. I. - Kilsgard, O. - Hellmark, T. - Bakoush, O. - Segelmark, M. - Ferno, M. - Olsson, H. - Malmstrom, J. - Wuhrer, M. - Leffler, H.
Journal: Biochim Biophys Acta

BACKGROUND: Changes in glycosylation of serum proteins are common, and various glycoforms are being explored as biomarkers in cancer and inflammation. We recently showed that glycoforms detected by endogenous galectins not only provide potential biomarkers, but also have different functions when they encounter galectins in tissue cells. Now we have explored the use of a combination of two galectins with different specificities, to further increase biomarker sensitivity and specificity. METHODS: Sera from 14 women with metastatic breast cancer, 12 healthy controls, 14 patients with IgA-nephritis (IgAN), and 12 patients with other glomerulonephritis were fractionated by affinity chromatography on immobilized human galectin-1 or galectin-8N, and the protein amounts of the bound and unbound fractions for each galectin were determined. RESULTS: Each galectin bound largely different fractions of the serum glycoproteins, including different glycoforms of haptoglobin. In the cancer sera, the level of galectin-1 bound glycoproteins was higher and galectin-8N bound glycoproteins lower compared to the other patients groups, whereas in IgAN sera the level of galectin-8N bound glycoproteins were higher. CONCLUSION: The ratio of galectin-1 bound/galectin-8N bound glycoproteins showed high discriminatory power between cancer patients and healthy, with AUC of 0.98 in ROC analysis, and thus provides an interesting novel cancer biomarker candidate. GENERAL SIGNIFICANCE: The galectin-binding ability of a glycoprotein is not only a promising biomarker candidate but may also have a specific function when the glycoprotein encounters the galectin in tissue cells, and thus be related to the pathophysiological state of the patient. This article is part of a Special Issue entitled Glycoproteomics.

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Adaptation of light-harvesting systems of Arthrospira platensis to light conditions, probed by time-resolved fluorescence spectroscopy.

Publication Date: 2012 Jan 20 PMID: 22285745
Authors: Akimoto, S. - Yokono, M. - Hamada, F. - Teshigahara, A. - Aikawa, S. - Kondo, A.
Journal: Biochim Biophys Acta

Cyanobacteria change the quantity and/or quality of their pigment-protein complexes in response to light conditions. In the present study, we analyzed excitation relaxation dynamics in the cyanobacterium, Arthrospira (Spirulina) platensis, grown under lights exhibiting different spectral profiles, by means of steady-state absorption and picosecond time-resolved fluorescence spectroscopies. It was found that F760, which is the PSI red-chlorophyll characteristic of A. platensis, contributes to slower energy-transfer phase in the PSI of A. platensis. Excitation energy transfers in phycobilisome and those from PSII to PSI were modified depending on the light quality. Existence of quencher was suggested in PSI of the blue-light grown cells. Phycobilisomes in the green-light grown cells and the far-red-light grown cells transferred excitation energy from phycobilisome to chlorophyll without loss of energy. In these cells, excitation energy was shared between two photosystems. Fast energy transfer was established in phycobilisome under the yellow-light condition where only the phycobilisome can absorb the cultivation light. Differences in light-harvesting and energy-transfer processes under different cultivation-light conditions are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Articifical Photosynthesis."

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Probing the interaction between heparan sulfate proteoglycan with biologically relevant molecules in mimetic models for cell membranes: A Langmuir film study.

Publication Date: 2012 Jan 21 PMID: 22285741
Authors: Caseli, L. - Cavalheiro, R. P. - Nader, H. B. - Lopes, C. C.
Journal: Biochim Biophys Acta

Investigating the role of proteoglycans associated to cell membranes is fundamental to comprehend biochemical process that occurs at the level of membrane surfaces. In this paper, we exploit syndecan-4, a heparan sulfate proteoglycan obtained from cell cultures, in lipid Langmuir monolayers at the air-water interface. The monolayer served as a model for half a membrane, and the molecular interactions involved could be evaluated with tensiometry and vibrational spectroscopy techniques. Polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) employed in a constant surface pressure regime showed that the main chemical groups for syndecan-4 were present at the air-water interface. Subsequent monolayer decompression and compression showed surface pressure-area isotherms with a large expansion for the lipid monolayers interacting with the cell culture reported to over-express syndecan-4, which was also an indication that the proteoglycan was inserted in the lipid monolayer. The introduction of biological molecules with affinity for syndecam-4, such as growth factors, which present a key role in biochemical process of cell signaling, changed the surface properties of the hybrid film, leading to a model, by which the growth factor binds to the sulfate groups present in the heparan sulfate chains. The polypeptide moiety of syndecan-4 responds to this interaction changing its conformation, which leads to lipid film relaxation and further monolayer condensation.

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Efficiency of detergents at maintaining membrane protein structures in their biologically relevant forms.

Publication Date: 2012 Jan 21 PMID: 22285740
Authors: Tulumello, D. V. - Deber, C. M.
Journal: Biochim Biophys Acta

High-resolution structural analysis of membrane proteins by X-ray crystallography or solution NMR spectroscopy often requires their solubilization in the membrane-mimetic environments of detergents. Yet the choice of a detergent suitable for a given study remains largely empirical. In the present work, we considered the micelle-crystallized structures of lactose permease (LacY), the sodium/galactose symporter (vSGLT), the vitamin B(12) transporter (BtuCD), and the arginine/agmatine antiporter (AdiC). Representative transmembrane (TM) segments were selected from these proteins based on their relative contact(s) with water, lipid, and/or within the protein, and were synthesized as Lys-tagged peptides. Each peptide was studied by circular dichroism and fluorescence spectroscopy in water, and in the presence of the detergents sodium dodecylsulfate (SDS, anionic); n-dodecyl phosphatidylcholine (DPC, zwitterionic); n-dodecyl-beta-d-maltoside (DDM, neutral); and n-octyl-beta-d-glucoside (OG, neutral, varying acyl tail length). We found that (i) the secondary structures of the TM segments were statistically indistinguishable in the four detergents studied; and (ii) a strong correlation exists between the extent of helical structure of each individual TM segment in detergents with its helicity level as it exists in the full-length protein, indicating that helix adoption is fundamentally the same in both environments. The denaturing properties of so-called 'harsh' detergents may thus largely be due to their interactions with non-membranous regions of proteins. Given the consistency of structural features observed for each TM segment in a variety of micellar media, the overall results suggest that the structure likely corresponds to its relevant biological form in the intact protein in its native lipid bilayer environment.

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pH-dependent channel gating in connexin26 hemichannels involves conformational changes in N-terminus.

Publication Date: 2012 Jan 5 PMID: 22285739
Authors: Wang, X. - Xu, X. - Ma, M. - Zhou, W. - Wang, Y. - Yang, L.
Journal: Biochim Biophys Acta

Connexin (Cx) hemichannels controlling an exchange of ions and metabolites between the cytoplasm and extracellular milieu can be modulated by the variation of intracellular pH during physiological and pathological conditions. To address the mechanism by which the pH exerts its effect on hemichannels, we have performed two 100-ns molecular dynamics simulations of the Cx26 channel in both acidic and neutral states. The results show that: 1) transmembrane domains undergo clockwise motions around the pore axis under both acidic and neutral conditions, while extracellular segments keep stable. 2) Under neutral condition, Cx26 has a tightly closed configuration that occurs through the assembly of N-terminal helix (NTH) region. This shows a constriction formed by the interhelical interactions of Asp2 and Met1 from neighboring NTH, which shapes the narrowest segment (pore radius<2A) of the pore, preventing the passage of ions from the extracellular side. This indicates that Asp2 may act as a channel gate. 3) Under the acidic condition, the constriction is relieved by the protonation of Asp2 causing interruption of interhelical interactions, Cx26 has a flexibly opening pore (pore radius>4.5A) around NTH region, allowing the passage of chloride ions unimpeded by the side-chain Asp2. While in the extracellular part two chloride ions interact with the side-chain Lys41 from three subunits. Finally, we provide a plausible mechanism of pH-dependent gating of hemichannel that involves protonation of the aspartic residues, suggesting that the pH sensitivity of hemichannel permeability is a sophisticated mechanism for cell regulating ion permeation.

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Polyamines induce aggregation of LHC II and quenching of fluorescence in vitro.

Publication Date: 2012 Jan 21 PMID: 22285699
Authors: Tsiavos, T. - Ioannidis, N. E. - Kotzabasis, K.
Journal: Biochim Biophys Acta

Dissipation of excess excitation energy within the light-harvesting complex of Photosystem II (LHC II) is a main process in plants, which is measured as the non-photochemical quenching of chlorophyll fluorescence or qE. We showed in previous works that polyamines stimulate qE in higher plants in vivo and in eukaryotic algae in vitro. In the present contribution we have tested whether polyamines can stimulate quenching in trimeric LHC II and monomeric LHCb proteins from higher plants. The tetramine spermine was the most potent quencher and induced aggregation of LHC II trimers, due to its highly cationic character. Two transients are evident at 100muM and 350muM for the fluorescence and absorbance signals of LHC II respectively. On the basis of observations within this work, some links between polyamines and the activation of qE in vivo is discussed.

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The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor alpha

Publication Date: 2012 Jan 21 PMID: 22285688
Authors: Wen, G. - Ringseis, R. - Rauer, C. - Eder, K.
Journal: Biochim Biophys Acta

Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and gamma-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)alpha directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARalpha target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5 -truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position -132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARalpha to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARalpha also binds in vivo to a nucleotide sequence spanning the PPRE at -132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARalpha target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARalpha target genes this finding confirm that PPARalpha plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.

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Chromatin dynamics in DNA double-strand break repair.

Publication Date: 2012 Jan 17 PMID: 22285574
Authors: Shi, L. - Oberdoerffer, P.
Journal: Biochim Biophys Acta

DNA double-strand breaks (DSBs) occur in the context of a highly organized chromatin environment and are, thus, a significant threat to the epigenomic integrity of eukaryotic cells. Changes in break-proximal chromatin structure are thought to be a prerequisite for efficient DNA repair and may help protect the structural integrity of the nucleus. Unlike most bona fide DNA repair factors, chromatin influences the repair process at several levels: the existing chromatin context at the site of damage directly affects the access and kinetics of the repair machinery; DSB induced chromatin modifications influence the choice of repair factors, thereby modulating repair outcome; lastly, DNA damage can have a significant impact on chromatin beyond the site of damage. We will discuss recent findings that highlight both the complexity and importance of dynamic and tightly orchestrated chromatin reorganization to ensure efficient DSB repair and nuclear integrity. This article is part of a Special Issue entitled: Chromatin in time and space.

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Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity.

Publication Date: 2012 Jan 21 PMID: 22285573
Authors: Wang, Q. - Weng, L. - Tian, X. - Counor, D. - Sun, J. - Mao, Y. - Deubel, V. - Okada, H. - Toyoda, T.
Journal: Biochim Biophys Acta

Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn(2+) and alkaline conditions (pH 8-10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (muM) values were 22.13+/-1.11 (ATP), 21.94+/-3.88 (CTP), 21.27+/-1.23 (GTP), and 9.91+/-0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216+/-0.017 (ATP), 0.781+/-0.020 (CTP), 0.597+/-0.049 (GTP), and 0.347+/-0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.

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Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins.

Publication Date: 2012 Jan 21 PMID: 22285364
Authors: Feng, Y. D. - Li, J. - Zhou, W. C. - Jia, Z. G. - Wei, Q.
Journal: Biochim Biophys Acta

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2-EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.

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Lysophosphatidylinositol signalling: New wine from an old bottle.

Publication Date: 2012 Jan 20 PMID: 22285325
Authors: Pineiro, R. - Falasca, M.
Journal: Biochim Biophys Acta

Lysophosphatidylinositol (LPI) is a bioactive lipid generated by phospholipase A2 which is believed to play an important role in several diseases. Indeed LPI can affect various functions such as cell growth, differentiation and motility, in a number of cell-types, including cancer cells, endothelial cells and nervous cells. Despite the fact that LPI-induced cellular functions had been known for more than twenty years, the recent discovery that in several cell-types the orphan G protein-coupled receptor GPR55 acts as the specific receptor for LPI has fuelled novel interest in this lysolipid. Different research groups, including our own, have recently suggested that LPI may be the specific and functional ligand for GPR55, triggering signalling cascades that are relevant to cell proliferation, migration, survival and tumourigenesis. Recently published data suggest that the LPI/GPR55 axis plays an important role in different physiological and pathological contexts. Here we review the available data supporting the role of LPI in cell signalling and pharmacology of its putative receptor GPR55.

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Fatty acid composition of membrane bilayers: Importance of diet polyunsaturated fat balance.

Publication Date: 2012 Jan 18 PMID: 22285120
Authors: Abbott, S. K. - Else, P. L. - Atkins, T. A. - Hulbert, A. J.
Journal: Biochim Biophys Acta

In one of the most extensive analyses to date we show that the balance of diet n-3 and n-6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under 'normal' conditions. Young adult male Sprague-Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8-88% of total FAs, monounsaturate (MUFA) 6-65%, total PUFA 4-81%, n-6 PUFA 3-70% and n-3 PUFA 1-70%. Diet PUFA included only essential FAs 18:2n-6 and 18:3n-3. Balance between n-3 and n-6 PUFA is defined as the PUFA balance (n-3 PUFA as % of total PUFA) and ranged 1-86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer-regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n-3 and n-6 PUFA had a biphasic influence on membrane composition. When n-3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications.

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Phosphoinositides and vesicular membrane traffic.

Publication Date: 2012 Jan 14 PMID: 22281700
Authors: Mayinger, P.
Journal: Biochim Biophys Acta

Phosphoinositide lipids were initially discovered as precursors for specific second messengers involved in signal transduction, but have now taken the center stage in controlling many essential processes at virtually every cellular membrane. In particular, phosphoinositides play a critical role in regulating membrane dynamics and vesicular transport. The unique distribution of certain phosphoinositides at specific intracellular membranes makes these molecules uniquely suited to direct organelle-specific trafficking reactions. In this regulatory role, phosphoinositides cooperate specifically with small GTPases from the Arf and Rab families. This review will summarize recent progress in the study of phosphoinositides in membrane trafficking and organellar organization and highlight the particular relevance of these signaling pathways in disease. This article is part of a Special Issue entitled Vesicular Transport.

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Postprandial metabolism of meal triglyceride in humans.

Publication Date: 2012 Jan 17 PMID: 22281699
Authors: Lambert, J. E. - Parks, E. J.
Journal: Biochim Biophys Acta

The intake of dietary fat above energy needs has contributed to the growing rates of obesity worldwide. The concept of disease development occurring in the fed state now has much support and dysregulation of substrate flux may occur due to poor handling of dietary fat in the immediate postprandial period. The present paper will review recent observations implicating cephalic phase events in the control of enterocyte lipid transport, the impact of varying the composition of meals on subsequent fat metabolism, and the means by which dietary lipid carried in chylomicrons can lead to elevated postprandial non-esterified fatty acid concentrations. This discussion is followed by an evaluation of the data on quantitative meal fat oxidation at the whole body level and an examination of dietary fat clearance to peripheral tissues - with particular attention paid to skeletal muscle and liver given the role of ectopic lipid deposition in insulin resistance. Estimates derived from data of dietary-TG clearance show good agreement with clearance to the liver equaling 8-12% of meal fat in lean subjects and this number appears higher (10-16%) in subjects with diabetes and fatty liver disease. Finally, we discuss new methods with which to study dietary fatty acid partitioning in vivo. Future research is needed to include a more comprehensive understanding of 1) the potential for differential oxidation of saturated versus unsaturated fatty acids which might lead to meaningful energy deficit and whether this parameter varies based on insulin sensitivity, 2) whether compartmentalization exists for diet-derived fatty acids within tissues vs. intracellular pools, and 3) the role of reduced peripheral fatty acid clearance in the development of fatty liver disease. Further advancements in the quantitation of dietary fat absorption and disposal will be central to the development of therapies designed to treat diet-induced obesity. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

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Robustness testing of the high throughput HPLC-based analysis of plasma N-glycans.

Publication Date: 2012 Jan 15 PMID: 22281527
Authors: Novokmet, M. - Pucic, M. - Redzic, I. - Muzinic, A. - Gornik, O.
Journal: Biochim Biophys Acta

BACKGROUND: Plasma glycan analysis using high throughput HPLC-based 96 well platform became a standard procedure for analyzing a large pool of samples for studies comprising thousands of observed individuals. An analytical method which is used to obtain such a huge amount of data should be well characterized and all potentially critical steps should be known. METHODS: Robustness of the high throughput method was tested by Plackett Burman two level, 11-factor, 12 experiment screening design. It provides valuable information about the few most important factors on which further optimization should be focused. According to a long-term laboratory experience, eleven potentially critical factors were chosen for initial screening. Response variable was calculated as coefficient of variance between area % of each peak in each reaction and the area % obtained after performing the procedure according to the laboratory standard operating procedure. RESULTS: Six out of 16, by HPLC separated, glycan groups revealed significant changes according to changes in factor levels. As expected due to their structural and chemical differences, glycan groups did not display uniform response to 11 factors, but effect estimates for six significant glycan groups showed the same direction regarding high and low factor levels. GENERAL SIGNIFICANCE: Screening experiment provided quality data which resolved the questions about optimal conditions and robustness of the high throughput glycan analysis. Fraction factorial design used in this study enabled us to test a great deal of critical steps in time, labor and money saving manner. This article is part of a Special Issue entitled Glycoproteomics.

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Bacillus cereus can attack the cell membranes of the alga Chara corallina by means of HlyII.

Publication Date: 2012 Jan 17 PMID: 22281415
Authors: Kataev, A. A. - Andreeva-Kovalevskaya, Z. I. - Solonin, A. S. - Ternovsky, V. I.
Journal: Biochim Biophys Acta

We studied the influence of Bacillus cereus bacteria on cells of the freshwater alga Chara corallina. These bacteria and recombinant Bacillus subtilis strains are capable of producing the secreted toxin HlyII, which changes the electrophysiological parameters of the algal electrically excitable plasma membrane by forming pores. Cooperative incubation of bacterial cells, which carry active hlyII gene, and Chara corallina cells caused a decrease in the resting potential (V(m)) and plasma membrane resistance (R(m)) of algal cells. The efficiency of each strain was commensurable with its ability to produce HlyII. Purified hemolysin II caused a similar effect on V(m) and R(m) of intact and perfused cells. This protein changed the kinetics and magnitude of transient voltage-dependent calcium and calcium-activated chloride currents owing to the formation of additional Ca(2+)-permeable pores in algal cell membrane. Occurrence of the cellulose cell wall with pores 2.1 to 4.6nm in diameter suggests that HlyII molecules reach the plasma membrane surface strictly as monomers.

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Respiratory oxidases.

Publication Date: 2012 Jan 24 PMID: 22280949
Authors: Ludwig, B.
Journal: Biochim Biophys Acta

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Taxane resistance in breast cancer: A closed HER2 circuit?

Publication Date: 2012 Jan 18 PMID: 22280939
Authors: de Hoon, J. P. - Veeck, J. - Vriens, B. E. - Calon, T. G. - van Engeland, M. - Tjan-Heijnen, V. C.
Journal: Biochim Biophys Acta

Microtubule inhibitors, such as the taxanes docetaxel and paclitaxel, are commonly used drugs for the treatment of breast cancer. Although highly active in a large fraction of individuals a considerable number of patients show poor response due to either intrinsic or acquired drug resistance. Extensive research in the past identified several taxane resistance-related mechanisms being activated by pathologically altered single gene function. To date, however, a clinically relevant predictive biomarker for taxanes has not been derived yet from this knowledge, most likely due to the manifold of resistance mechanisms that may combine in one tumor, thereby fostering escape from taxane cytotoxicity. Here, we aimed to comprehensively review the current literature on taxane resistance mechanisms in breast cancer. Interestingly, besides altered microtubule physiology, we identified the HER2 signaling cascade as a major dominator influencing several routes of cytotoxicity escape, such as cell survival, apoptosis, drug efflux, and drug metabolism. Furthermore, the transcription factor YBX-1, activated by HER2, facilitates a sustaining HER2 signaling feedback loop contributing to the establishment of cellular survival detours. In conclusion, taxane resistance in breast cancer follows a multiplex establishment of drug cytotoxicity escape routes, which may be most efficiently therapeutically targeted by interference with their mutually governing signaling nodes.

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Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis.

Publication Date: 2012 Jan 17 PMID: 22274567
Authors: Nolandt, O. V. - Walther, T. H. - Grage, S. L. - Ulrich, A. S.
Journal: Biochim Biophys Acta

A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) with the short-chain detergent n-dodecylphosphocholine (DPC) is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples. Magnetically aligned DMPC/DPC bicelles are stable over a range of concentrations, with an optimum lipid ratio of q=3:1, and they can be flipped with lanthanide ions. The advantage of DMPC/DPC over established bicelle systems lies in the possibility to use one and the same detergent for purification and NMR analysis of the membrane protein, without any need for detergent exchange. Furthermore, the same batch of protein can be studied in both micelles and bicelles, using liquid-state and solid-state NMR, respectively. The applicability of the DMPC/DPC bicelles is demonstrated here with the (15)N-labeled transmembrane protein TatA.

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Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain.

Publication Date: 2012 Jan 18 PMID: 22274566
Authors: Pandit, K. R. - Klauda, J. B.
Journal: Biochim Biophys Acta

Nearly all molecular dynamic simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.

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CoQ(10) deficiencies and MNGIE: Two treatable mitochondrial disorders.

Publication Date: 2012 Jan 18 PMID: 22274133
Authors: Hirano, M. - Garone, C. - Quinzii, C. M.
Journal: Biochim Biophys Acta

BACKGROUND: Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). SCOPE OF REVIEW: Here, we describe clinical and molecular features of CoQ(10) deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ(10) deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ(10) biosynthesis, often improve with CoQ(10) supplementation. In vitro and in vivo studies of CoQ(10) deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ(10) deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE. MAJOR CONCLUSIONS: CoQ(10) deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules. GENERAL SIGNIFICANCE: Studies of CoQ(10) deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies. This article is part of a Special Issue entitled: Biochemistry of Mitochondria, Life and Intervention 2010.

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d-ribose in glycation and protein aggregation.

Publication Date: 2012 Jan 17 PMID: 22274132
Authors: Wei, Y. - Han, C. S. - Zhou, J. - Liu, Y. - Chen, L. - He, R. Q.
Journal: Biochim Biophys Acta

BACKGROUND: d-ribose is a naturally occurring pentose monosaccharide present in all living cells and their microenvironments and is a key component of numerous biomolecules involved in many important metabolic pathways. It also participates in the glycation of proteins producing advanced glycation end products (AGEs) that lead to cell dysfunction and death. As recent studies show, ribosylation, a rapid process, causes protein aggregation in vitro and in vivo. SCOPE OF REVIEW: This review summarizes the relationship between ribosylation, protein aggregation, cell death and cognitive impairments. MAJOR CONCLUSION: d-ribose is active in glycation and induces protein aggregation, rapidly producing AGEs in vitro and in vivo. GENERAL SIGNIFICANCE: Ribosylation, leading to the production of significant amounts of AGEs both extracellularly and intracellularly, may be involved in cell dysfunction and subsequent cognitive impairments. This review may be a useful reference for studies on the pharmacokinetics of d-ribose action.

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Mechanisms of FGFR-mediated carcinogenesis.

Publication Date: 2012 Jan 18 PMID: 22273505
Authors: Ahmad, I. - Iwata, T. - Leung, H. Y.
Journal: Biochim Biophys Acta

In this review, the evidence for a role of fibroblast growth factor receptor (FGFR) mediated signalling in carcinogenesis are considered and relevant underlying mechanisms highlighted. FGF signalling mediated by FGFR follows a classic receptor tyrosine kinase signalling pathway and its deregulation at various points of its cascade could result in malignancy. Here we review the accumulating reports that revealed the association of FGF/FGFRs to various types of cancer at a genetic level, along with in vitro and in vivo evidences available so far, which indicates the functional involvement of FGF signalling in tumour formation and progression. An increasing number of drugs against the FGF pathways is currently in clinical testing. We will discuss the strategies for future FGF research in cancer and translational approaches.

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Radical AdoMet enzymes in complex metal cluster biosynthesis.

Publication Date: 2012 Jan 14 PMID: 22269887
Authors: Duffus, B. R. - Hamilton, T. L. - Shepard, E. M. - Boyd, E. S. - Peters, J. W. - Broderick, J. B.
Journal: Biochim Biophys Acta

Radical S-adenosylmethionine (AdoMet) enzymes comprise a large superfamily of proteins that engage in a diverse series of biochemical transformations through generation of the highly reactive 5'-deoxyadenosyl radical intermediate. Recent advances into the biosynthesis of unique iron-sulfur (FeS)-containing cofactors such as the H-cluster in [FeFe]-hydrogenase, the FeMo-co in nitrogenase, as well as the iron-guanylylpyridinol (FeGP) cofactor in [Fe]-hydrogenase have implicated new roles for radical AdoMet enzymes in the biosynthesis of complex inorganic cofactors. Radical AdoMet enzymes in conjunction with scaffold proteins engage in modifying ubiquitous FeS precursors into unique clusters, through novel amino acid decomposition and sulfur insertion reactions. The ability of radical AdoMet enzymes to modify common metal centers to unusual metal cofactors may provide important clues into the stepwise evolution of these and other complex bioinorganic catalysts. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

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Lipids in autophagy: Constituents, signaling molecules and cargo with relevance to disease.

Publication Date: 2012 Jan 14 PMID: 22269166
Authors: Knaevelsrud, H. - Simonsen, A.
Journal: Biochim Biophys Acta

The balance between protein and lipid biosynthesis and their eventual degradation is a critical component of cellular health. Autophagy, the catabolic process by which cytoplasmic material becomes degraded in lysosomes, can be induced by various physiological stimuli to maintain cellular homeostasis. Autophagy was for a long time considered a non-selective bulk process, but recent data have shown that unwanted components such as aberrant protein aggregates, dysfunctional organelles and invading pathogens can be selectively eliminated by autophagy. Recently, also intracellular lipid droplets were described as specific autophagic cargo, indicating that autophagy plays a role in lipid metabolism and storage (Singh et al., 2009 [1]). Moreover, over the past several years, it has become increasingly evident that lipids and lipid-modifying enzymes play important roles in the autophagy process itself, both at the level of regulation of autophagy and as membrane constituents required for formation of autophagic vesicles. In this review, we will discuss the interplay between lipids and autophagy, as well as the role of lipid-binding proteins in autophagy. We also comment on the possible implications of this mutual interaction in the context of disease. This article is part of a Special Issue entitled Vesicular Transport.

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Temperature-sensitive PSII and promiscuous PSI as a possible solution for sustainable photosynthetic hydrogen production.

Publication Date: 2012 Jan 13 PMID: 22269125
Authors: Mazor, Y. - Toporik, H. - Nelson, N.
Journal: Biochim Biophys Acta

Sustainable hydrogen production in cyanobacteria becomes feasible as a result of our recent studies of the structure of photosystem I encoding operon in a marine phage. We demonstrated that the fused PsaJF subunit from the phage, substituted for the two separate subunits in Synechocystis, enabled the mutated PSI to accept electrons from additional electron donors such as respiratory cytochromes. In this way, a type of photorespiration was created in which the cell consumes organic material through respiratory processes and PSI serves as a terminal electron acceptor, substituting for cytochrome oxidase. We designed a hydrogen-producing bioreactor in which this type of photorespiration could utilize the organic material of the cell as an electron source for H(2) production. We propose, in parallel, to engineer cyanobacterial and/or algal strains with a temperature-sensitive PSII and enhanced respiration rates to achieve efficient and sustainable hydrogen production. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial Photosynthesis.

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Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus.

Publication Date: 2012 Jan 14 PMID: 22266403
Authors: Gasparetti, C. - Nordlund, E. - Janis, J. - Buchert, J. - Kruus, K.
Journal: Biochim Biophys Acta

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5mM dopachrome the oxygen consumption rate of TrT on 8mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.

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Pressure dependence of activity and stability of dihydrofolate reductases of the deep-sea bacterium Moritella profunda and Escherichia coli.

Publication Date: 2012 Jan 14 PMID: 22266402
Authors: Ohmae, E. - Murakami, C. - Tate, S. I. - Gekko, K. - Hata, K. - Akasaka, K. - Kato, C.
Journal: Biochim Biophys Acta

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia coli (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 degrees C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53ml/mol at 25 degrees C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

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Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics.

Publication Date: 2012 Jan 14 PMID: 22266401
Authors: Eklund, S. - Lindas, A. C. - Hamnevik, E. - Widersten, M. - Tomkinson, B.
Journal: Biochim Biophys Acta

Tripeptidyl-peptidase II (TPP II) is a subtilisin-like serine protease which forms a large enzyme complex (>4MDa). It is considered a potential drug target due to its involvement in specific physiological processes. However, information is scarce concerning the kinetic characteristics of TPP II and its active site features, which are important for design of efficient inhibitors. To amend this, we probed the active site by determining the pH dependence of TPP II catalysis. Access to pure enzyme is a prerequisite for kinetic investigations and herein we introduce the first efficient purification system for heterologously expressed mammalian TPP II. The pH dependence of kinetic parameters for hydrolysis of two different chromogenic substrates, Ala-Ala-Phe-pNA and Ala-Ala-Ala-pNA, was determined for murine, human and Drosophila melanogaster TPP II as well as mutant variants thereof. The investigation demonstrated that TPP II, in contrast to subtilisin, has a bell-shaped pH dependence of k(cat)(app)/K(M) probably due to deprotonation of the N-terminal amino group of the substrate at higher pH. Since both the K(M) and k(cat)(app) are lower for cleavage of AAA-pNA than for AAF-pNA we propose that the former can bind non-productively to the active site of the enzyme, a phenomenon previously observed with some substrates for subtilisin. Two mutant variants, H267A and D387G, showed bell-shaped pH-dependence of k(cat)(app), possibly due to an impaired protonation of the leaving group. This work reveals previously unknown differences between TPP II orthologues and subtilisin as well as features that might be conserved within the entire family of subtilisin-like serine peptidases.

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Net light-induced oxygen evolution in photosystem I deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803.

Publication Date: 2012 Jan 12 PMID: 22266340
Authors: Wang, Q. J. - Singh, A. - Li, H. - Nedbal, L. - Sherman, L. A. - Govindjee - Whitmarsh, J.
Journal: Biochim Biophys Acta

Oxygenic photosynthesis in cyanobacteria, algae, and plants requires photosystem II (PSII) to extract electrons from H(2)O and depends on photosystem I (PSI) to reduce NADP(+). Here we demonstrate that mixotrophically-grown mutants of the cyanobacterium Synechocystis sp. PCC 6803 that lack PSI (DeltaPSI) are capable of net light-induced O(2) evolution in vivo. The net light-induced O(2) evolution requires glucose and can be sustained for more than 30min. Utilizing electron transport inhibitors and chlorophyll a fluorescence measurements, we show that in these mutants PSII is the source of the light-induced O(2) evolution, and that the plastoquinone pool is reduced by PSII and subsequently oxidized by an unidentified electron acceptor that does not involve the plastoquinol oxidase site of the cytochrome b(6)f complex. Moreover, both O(2) evolution and chlorophyll a fluorescence kinetics of the DeltaPSI mutants are highly sensitive to KCN, indicating the involvement of a KCN-sensitive enzyme(s). Experiments using (14)C-labeled bicarbonate show that the DeltaPSI mutants assimilate more CO(2) in the light compared to the dark. However, the rate of the light-minus-dark CO(2) assimilation accounts for just over half of the net light-induced O(2) evolution rate, indicating the involvement of unidentified terminal electron acceptors. Based on these results we suggest that O(2) evolution in DeltaPSI cells can be sustained by an alternative electron transport pathway that results in CO(2) assimilation and that includes PSII, the platoquinone pool, and a KCN-sensitive enzyme.

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Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase.

Publication Date: 2012 Jan 13 PMID: 22266334
Authors: Nelson, J. - Francom, L. L. - Anderson, L. - Damm, K. - Baker, R. - Chen, J. - Franklin, S. - Hamaker, A. - Izidoro, I. - Moss, E. - Orton, M. - Stevens, E. - Yeung, C. - Judd, A. M. - Bell, J. D.
Journal: Biochim Biophys Acta

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.

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The participation of plasma membrane hemichannels to purinergic signaling.

Publication Date: 2012 Jan 12 PMID: 22266266
Authors: Baroja-Mazo, A. - Barbera-Cremades, M. - Pelegrin, P.
Journal: Biochim Biophys Acta

The field of hemichannels is closely related to the purinergic signaling and both areas have been growing in parallel. Hemichannels open in response to a wide range of stressful conditions, such as ischemia, pressure or swelling. Hemichannels represent an important mechanism for the cellular release of adenosine 5'-triphosphate (ATP), which is an agonist of the P2Y and P2X family of purinergic receptors. Therefore, hemichannels are key molecules in the regulation of purinergic receptor activation, during physiological and pathophysiological conditions. Furthermore, purinergic receptor activation can also lead to the opening of hemichannels and the subsequent amplification of purinergic signaling via a positive signaling feedback loop, giving rise to the concept of ATP-induced ATP release. Purinergic receptor signaling is involved in regulating many physiological and pathophysiological processes. P2Y receptors activate inositol trisphosphate and transiently increase intracellular calcium. This signaling opens both connexin and pannexin channels, therefore contributing to the expansion of calcium waves across astrocytes and epithelial cells. In addition, several of the P2X receptor subtypes, including the P2X2, P2X4 and P2X7 receptors, activate select cellular permeation pathways to large molecules, including the pannexin-1 channels, which are involved in the initiation of inflammatory responses and cell death. Consequently, the interplay between purinergic receptors and hemichannels could represent a novel target with substantial therapeutic implications in areas such as chronic pain, inflammation or atherosclerosis. This article is part of a Special Issue entitled: The communicating junctions, roles and dysfunctions (Pt II).

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Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C(201)-Y or C(489)-S substitution associated with severe hypophosphatasia.

Publication Date: 2012 Jan 14 PMID: 22266140
Authors: Satou, Y. - Al-Shawahi, H. A. - Sultana, S. - Makita, S. - Sohda, M. - Oda, K.
Journal: Biochim Biophys Acta

Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66kDa immature form was predominant with a marginal amount of a 80kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80kDa mature form, though most of the 66kDa form was found to be endo-beta-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme.

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Next-generation sequencing identifies TGF-beta1-associated gene expression profiles in renal epithelial cells reiterated in human diabetic nephropathy.

Publication Date: 2012 Jan 14 PMID: 22266139
Authors: Brennan, E. P. - Morine, M. J. - Walsh, D. W. - Roxburgh, S. A. - Lindenmeyer, M. T. - Brazil, D. P. - Gaora, P. O. - Roche, H. M. - Sadlier, D. M. - Cohen, C. D. - Godson, C. - Martin, F.
Journal: Biochim Biophys Acta

Transforming growth factor-beta (TGF-beta1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-beta1 was assessed using RNA-seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-beta1 responsive gene set predicted activation of multiple transcriptional networks, including NFkappaB. Comparison of RNA-seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI - encoding transforming growth factor, beta-induced protein were induced by TGF-beta1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-beta1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker - E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-beta1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.

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S100 proteins in cartilage: Role in arthritis.

Publication Date: 2012 Jan 12 PMID: 22266138
Authors: Yammani, R. R.
Journal: Biochim Biophys Acta

S100 proteins are low molecular weight calcium binding proteins expressed in vertebrates. The family constitutes 21 known members that are expressed in several tissues and cell types and play a major role in various cellular functions. Uniquely, members of the S100 family have both intracellular and extracellular functions. Several members of the S100 family (S100A1, S100A2, S100A4, S1008, S100A9, S100A11, and S100B) have been identified in human articular cartilage, and their expression is upregulated in diseased tissue. These S100 proteins elicit a catabolic signaling pathway via receptor for advanced glycation end products (RAGE) in cartilage and may promote progression of arthritis. This review summarizes our current understanding of the role of S100 proteins in cartilage biology and in the development of arthritis.

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Ways and means of eukaryotic mRNA decay.

Publication Date: 2012 Jan 12 PMID: 22266130
Authors: Balagopal, V. - Fluch, L. - Nissan, T.
Journal: Biochim Biophys Acta

Messenger RNA degradation is an important point of control for gene expression. Genome-wide studies on mRNA stability have demonstrated its importance in adaptation and stress response. Most of the key players in mRNA decay appear to have been identified. The study of these proteins brings insight into the mechanism of general and specific targeting of transcripts for degradation. Recruitment and assembly of mRNP complexes enhance and bring specificity to mRNA decay. mRNP complexes can form larger structures that have been found to be ubiquitous in nature. Discovery of P-Bodies, an archetype of this sort of aggregates, has generated interest in the question of where mRNA degrades. This is currently an open question under extensive investigation. This review will discuss in detail the recent developments in the regulation of mRNA decay focusing on yeast as a model system. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

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Connecting vesicular transport with lipid synthesis: FAPP2.

Publication Date: 2012 Jan 14 PMID: 22266015
Authors: D'Angelo, G. - Rega, L. R. - De Matteis, M. A.
Journal: Biochim Biophys Acta

Next to the protein-based machineries composed of small G-proteins, coat complexes, SNAREs and tethering factors, the lipid-based machineries are emerging as important players in membrane trafficking. As a component of these machineries, lipid transfer proteins have recently attracted the attention of cell biologists for their involvement in trafficking along different segments of the secretory pathway. Among these, the four-phosphate adaptor protein 2 (FAPP2) was discovered as a protein that localizes dynamically with the trans-Golgi network and regulates the transport of proteins from the Golgi complex to the cell surface. Later studies have highlighted a role for FAPP2 as lipid transfer protein involved in glycosphingolipid metabolism at the Golgi complex. Here we discuss the available evidence on the function of FAPP2 in both membrane trafficking and lipid metabolism and propose a mechanism of action of FAPP2 that integrates its activities in membrane trafficking and in lipid transfer. This article is part of a Special Issue entitled Vesicular Transport.

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Potentiation of dietary restriction-induced lifespan extension by polyphenols.

Publication Date: 2012 Jan 11 PMID: 22265987
Authors: Aires, D. J. - Rockwell, G. - Wang, T. - Frontera, J. - Wick, J. - Wang, W. - Tonkovic-Capin, M. - Lu, J. - , E. L - Zhu, H. - Swerdlow, R. H.
Journal: Biochim Biophys Acta

Dietary restriction (DR) extends lifespan across multiple species including mouse. Antioxidant plant extracts rich in polyphenols have also been shown to increase lifespan. We hypothesized that polyphenols might potentiate DR-induced lifespan extension. Twenty week old C57BL/6 mice were placed on one of three diets: continuous feeding (control), alternate day chow (Intermittent fed, IF), or IF supplemented with polyphenol antioxidants (PAO) from blueberry, pomegranate, and green tea extracts (IF+PAO). Both IF and IF+PAO groups outlived the control group and the IF+PAO group outlived the IF group (all p<0.001). In the brain, IF induced the expression of inflammatory genes and p38 MAPK phosphorylation, while the addition of PAO reduced brain inflammatory gene expression and p38 MAPK phosphorylation. Our data indicate that while IF overall promotes longevity, some aspects of IF-induced stress may paradoxically lessen this effect. Polyphenol compounds, in turn, may potentiate IF-induced longevity by minimizing specific components of IF-induced cell stress.

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Reduction in neuronal L-type calcium channel activity in a double knock-in mouse model of Alzheimer's disease.

Publication Date: 2012 Jan 10 PMID: 22265986
Authors: Thibault, O. - Pancani, T. - Landfield, P. W. - Norris, C. M.
Journal: Biochim Biophys Acta

Increased function of neuronal L-type voltage-sensitive Ca(2+) channels (L-VSCCs) is strongly linked to impaired memory and altered hippocampal synaptic plasticity in aged rats. However, no studies have directly assessed L-VSCC function in any of the common mouse models of Alzheimer's disease where neurologic deficits are typically more robust. Here, we used cell-attached patch-clamp recording techniques to measure L-VSCC activity in CA1 pyramidal neurons of partially dissociated hippocampal "zipper" slices prepared from 14-month-old wild-type mice and memory-impaired APP/PS1 double knock-in mice. Surprisingly, the functional channel density of L-VSCCs was significantly reduced in the APP/PS1 group. No differences in voltage dependency and unitary conductance of L-VSCCs were observed. The results suggest that mechanisms for Ca(2+) dysregulation can differ substantially between animal models of normal aging and models of pathological aging.

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Evidence for the critical roles of NF-kappaB p65 and specificity proteins in the apoptosis-inducing activity of proteasome inhibitors in leukemia cells.

Publication Date: 2012 Jan 10 PMID: 22265847
Authors: Reuter, S. - Gupta, S. C. - Kannappan, R. - Aggarwal, B. B.
Journal: Biochim Biophys Acta

Although proteasome inhibitors, such as Bortezomib, have been approved for the treatment of multiple myeloma and mantle cell lymphoma, the mechanism by which they induce apoptosis is still incompletely understood. In the present study, we demonstrate that genetic deletion of the NF-kappaB p65 subunit abolished the ability of Bortezomib to induce apoptosis, indicating that p65 is needed for apoptosis. Although Bortezomib inhibited TNF-induced NF-kappaB activation through suppression of IkappaBalpha degradation, it also induced proteolytic degradation of constitutive NF-kappaB proteins, including p65, IkappaBalpha and p105. These effects were also observed with two other proteasome inhibitors, N-acetyl-leucylleucyl-norleucinal (ALLN) and MG132. The p65 is known to be linked with Specific proteins (Sp), and we found that proteasome inhibition also induced degradation of Sp-1, Sp-3, and Sp-4 proteins. Bortezomib induced apoptosis in cells expressing caspase-3 but not in cells that lack caspase-3, indicating the critical role for this enzyme in the apoptotic action of Bortezomib. Furthermore, inhibition of pan-caspases abolished Bortezomib-induced degradation of p65, p105 and Sp proteins, but not that of IkappaBalpha. Overall, our results demonstrate for the first time a critical role for the degradation of NF-kappaB and Sp proteins by caspases in the apoptosis-inducing activity of proteasome inhibitors, such as Bortezomib.

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Vesicle-mediated ER export of proteins and lipids.

Publication Date: 2012 Jan 11 PMID: 22265716
Authors: Gillon, A. D. - Latham, C. F. - Miller, E. A.
Journal: Biochim Biophys Acta

In eukaryotic cells, the endoplasmic reticulum (ER) is a major site of synthesis of both lipids and proteins, many of which must be transported to other organelles. The COPII coat-comprising Sar1, Sec23/24, Sec13/31-generates transport vesicles that mediate the bulk of protein/lipid export from the ER. The coat exhibits remarkable flexibility in its ability to specifically select and accommodate a large number of cargoes with diverse properties. In this review, we discuss the fundamentals of COPII vesicle production and describe recent advances that further our understanding of just how flexible COPII cargo recruitment and vesicle formation may be. Large or bulky cargo molecules (e.g. collagen rods and lipoprotein particles) exceed the canonical size for COPII vesicles and seem to rely on the additional action of recently identified accessory molecules. Although the bulk of the research has focused on the fate of protein cargo, the mechanisms and regulation of lipid transport are equally critical to cellular survival. From their site of synthesis in the ER, phospholipids, sphingolipids and sterols exit the ER, either accompanying cargo in vesicles or directly across the cytoplasm shielded by lipid-transfer proteins. Finally, we highlight the current challenges to the field in addressing the physiological regulation of COPII vesicle production and the molecular details of how diverse cargoes, both proteins and lipids, are accommodated. This article is part of a Special Issue entitled Vesicular Transport.

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GPI-anchor remodeling: Potential functions of GPI-anchors in intracellular trafficking and membrane dynamics.

Publication Date: 2012 Jan 11 PMID: 22265715
Authors: Fujita, M. - Kinoshita, T.
Journal: Biochim Biophys Acta

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. GPI is synthesized and transferred to proteins in the endoplasmic reticulum. GPI-anchored proteins are then transported from the endoplasmic reticulum to the plasma membrane through the Golgi apparatus. GPI-anchor functions as a sorting signal for transport of GPI-anchored proteins in the secretory and endocytic pathways. After GPI attachment to proteins, the structure of the GPI-anchor is remodeled, which regulates the trafficking and localization of GPI-anchored proteins. Recently, genes required for GPI remodeling were identified in yeast and mammalian cells. Here, we describe the structural remodeling and function of GPI-anchors, and discuss how GPI-anchors regulate protein sorting, trafficking, and dynamics. This article is part of a Special Issue entitled Vesicular Transport.

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Transcriptional control of mitochondrial biogenesis and its interface with inflammatory processes.

Publication Date: 2012 Jan 14 PMID: 22265687
Authors: Piantadosi, C. A. - Suliman, H. B.
Journal: Biochim Biophys Acta

BACKGROUND: Cells avoid major mitochondrial damage and energy failure during systemic inflammatory states, such as severe acute infections, by specific targeting of the inflammatory response and by inducing anti-inflammatory and anti-oxidant defenses. Recent evidence indicates that these cell defenses also include mitochondrial biogenesis and the clearance of damaged mitochondria through autophagy. SCOPE OF REVIEW: This review addresses a group of transcriptional signaling mechanisms that engage mitochondrial biogenesis, including energy-sensing and redox-regulated transcription factors and co-activators, after major inflammatory events. MAJOR CONCLUSIONS: Stimulation of the innate immune system by activation of toll-like receptors (TLR) generates pro-inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha)and interleukin-1beta (IL-1beta), necessary for optimal host defense, but which also contribute to mitochondrial damage through oxidative stress and other mechanisms. To protect its energy supply, host cells sense mitochondrial damage and initiate mitochondrial biogenesis under the control of an inducible transcriptional program that also activates anti-oxidant and anti-inflammatory gene expression. This multifunctional network not only increases cellular resistance to metabolic failure, oxidative stress, and cell death, but promotes immune tolerance as shown in the graphical abstract. GENERAL SIGNIFICANCE: The post-inflammatory induction of mitochondrial biogenesis supports metabolic function and cell viability while helping to control inflammation. In clinical settings, patients recovering from severe systemic infections may develop transient immune suppression, placing them at risk for recurrent infection, but there may be therapeutic opportunities to enhance mitochondrial quality control that would improve the resolution of life-threatening host responses to such infections.

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Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes.

Publication Date: 2012 Jan 14 PMID: 22265686
Authors: Hoshi, H. - Shimawaki, K. - Takegawa, Y. - Ohyanagi, T. - Amano, M. - Hinou, H. - Nishimura, S. I.
Journal: Biochim Biophys Acta

BACKGROUND: Cell surface proteoglycans play vital functional roles in various biological processes such as cell proliferation, differentiation, adhesion, inflammation, immune response, sustentation of cartilage tissue and intensity of tissues. We show here that serglycin-like synthetic glycopeptides function efficiently as a molecular shuttle to hijack glycosaminoglycan (GAG) biosynthetic pathway within cells across the plasma membrane. METHODS: Fluorescence (FITC)-labeled tetrapeptide (H-Ser(1)-Gly(2)-Ser(3)-Gly(4)-OH) carrying Galbeta(14)Xylbeta1 defined as proteoglycan initiator (PGI) monomer and its tandem repeating PGI polymer was employed for direct imaging of cellular uptake and intracellular traffic by confocal laser-scanning microscopy. Novel method for enrichment analysis of GAG-primed PGIs by combined use of anti-FITC antibody and LC/mass spectrometry was established. RESULTS: PGI monomer was incorporated promptly into human articular chondrocytes and distributed in whole cytoplasm including ER/Golgi while PGI polymer localized specifically in nucleus. It was demonstrated that PGIs become good substrates for GAG biosynthesis within the cells and high molecular weight GAGs primed by PGIs is chondroitin sulfate involving N-acetyl-d-galactosamine residues substituted by 4-O-sulfate or 6-O-sulfate group as major components. PGIs activated chondrocytes proliferation and induced up-regulation of the expression level of type II collagen, suggesting that PGIs can function as new class cytokine-like molecules to stimulate cell growth. CONCLUSION: Synthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and structural characterization of GAG-primed PGIs by an antibody-based enrichment protocol. GENERAL SIGNIFICANCE: Novel glycomics designated for investigating proteoglycan biosynthesis, namely real-time GAGomics using synthetic glycopeptides as PGIs, should facilitate greatly dynamic profiling of GAGs in the living cells. This article is part of a Special Issue entitled Glycoproteomics.

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Preface for the special issue of imaging brain aging and neurodegenerative disease.

Publication Date: 2012 Mar PMID: 22265024
Authors: Gold, B. T. - Keller, J. N.
Journal: Biochim Biophys Acta

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Differential effects of alpha-tocopherol and N-acetyl-cysteine on advanced glycation end product-induced oxidative damage and neurite degeneration in SH-SY5Y cells.

Publication Date: 2012 Jan 10 PMID: 22261284
Authors: Pazdro, R. - Burgess, J. R.
Journal: Biochim Biophys Acta

Advanced glycation end products (AGEs) result from non-enzymatic glycation of proteins and cause cellular oxidative stress in a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent manner. Due to these effects, AGEs are implicated as a causal factor in diabetic complications. Several antioxidants, including vitamin E, improve cell viability and diminish markers of oxidative damage in cells exposed to AGEs. However, vitamin E has been studied in cell culture systems with primary focus on apoptosis and lipid peroxidation, while its influences on AGE-induced protein and DNA oxidation, intracellular antioxidant status and cell morphology remain largely unknown. Here, we verify the suppression of AGE-induced cell death and lipid peroxidation by 200muM alpha-tocopherol in SH-SY5Y cells. We report the partial inhibition of DNA oxidation and a decrease in protein carbonyl formation by alpha-tocopherol with no effects on intracellular GSH concentrations. We observed that 2mM N-acetyl cysteine (NAC) also had a suppressive effect on DNA and protein oxidation, but unlike alpha-tocopherol, it caused a marked increase in intracellular GSH. Finally, we compared the ability of both antioxidants to maintain neurites in SH-SY5Y cells and found that alpha-tocopherol had no effect on neurite loss due to AGEs, while NAC fully maintained cell morphology. Thus, while alpha-tocopherol suppressed AGE-induced macromolecule damage, it was ineffective against neurite degeneration. These results may implicate thiol oxidation and maintenance as a major regulator of neurite degeneration in this model.

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delta-Catenin promotes E-cadherin processing and activates beta-catenin-mediated signaling: Implications on human prostate cancer progression.

Publication Date: 2012 Jan 11 PMID: 22261283
Authors: Kim, H. - He, Y. - Yang, I. - Zeng, Y. - Kim, Y. - Seo, Y. W. - Murnane, M. J. - Jung, C. - Lee, J. H. - Min, J. J. - Kwon, D. D. - Kim, K. K. - Lu, Q. - Kim, K.
Journal: Biochim Biophys Acta

delta-Catenin binds the juxtamembrane domain of E-cadherin and is known to be overexpressed in some human tumors. However, the functions of delta-catenin in epithelial cells and carcinomas remain elusive. We found that prostate cancer cells overexpressing delta-catenin show an increase in multi-layer growth in culture. In these cells, delta-catenin colocalizes with E-cadherin at the plasma membrane, and the E-cadherin processing is noticeably elevated. E-Cadherin processing induced by delta-catenin is serum-dependent and requires MMP- and PS-1/gamma-secretase-mediated activities. A deletion mutant of delta-catenin that deprives the ability of delta-catenin to bind E-cadherin or to recruit PS-1 to E-cadherin totally abolishes the delta-catenin-induced E-cadherin processing and the multi-layer growth of the cells. In addition, prostate cancer cells overexpressing delta-catenin display an elevated total beta-catenin level and increase its nuclear distribution, resulting in the activation of beta-catenin/LEF-1-mediated transcription and their downstream target genes as well as androgen receptor-mediated transcription. Indeed, human prostate tumor xenograft in nude mice, which is derived from cells overexpressing delta-catenin, shows increased beta-catenin nuclear localization and more rapid growth rates. Moreover, the metastatic xenograft tumor weights positively correlate with the level of 29kD E-cadherin fragment, and primary human prostate tumor tissues also show elevated levels of delta-catenin expression and the E-cadherin processing. Taken together, these results suggest that delta-catenin plays an important role in prostate cancer progression through inducing E-cadherin processing and thereby activating beta-catenin-mediated oncogenic signals.

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Genetic variation of GPLD1 associates with serum GPI-PLD levels: A preliminary study.

Publication Date: 2012 Jan 8 PMID: 22260953
Authors: Deeg, M. A. - Xuei, X. - Eckert, G. - Considine, R. V. - Li, Y. G. - Pratt, J. H.
Journal: Biochim Biophys Acta

HDL is a heterogeneous mixture of lipoprotein particles varying in composition, size, and function. We and others have described a small (7.0nm), minor (0.1% of total apolipoprotein AI) particle containing apolipoprotein AI, AIV and glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in humans the function of which is not entirely known. Circulating GPI-PLD levels are regulated by multiple factors including genetics. To determine if genetic variation in GPLD1 affects circulating GPI-PLD levels, we examined the relationship between 32 SNPS upstream, within, and downstream of GPLD1 and circulating GPI-PLD levels in Caucasians (n=77) and African-Americans (n=99). The genotype distribution among races differed at 13 SNPs. Nine SNPS were associated with circulating GPI-PLD levels in Caucasians but not African-Americans. These results suggest that genetic variation of GPLD1 appears to associate with circulating GPI-PLD levels. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

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Orientation and depth of surfactant protein B C-terminal helix in lung surfactant bilayers.

Publication Date: 2012 Jan 9 PMID: 22252270
Authors: Bertani, P. - Vidovic, V. - Yang, T. C. - Rendell, J. - Gordon, L. M. - Waring, A. J. - Bechinger, B. - Booth, V.
Journal: Biochim Biophys Acta

SP-B(CTERM) is a cationic amphipathic helical peptide and functional fragment composed of residues 63 to 78 of surfactant protein B (SP-B). Static oriented and magic angle spinning solid state NMR, along with molecular dynamics simulation was used to investigate its structure, orientation, and depth in lipid bilayers of several compositions, namely POPC, DPPC, DPPC/POPC/POPG, and bovine lung surfactant extract (BLES). In all lipid environments the peptide was oriented parallel to the membrane surface. While maintaining this approximately planar orientation, SP-B(CTERM) exhibited a flexible topology controlled by subtle variations in lipid composition. SP-B(CTERM)-induced lipid realignment and/or conformational changes at the level of the head group were observed using (31)P solid-state NMR spectroscopy. Measurements of the depth of SP-B(CTERM) indicated the peptide center positions ~8A more deeply than the phosphate headgroups, a topology that may allow the peptide to promote functional lipid structures without causing micellization upon compression.

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Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

Publication Date: 2012 Jan 11 PMID: 22251894
Authors: Vancraenenbroeck, R. - Lobbestael, E. - Weeks, S. D. - Strelkov, S. V. - Baekelandt, V. - Taymans, J. M. - De Maeyer, M.
Journal: Biochim Biophys Acta

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% alpha-helices and 21% beta-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions.

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Solution structure of native and recombinant expressed toxin CssII from the venom of the scorpion Centruroides suffusus suffusus, and their effects on Nav1.5 Sodium channels.

Publication Date: 2012 Jan 11 PMID: 22251893
Authors: Saucedo, A. L. - Del Rio-Portilla, F. - Picco, C. - Estrada, G. - Prestipino, G. - Possani, L. D. - Delepierre, M. - Corzo, G.
Journal: Biochim Biophys Acta

The three-dimensional structures of the long-chain mammalian scorpion beta-toxin CssII from Centruroides suffusus suffusus and of its recombinant form, HisrCssII, were determined by NMR. The neurotoxin CssII (nCssII) is a 66 amino acid long peptide with four disulfide bridges; it is the most abundant and deadly toxin from the venom of this scorpion. Both native and recombinant CssII structures were determined by nuclear magnetic resonance using a total of 828 sequential distance constraints derived from the volume integration of the cross peaks observed in 2D NOESY spectra. Both nCssII and HisrCssII structures display a mixed alpha/beta fold stabilized by four disulfide bridges formed between pairs of cysteines: C1-C8, C2-C5, C3-C6, and C4-C7 (the numbers indicate the relative positions of the cysteine residues in the primary structure), with a distortion induced by two cis-prolines in its C-terminal part. The native CssII electrostatic surface was compared to both the recombinant one and to the Cn2 toxin, from the scorpion Centruroides noxius, which is also toxic to mammals. Structural features such N- and C-terminal differences could influence toxin specificity and affinity towards isoforms of different sub-types of Na(v) channels.

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Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity.

Publication Date: 2012 Jan 9 PMID: 22251892
Authors: Es-Haghi, A. - Shariatizi, S. - Ebrahim-Habibi, A. - Nemat-Gorgani, M.
Journal: Biochim Biophys Acta

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.

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Characterization of a novel tyrosinase inhibitor, (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384).

Publication Date: 2012 Jan 9 PMID: 22251576
Authors: Han, Y. K. - Park, Y. J. - Ha, Y. M. - Park, D. - Lee, J. Y. - Lee, N. - Yoon, J. H. - Moon, H. R. - Chung, H. Y.
Journal: Biochim Biophys Acta

BACKGROUND: We synthesized (2RS,4R)-2-(2,4-dihydroxyphenyl)thiazolidine-4-carboxylic acid (MHY384) as a potential tyrosinase inhibitor and investigated its antityrosinase activity. METHODS: The structure of MHY384 was established using (1)H and (13)C NMR spectroscopy and mass spectral analyses. To investigate dual mechanisms of action of MHY384 for the inhibition of melanin synthesis, we confirmed the inhibitory effect of tyrosinase catalytic activity of MHY384. Then, we confirmed the inhibitory effect of MHY384 on transcription of tyrosinase mRNA through alpha-MSH-induced cAMP-PKA-MITF signaling. In addition, we supported the inhibitory mechanism of MHY384 against tyrosinase using a kinetic study and docking programs. RESULTS: To determine how MHY384 regulates melanogenesis, we measured melanin levels and expression of the genes for microphthalmia-associated transcription factor (MITF) and tyrosinase in alpha-melanocyte-stimulating hormone (alpha-MSH)-induced B16F10 melanoma cells. MHY384 potently inhibited tyrosinase activity and melanin production in B16F10 melanoma cells. Through docking models, we were able to construct the tertiary structure of mushroom tyrosinase and simulate its docking with MHY384. The result supports that MHY384 strongly interacts with tyrosinase residues in the active site and it can directly inhibit tyrosinase. To investigate additional mechanisms of action of MHY384, we confirmed that the inhibition of tyrosinase activity was found to be due to the modulation of the expression of tyrosinase and its transcription factor, MITF, through cAMP, which regulates protein kinase A. CONCLUSIONS: This study strongly indicates that the depigmenting effect of MHY384 results from the down-regulation of MITF and tyrosinase through direct tyrosinase inhibition. GENERAL SIGNIFICANCE: Our findings suggest that MHY384 can be an effective skin-whitening agent.

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The roles of Rhodobacter sphaeroides copper chaperones PCu(A)C and Sco (PrrC) in the assembly of the copper centers of the aa(3)-type and the cbb(3)-type cytochrome c oxidases.

Publication Date: 2012 Jan 8 PMID: 22248670
Authors: Thompson, A. K. - Gray, J. - Liu, A. - Hosler, J. P.
Journal: Biochim Biophys Acta

The alpha proteobacter Rhodobacter sphaeroides accumulates two cytochrome c oxidases (CcO) in its cytoplasmic membrane during aerobic growth: a mitochondrial-like aa(3)-type CcO containing a di-copper Cu(A) center and mono-copper Cu(B), plus a cbb(3)-type CcO that contains Cu(B) but lacks Cu(A). Three copper chaperones are located in the periplasm of R. sphaeroides, PCu(A)C, PrrC (Sco) and Cox11. Cox11 is required to assemble Cu(B) of the aa(3)-type but not the cbb(3)-type CcO. PrrC is homologous to mitochondrial Sco1; Sco proteins are implicated in Cu(A) assembly in mitochondria and bacteria, and with Cu(B) assembly of the cbb(3)-type CcO. PCu(A)C is present in many bacteria, but not mitochondria. PCu(A)C of Thermus thermophilus metallates a Cu(A) center in vitro, but its in vivo function has not been explored. Here, the extent of copper center assembly in the aa(3)- and cbb(3)-type CcOs of R. sphaeroides has been examined in strains lacking PCu(A)C, PrrC, or both. The absence of either chaperone strongly lowers the accumulation of both CcOs in the cells grown in low concentrations of Cu(2+). The absence of PrrC has a greater effect than the absence of PCu(A)C and PCu(A)C appears to function upstream of PrrC. Analysis of purified aa(3)-type CcO shows that PrrC has a greater effect on the assembly of its Cu(A) than does PCu(A)C, and both chaperones have a lesser but significant effect on the assembly of its Cu(B) even though Cox11 is present. Scenarios for the cellular roles of PCu(A)C and PrrC are considered. The results are most consistent with a role for PrrC in the capture and delivery of copper to Cu(A) of the aa(3)-type CcO and to Cu(B) of the cbb(3)-type CcO, while the predominant role of PCu(A)C may be to capture and deliver copper to PrrC and Cox11. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

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Low pH induced structural reorganization in thylakoid membranes.

Publication Date: 2012 Jan 8 PMID: 22248669
Authors: Jajoo, A. - Szabo, M. - Zsiros, O. - Garab, G.
Journal: Biochim Biophys Acta

By using low temperature fluorescence spectroscopy, it has been shown that exposing chloroplast thylakoid membranes to acidic pH reversibly decreases the fluorescence of photosystem II while the fluorescence of photosystem I increases [P. Singh-Rawal et al. (2010) Evidence that pH can drive state transitions in isolated thylakoid membranes from spinach, Photochem Photobiol Sci, 9 830-837]. In order to shed light on the origin of these changes, we performed circular dichroism (CD) spectroscopy on freshly isolated pea thylakoid membranes. We show that the magnitude of the psi-type CD, which is associated with the presence of chirally ordered macroarrays of the chromophores in intact thylakoid membranes, decreases gradually and reversibly upon gradually lowering the pH of the medium from 7.5 to 4.5 (psi, polymer or salt induced). The same treatment, as shown on thylakoid membranes washed in hypotonic low salt medium possessing no psi-type bands, induces no discernible change in the excitonic CD. These data show that while no change in the pigment-pigment interactions and thus in the molecular organization of the bulk protein complexes can be held responsible for the observed changes in the fluorescence, acidification of the medium significantly alters the macro-organization of the complexes, hence providing an explanation for the pH-induced redistribution of the excitation energy between the two photosystems. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial Photosynthesis.

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Regulated pre-mRNA splicing: The ghostwriter of the eukaryotic genome.

Publication Date: 2012 Jan 9 PMID: 22248620
Authors: Johnson, T. L. - Vilardell, J.
Journal: Biochim Biophys Acta

Intron removal is at the heart of mRNA synthesis. It is mediated by one of the cell's largest complexes, the spliceosome. Yet, the fundamental chemistry involved is simple. In this review we will address how the spliceosome acts in diverse ways to optimize gene expression in order to meet the cell's needs. This is done largely by regulating the splicing of key transcripts encoding products that control gene expression pathways. This widespread role is evident even in the yeast Saccharomyces cerevisiae, where many introns appear to have been lost; yet how this control is being achieved is known only in a few cases. Here we explore the relevant examples and posit hypotheses whereby regulated splicing fine-tunes gene expression pathways to maintain cell homeostasis. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

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Nuclear export as a key arbiter of "mRNA identity" in eukaryotes.

Publication Date: 2012 Jan 9 PMID: 22248619
Authors: Palazzo, A. F. - Akef, A.
Journal: Biochim Biophys Acta

Over the past decade, various studies have indicated that most of the eukaryotic genome is transcribed at some level. The pervasiveness of transcription might seem surprising when one considers that only a quarter of the human genome comprises genes (including exons and introns) and less than 2% codes for protein. This conundrum is partially explained by the unique evolutionary pressures that are imposed on species with small population sizes, such as eukaryotes. These conditions promote the expansion of introns and non-functional intergenic DNA, and the accumulation of cryptic transcriptional start sites. As a result, the eukaryotic gene expression machinery must effectively evaluate whether or not a transcript has all the hallmarks of a protein-coding mRNA. If a transcript contains these features, then positive feedback loops are activated to further stimulate its transcription, processing, nuclear export and ultimately, translation. However if a transcript lacks features associated with "mRNA identity", then the RNA is degraded and/or used to inhibit further transcription and translation of the gene. Here we discuss how mRNA identity is assessed by the nuclear export machinery in order to extract meaningful information from the eukaryotic genome. In the process, we provide an explanation of why certain sequences that are enriched in protein-coding genes, such as the signal sequence coding region, promote mRNA nuclear export in vertebrates. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

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Antigen-antibody interface properties: Composition, residue interactions, and features of 53 non-redundant structures.

Publication Date: 2012 Jan 10 PMID: 22246133
Authors: Ramaraj, T. - Angel, T. - Dratz, E. A. - Jesaitis, A. - Mumey, B.
Journal: Biochim Biophys Acta

The structures of protein antigen-antibody (Ag-Ab) interfaces contain information about how Ab recognize Ag as well as how Ag are folded to present surfaces for Ag recognition. As such, the Ab surface holds information about Ag folding that resides with the Ab-Ag interface residues and how they interact. In order to gain insight into the nature of such interactions, a data set comprised of 53 non-redundant 3D structures of Ag-Ab complexes was analyzed. We assessed the physical and biochemical features of the Ag-Ab interfaces and the degree to which favored interactions exist between amino acid residues on the corresponding interface surfaces. Amino acid compositional analysis of the interfaces confirmed the dominance of TYR in the Ab paratope-containing surface (PCS), with almost two fold greater abundance than any other residue. Additionally TYR had a much higher than expected presence in the PCS compared to the surface of the whole antibody (defined as the occurrence propensity), along with aromatics PHE, TRP, and to a lesser degree HIS and ILE. In the Ag epitope-containing surface (ECS), there were slightly increased occurrence propensities of TRP and TYR relative to the whole Ag surface, implying an increased significance over the compositionally most abundant LYS>ASN>GLU>ASP>ARG. This examination encompasses a large, diverse set of unique Ag-Ab crystal structures that help explain the biological range and specificity of Ag-Ab interactions. This analysis may also provide a measure of the significance of individual amino acid residues in phage display analysis of Ag binding.

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Alcohol induced structural and dynamic changes in beta-lactoglobulin in aqueous solution: A neutron scattering study.

Publication Date: 2012 Jan 8 PMID: 22246132
Authors: Yoshida, K. - Vogtt, K. - Izaola, Z. - Russina, M. - Yamaguchi, T. - Bellissent-Funel, M. C.
Journal: Biochim Biophys Acta

Structural and dynamic properties of beta-lactoglobulin (beta-LG) were revealed as a function of alcohol concentration in ethanol- and trifluoroethanol(TFE)-water mixtures with circular dichroism (CD), small-angle neutron scattering (SANS) and quasi-elastic neutron scattering (QENS). The CD spectra showed that an increase in TFE concentration promotes the formation of the beta-sheet structure of beta-LG. The SANS-intensities were fitted using form factors for two attached spheres for the native and native-like states of the protein. At higher alcohol concentrations, where aggregation takes place, a form factor modelling diffusion limited colloidal aggregation (DLCA) was employed. The QENS-data were analyzed in terms of internal motions for all alcohol concentrations. While low concentrations of TFE (10% (v/v)) lead to an increase of the mean square amplitudes of vibrations and a retention of a native-like structure - but not to an increase of the characteristic radius of proton diffusion processes a. Addition of 20% (v/v) of TFE induces aggregation, going along with a further increase of . Further increase of TFE concentration to 30% (v/v) changes the nanoscale structure of the oligomeric nucleate, but induces no further significant changes in . The present study underlines the necessity of methods sensitive to the dynamics of a system to obtain a complete picture of a molecular process.

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Long chain polyunsaturated fatty acid synthesis in a marine vertebrate: Ontogenetic and nutritional regulation of a fatty acyl desaturase with Delta4 activity.

Publication Date: 2012 Jan 8 PMID: 22245719
Authors: Morais, S. - Castanheira, F. - Martinez-Rubio, L. - Conceicao, L. E. - Tocher, D. R.
Journal: Biochim Biophys Acta

Solea senegalensis is an unusual marine teleost as it has very low dietary requirement for long-chain polyunsaturated fatty acids (LC-PUFA) during early development. Aquaculture is rapidly becoming the main source of health-beneficial fish products for human consumption. This, associated with limited supply of LC-PUFA-rich ingredients for fish feeds, render S. senegalensis a highly interesting species in which to study the LC-PUFA biosynthesis pathway. We have cloned and functionally characterized fatty acyl desaturase and elongase cDNAs corresponding to Delta4fad (with some Delta5 activity for the n-3 series) and elovl5 with the potential to catalyze docosahexaenoic acid (DHA) biosynthesis from eicosapentaenoic acid (EPA). Changes in expression of both transcripts were determined during embryonic and early larval development, and transcriptional regulation in response to higher or lower dietary n-3 LC-PUFA was assessed during larval and post-larval stages. There was a marked pattern of regulation during early ontogenesis, with both transcripts showing peak expression coinciding with the start of exogenous feeding. Although elovl5 transcripts were present in fertilized eggs, Delta4fad only appeared at hatching. However, eggs have high proportions of DHA (~20%) and high DHA/EPA ratio (~11) to meet the high demands for early embryonic development. The fatty acid profile of larvae after the start of exogenous feeding closely reflected dietary composition. Nonetheless, Delta4fad was significantly up-regulated in response to LC-PUFA-poor diets, which may suggest biological relevance of this pathway in reducing LC-PUFA dietary requirements in this species, compared to other marine teleosts. These results indicate that sole is capable of synthesizing DHA from EPA through a Sprecher-independent pathway.

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Chromatin changes in the development and pathology of the Fragile X-associated disorders and Friedreich ataxia.

Publication Date: 2012 Jan 5 PMID: 22245581
Authors: Kumari, D. - Lokanga, R. - Yudkin, D. - Zhao, X. N. - Usdin, K.
Journal: Biochim Biophys Acta

The Fragile X-associated disorders (FXDs) and Friedreich ataxia (FRDA) are genetic conditions resulting from expansion of a trinucleotide repeat in a region of the affected gene that is transcribed but not translated. In the case of the FXDs, pathology results from expansion of CGG*CCG-repeat tract in the 5' UTR of the FMR1 gene, while pathology in FRDA results from expansion of a GAA*TTC-repeat in intron 1 of the FXN gene. Expansion occurs during gametogenesis or early embryogenesis by a mechanism that is not well understood. Associated Expansion then produces disease pathology in various ways that are not completely understood either. In the case of the FXDs, alleles with 55-200 repeats express higher than normal levels of a transcript that is thought to be toxic, while alleles with >200 repeats are silenced. In addition, alleles with >200 repeats are associated with a cytogenetic abnormality known as a fragile site, which is apparent as a constriction or gap in the chromatin that is seen when cells are grown in presence of inhibitors of thymidylate synthase. FRDA alleles show a deficit of the FXN transcript. This review will address the role of repeat-mediated chromatin changes in these aspects of FXD and FRDA disease pathology. This article is part of a Special Issue entitled: Chromatin in time and space.

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Identification of a nuclear carbonic anhydrase in Caenorhabditis elegans.

Publication Date: 2012 Jan 5 PMID: 22245567
Authors: Sherman, T. A. - Rongali, S. - Matthews, T. A. - Pfeiffer, J. - Nehrke, K.
Journal: Biochim Biophys Acta

BACKGROUND: Carbonic anhydrases (CA) catalyze the inter-conversion of CO(2) with HCO(3) and H(+), and are involved in a wide variety of physiologic processes such as anion transport, pH regulation, and water balance. In mammals there are sixteen members of the classical alpha-type CA family, while the simple genetic model organism Caenorhabditis elegans codes for six alphaCA isoforms (cah-1 through cah-6). METHODS: Fluorescent reporter constructs were used to analyze gene promoter usage, splice variation, and protein localization in transgenic worms. Catalytic activity of recombinant CA proteins was assessed using Hansson's histochemistry. CA's ability to regulate pH as a function of CO(2) and HCO(3) was measured using dynamic fluorescent imaging of genetically-targeted biosensors. RESULTS: Each of the six CA genes was found to be expressed in a distinct repertoire of cell types. Surprisingly, worms also expressed a catalytically-active CA splice variant, cah-4a, in which an alternative first exon targeted the protein to the nucleus. Cah-4a expression was restricted mainly to the nervous system, where it was found in nearly all neurons, and recombinant CAH-4A protein could regulate pH in the nucleus. CONCLUSIONS: In addition to establishing C. elegans as a platform for studying alphaCA function, this is the first example of a nuclear-targeted alphaCA in any organism to date. GENERAL SIGNIFICANCE: A classical alphaCA isoform is targeted exclusively to the nucleus where its activity may impact nuclear physiologic and pathophysiologic responses.

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The "beta-clasp" model of apolipoprotein A-I - A lipid-free solution structure determined by electron paramagnetic resonance spectroscopy.

Publication Date: 2012 Jan 8 PMID: 22245143
Authors: Lagerstedt, J. O. - Budamagunta, M. S. - Liu, G. S. - Devalle, N. C. - Voss, J. C. - Oda, M. N.
Journal: Biochim Biophys Acta

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and plays a central role in cholesterol metabolism. The lipid-free/lipid-poor form of apoA-I is the preferred substrate for the ATP-binding cassette transporter A1 (ABCA1). The interaction of apoA-I with ABCA1 leads to the formation of cholesterol laden high density lipoprotein (HDL) particles, a key step in reverse cholesterol transport and the maintenance of cholesterol homeostasis. Knowledge of the structure of lipid-free apoA-I is essential to understanding its critical interaction with ABCA1 and the molecular mechanisms underlying HDL biogenesis. We therefore examined the structure of lipid-free apoA-I by electron paramagnetic resonance spectroscopy (EPR). Through site directed spin label EPR, we mapped the secondary structure of apoA-I and identified sites of spin coupling as residues 26, 44, 64, 167, 217 and 226. We capitalize on the fact that lipid-free apoA-I self-associates in an anti-parallel manner in solution. We employed these sites of spin coupling to define the central plane in the dimeric apoA-I complex. Applying both the constraints of dipolar coupling with the EPR-derived pattern of solvent accessibility, we assembled the secondary structure into a tertiary context, providing a solution structure for lipid-free apoA-I. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

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Nuclear organization and chromatin dynamics in yeast: Biophysical models or biologically driven interactions?

Publication Date: 2012 Jan 5 PMID: 22245105
Authors: Albert, B. - Leger-Silvestre, I. - Normand, C. - Gadal, O.
Journal: Biochim Biophys Acta

Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

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O(6)-Methylguanine-DNA methyltransferase in glioma therapy: Promise and problems.

Publication Date: 2012 Jan 8 PMID: 22244911
Authors: Silber, J. R. - Bobola, M. S. - Blank, A. - Chamberlain, M. C.
Journal: Biochim Biophys Acta

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.

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Molecular and structural basis for N-glycan-dependent determination of glycoprotein fates in cells.

Publication Date: 2012 Jan 5 PMID: 22240168
Authors: Kamiya, Y. - Satoh, T. - Kato, K.
Journal: Biochim Biophys Acta

BACKGROUND: N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway. SCOPE OF REVIEW: This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins. MAJOR CONCLUSIONS: Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway. GENERAL SIGNIFICANCE: Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics.

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Brown adipose tissue mitochondria oxidizing fatty acids generate high levels of reactive oxygen species irrespective of the uncoupling protein-1 activity state.

Publication Date: 2012 Mar PMID: 22226918
Authors: Schonfeld, P. - Wojtczak, L.
Journal: Biochim Biophys Acta

Mitochondria from brown adipose tissue (BATM) have a high enzymatic capacity for fatty acid oxidation and therefore are an ideal model to examine the sites of reactive oxygen species (ROS) generation during fatty acid oxidation. ROS generation by BATM (isolated from 3-week-old rats) was measured during acylcarnitine oxidation as release of H(2)O(2) into the medium and as inactivation of the matrix enzyme aconitase. The following results were obtained: (1) BATM release large amounts of H(2)O(2) in the coupled as well as in the uncoupled states, several times more than skeletal muscle mitochondria. (2) H(2)O(2) release is especially large with acylcarnitines of medium-chain fatty acids (e.g. octanoylcarnitine). (3) Reverse electron transport does not contribute in a significant extent to the overall ROS generation. (4) Despite the large release of H(2)O(2), the ROS-sensitive matrix enzyme aconitase is not inactivated during acylcarnitine oxidation. (5) In contrast to acylcarnitines, oxidation of alpha-glycerophosphate by BATM is characterized by large H(2)O(2) release and a pronounced aconitase inactivation. We hypothesize that acylcarnitine-supported ROS generation in BATM may be mainly associated with acyl-CoA dehydrogenase and electron transferring flavoprotein-ubiquinone reductase rather than with complexes of the respiratory chain.

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Membrane protein structure and function.

Publication Date: 2012 Feb PMID: 22225970
Authors: Allen, T. W. - Separovic, F.
Journal: Biochim Biophys Acta

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High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases.

Publication Date: 2012 Mar PMID: 22222354
Authors: Leidel, N. - Popovic-Bijelic, A. - Havelius, K. G. - Chernev, P. - Voevodskaya, N. - Graslund, A. - Haumann, M.
Journal: Biochim Biophys Acta

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(muO)(2)Fe(III)L(4) (metal-metal distance of ~2.75A, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(muO)(muOH)Fe(III)L(4) (~2.90A) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55A) with a L(4)Fe(IV)(muO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1A) and Mn,Fe(III)Fe(II) species (~3.3-3.4A) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(muO)(muOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.

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Changes in mitochondrial glutathione levels and protein thiol oxidation in yfh1 yeast cells and the lymphoblasts of patients with Friedreich's ataxia.

Publication Date: 2012 Feb PMID: 22200491
Authors: Bulteau, A. L. - Planamente, S. - Jornea, L. - Dur, A. - Lesuisse, E. - Camadro, J. M. - Auchere, F.
Journal: Biochim Biophys Acta

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by low levels of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulfur cluster defects and high sensitivity to oxidative stress. Frataxin deficiency is also associated with severe impairment of glutathione homeostasis and changes in glutathione-dependent antioxidant defenses. The potential biological consequences of oxidative stress and changes in glutathione levels associated with frataxin deficiency include the oxidation of susceptible protein thiols and reversible binding of glutathione to the SH of proteins by S-glutathionylation. In this study, we isolated mitochondria from frataxin-deficient yfh1 yeast cells and lymphoblasts of FRDA patients, and show evidence for a severe mitochondrial glutathione-dependent oxidative stress, with a low GSH/GSSG ratio, and thiol modifications of key mitochondrial enzymes. Both yeast and human frataxin-deficient cells had abnormally high levels of mitochondrial proteins binding an anti-glutathione antibody. Moreover, proteomics and immunodetection experiments provided evidence of thiol oxidation in alpha-ketoglutarate dehydrogenase (KGDH) or subunits of respiratory chain complexes III and IV. We also found dramatic changes in GSH/GSSG ratio and thiol modifications on aconitase and KGDH in the lymphoblasts of FRDA patients. Our data for yeast cells also confirm the existence of a signaling and/or regulatory process involving both iron and glutathione.

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Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells.

Publication Date: 2012 Mar PMID: 22198343
Authors: Yin, F. - Sancheti, H. - Cadenas, E.
Journal: Biochim Biophys Acta

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP(+) to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H(2)O(2) levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics - as a consequence of NNT knockdown - cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.

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Plastoquinol is more active than alpha-tocopherol in singlet oxygen scavenging during high light stress of Chlamydomonas reinhardtii.

Publication Date: 2012 Mar PMID: 22192719
Authors: Nowicka, B. - Kruk, J.
Journal: Biochim Biophys Acta

In the present study, we have performed comparative analysis of different prenyllipids in Chlamydomonas reinhardtii cultures during high light stress under variety of conditions (presence of inhibitors, an uncoupler, heavy water). The obtained results indicate that plastoquinol is more active than alpha-tocopherol in scavenging of singlet oxygen generated in photosystem II. Besides plastoquinol, also its oxidized form, plastoquinone shows antioxidant action during the stress conditions, resulting in formation of plastoquinone-C, whose level can be regarded as an indicator of singlet oxygen oxidative stress in vivo. The pronounced stimulation of alpha-tocopherol consumption and alpha-tocopherolquinone formation by an uncoupler, FCCP, together with the results of additional model system studies, led to the suggestion that alpha-tocopherol can be recycled in thylakoid membranes under high light conditions from 8a-hydroperoxy-alpha-tocopherone, the primary oxidation product of alpha-tocopherol by singlet oxygen.

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Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species.

Publication Date: 2012 Mar PMID: 22182773
Authors: Mizoguchi, T. - Isaji, M. - Harada, J. - Tamiaki, H.
Journal: Biochim Biophys Acta

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

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Damage to mitochondrial complex I during cardiac ischemia reperfusion injury is reduced indirectly by anti-anginal drug ranolazine.

Publication Date: 2012 Mar PMID: 22178605
Authors: Gadicherla, A. K. - Stowe, D. F. - Antholine, W. E. - Yang, M. - Camara, A. K.
Journal: Biochim Biophys Acta

Ranolazine, an anti-anginal drug, is a late Na(+) channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine's protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30min ischemia and then isolated cardiac mitochondria at the end of 30min ischemia and/or 30min ischemia followed by 10min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe-S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe-S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I.

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Thermodynamic and structural analysis of homodimeric proteins: Model of beta-lactoglobulin.

Publication Date: 2012 Feb PMID: 22172914
Authors: Burgos, I. - Dassie, S. A. - Villarreal, M. A. - Fidelio, G. D.
Journal: Biochim Biophys Acta

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing beta-lactoglobulin A from bovine milk at pH=6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of beta-lactoglobulin (N(2)) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that beta-lactoglobulin follows the conformational path induced by temperature:N(2)right harpoon over left harpoon2Nright harpoon over left harpoon2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.

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Influence of the pathogenic mutations T188K/R/A on the structural stability and misfolding of human prion protein: Insight from molecular dynamics simulations.

Publication Date: 2012 Feb PMID: 22155634
Authors: Guo, J. - Ning, L. - Ren, H. - Liu, H. - Yao, X.
Journal: Biochim Biophys Acta

BACKGROUND: Prion diseases are associated with a conformational switch for PrP from PrP(C) to PrP(Sc). Many genetic mutations are linked with prion diseases, such as mutations T188K/R/A with fCJD. SCOPE OF REVIEW: MD simulations for the WT PrP and its mutants were performed to explore the underlying dynamic effects of T188 mutations on human PrP. Although the globular domains are fairly conserved, the three mutations have diverse effects on the dynamics properties of PrP, including the shift of H1, the elongation of native beta-sheet and the conversion of S2-H2 loop to a 3(10) helix. MAJOR CONCLUSIONS: Our present study indicates that the three mutants for PrP may undergo different pathogenic mechanisms and the realistic atomistic simulations can provide insights into the effects of disease-associated mutations on PrP dynamics and stability, which can enhance our understanding of how mutations induce the conversion from PrP(C) to PrP(Sc). General significance Our present study helps to understand the effects of T188K/R/A mutations on human PrP: despite the three pathogenic mutations almost do not alter the native structure of PrP, but perturb its stability. This instability may further modulate the oligomerization pathways and determine the features of the PrP(Sc) assemblies.

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Inhibiting effect of alpha(s1)-casein on Abeta(1-40) fibrillogenesis.

Publication Date: 2012 Feb PMID: 22155633
Authors: Carrotta, R. - Canale, C. - Diaspro, A. - Trapani, A. - Biagio, P. L. - Bulone, D.
Journal: Biochim Biophys Acta

BACKGROUND: alpha(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of alpha(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of alpha(s1)-casein on the fibrillogenesis of 1-40 beta-amyloid peptide, involved in Alzheimer's disease. METHODS: The aggregation kinetics of beta-peptide in presence and absence of alpha(s1)-casein was followed under shear at 37 degrees C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of alpha(s1)-casein-Abeta(1-40) interaction. RESULTS AND DISCUSSIONS: alpha(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. alpha(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. GENERAL SIGNIFICANCE: Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.

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The effect of the Asp175Asn and Glu180Gly TPM1 mutations on actin-myosin interaction during the ATPase cycle.

Publication Date: 2012 Feb PMID: 22155441
Authors: Rysev, N. A. - Karpicheva, O. E. - Redwood, C. S. - Borovikov, Y. S.
Journal: Biochim Biophys Acta

Hypertrophic cardiomyopathy (HCM), characterized by cardiac hypertrophy and contractile dysfunction, is a major cause of heart failure. HCM can result from mutations in the gene encoding cardiac alpha-tropomyosin (TM). To understand how the HCM-causing Asp175Asn and Glu180Gly mutations in alpha-tropomyosin affect on actin-myosin interaction during the ATPase cycle, we labeled the SH1 helix of myosin subfragment-1 and the actin subdomain-1 with the fluorescent probe N-iodoacetyl-N'-(5-sulfo-1-naphtylo)ethylenediamine. These proteins were incorporated into ghost muscle fibers and their conformational states were monitored during the ATPase cycle by measuring polarized fluorescence. For the first time, the effect of these alpha-tropomyosins on the mobility and rotation of subdomain-1 of actin and the SH1 helix of myosin subfragment-1 during the ATP hydrolysis cycle have been demonstrated directly by polarized fluorimetry. Wild-type alpha-tropomyosin increases the amplitude of the SH1 helix and subdomain-1 movements during the ATPase cycle, indicating the enhancement of the efficiency of the work of cross-bridges. Both mutant TMs increase the proportion of the strong-binding sub-states, with the effect of the Glu180Gly mutation being greater than that of Asp175Asn. It is suggested that the alteration in the concerted conformational changes of actomyosin is likely to provide the structural basis for the altered cardiac muscle contraction.

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Green leaf divinyl ether synthase: Gene detection, molecular cloning and identification of a unique CYP74B subfamily member.

Publication Date: 2012 Feb PMID: 22155387
Authors: Gogolev, Y. V. - Gorina, S. S. - Gogoleva, N. E. - Toporkova, Y. Y. - Chechetkin, I. R. - Grechkin, A. N.
Journal: Biochim Biophys Acta

Enzymes of the CYP74 family (P450 superfamily) play a key role in the plant lipoxygenase signalling cascade. Recently we detected a pathogen inducible divinyl ether synthase (DES) in flax leaves [Chechetkin, Blufard, Hamberg, Grechkin, 2008]. This prompted us to examine the CYP74 genes in the flax leaf transcriptome. Since the flax genome is not sequenced, we used the PCR approach with degenerate primers related to the conserved domains of selected CYP74 genes; this revealed several CYP74 transcripts in flax leaves. One transcript belongs to the previously described allene oxide synthase (LuAOS, CYP74A, GenBank ID: U00428.1). Another one contains the ORF (1473bp) of an unknown CYP74B16 gene. Three more nearly identical sequences, including one expressed pseudogene, were also identified. The recombinant CYP74B16 protein expressed in Escherichia coli had 491 amino acid residues and MW of 56kDa. The preferred substrate of this enzyme is the 13-hydroperoxide of alpha-linolenic acid, and the reaction product was identified by mass spectrometry, NMR and UV spectroscopy as the divinyl ether (9Z,11E)-12-[(1'Z,3'Z)-hexadienyloxy]-9,11-dodecadienoic acid, (omega5Z)-etherolenic acid. All previously known CYP74B subfamily enzymes are hydroperoxide lyases. The novel flax enzyme CYP74B16 (LuDES) is an unprecedented DES member of the CYP74B subfamily.

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Dynamics of arachidonic acid mobilization by inflammatory cells.

Publication Date: 2012 Feb PMID: 22155285
Authors: Astudillo, A. M. - Balgoma, D. - Balboa, M. A. - Balsinde, J.
Journal: Biochim Biophys Acta

The development of mass spectrometry-based techniques is opening new insights into the understanding of arachidonic acid (AA) metabolism. AA incorporation, remodeling and release are collectively controlled by acyltransferases, phospholipases and transacylases that exquisitely regulate the distribution of AA between the different glycerophospholipid species and its mobilization during cellular stimulation. Traditionally, studies involving phospholipid AA metabolism were conducted by using radioactive precursors and scintillation counting from thin layer chromatography separations that provided only information about lipid classes. Today, the input of lipidomic approaches offers the possibility of characterizing and quantifying specific molecular species with great accuracy and within a biological context associated to protein and/or gene expression in a temporal frame. This review summarizes recent results applying mass spectrometry-based lipidomic approaches to the identification of AA-containing glycerophospholipids, phospholipid AA remodeling and synthesis of oxygenated metabolites.

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Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase.

Publication Date: 2012 Feb PMID: 22155276
Authors: Amini-Bayat, Z. - Hosseinkhani, S. - Jafari, R. - Khajeh, K.
Journal: Biochim Biophys Acta

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D(474) and K(445) accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.

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Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803.

Publication Date: 2012 Feb PMID: 22155275
Authors: Kim, S. G. - Chung, J. S. - Sutton, R. B. - Lee, J. S. - Lopez-Maury, L. - Lee, S. Y. - Florencio, F. J. - Lin, T. - Zabet-Moghaddam, M. - Wood, M. J. - Nayak, K. - Madem, V. - Tripathy, J. N. - Kim, S. K. - Knaff, D. B.
Journal: Biochim Biophys Acta

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E(m)) value of -165mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E(m) value of -220mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8A resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.

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Red antenna states of Photosystem I trimers from Arthrospira platensis revealed by single-molecule spectroscopy.

Publication Date: 2012 Mar PMID: 22155210
Authors: Brecht, M. - Hussels, M. - Schlodder, E. - Karapetyan, N. V.
Journal: Biochim Biophys Acta

Single-molecule fluorescence spectroscopy at 1.4K was used to investigate the spectral properties of red (long-wavelength) chlorophylls in trimeric Photosystem I (PSI) complexes from the cyanobacterium Arthrospira platensis. Three distinct red antenna states could be identified in the fluorescence spectra of single PSI trimers from A. platensis in the presence of oxidized P700. Two of them are responsible for broad emission bands centered at 726 and 760nm. These bands are similar to those found in bulk fluorescence spectra measured at cryogenic temperatures. The broad fluorescence bands at congruent with726 and congruent with760nm belong to individual emitters that are broadened by strong electron-phonon coupling giving rise to a large Stokes-shift of about 20nm and rapid spectral diffusion. An almost perpendicular orientation of the transition dipole moments of F726 and F760 has to be assumed because direct excitation energy transfer does not occur between F726 and F760. For the first time a third red state assigned to the pool absorbing around 708nm could be detected by its zero-phonon lines. The center of the zero-phonon line distribution is found at congruent with714nm. The spectral properties of the three red antenna states show a high similarity to the red antenna states found in trimeric PSI of Thermosynechoccocus elongatus. Based on these findings a similar organization of the red antenna states in PSI of these two cyanobacteria is discussed.

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Enhanced charge-independent mitochondrial free Ca(2+) and attenuated ADP-induced NADH oxidation by isoflurane: Implications for cardioprotection.

Publication Date: 2012 Mar PMID: 22155157
Authors: Agarwal, B. - Camara, A. K. - Stowe, D. F. - Bosnjak, Z. J. - Dash, R. K.
Journal: Biochim Biophys Acta

Modulation of mitochondrial free Ca(2+) ([Ca(2+)](m)) is implicated as one of the possible upstream factors that initiates anesthetic-mediated cardioprotection against ischemia-reperfusion (IR) injury. To unravel possible mechanisms by which volatile anesthetics modulate [Ca(2+)](m) and mitochondrial bioenergetics, with implications for cardioprotection, experiments were conducted to spectrofluorometrically measure concentration-dependent effects of isoflurane (0.5, 1, 1.5, 2mM) on the magnitudes and time-courses of [Ca(2+)](m) and mitochondrial redox state (NADH), membrane potential (DeltaPsi(m)), respiration, and matrix volume. Isolated mitochondria from rat hearts were energized with 10mM Na(+)- or K(+)-pyruvate/malate (NaPM or KPM) or Na(+)-succinate (NaSuc) followed by additions of isoflurane, 0.5mM CaCl(2) ( approximately 200nM free Ca(2+) with 1mM EGTA buffer), and 250muM ADP. Isoflurane stepwise: (a) increased [Ca(2+)](m) in state 2 with NaPM, but not with KPM substrate, despite an isoflurane-induced slight fall in DeltaPsi(m) and a mild matrix expansion, and (b) decreased NADH oxidation, respiration, DeltaPsi(m), and matrix volume in state 3, while prolonging the duration of state 3 NADH oxidation, respiration, DeltaPsi(m), and matrix contraction with PM substrates. These findings suggest that isoflurane's effects are mediated in part at the mitochondrial level: (1) to enhance the net rate of state 2 Ca(2+) uptake by inhibiting the Na(+)/Ca(2+) exchanger (NCE), independent of changes in DeltaPsi(m) and matrix volume, and (2) to decrease the rates of state 3 electron transfer and ADP phosphorylation by inhibiting complex I. These direct effects of isoflurane to increase [Ca(2+)](m), while depressing NCE activity and oxidative phosphorylation, could underlie the mechanisms by which isoflurane provides cardioprotection against IR injury at the mitochondrial level.

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Large enhancement of skeletal muscle cell glucose uptake and suppression of hepatocyte glucose-6-phosphatase activity by weak uncouplers of oxidative phosphorylation.

Publication Date: 2012 Feb PMID: 22155143
Authors: Martineau, L. C.
Journal: Biochim Biophys Acta

BACKGROUND: Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects. METHODS: The reference weak uncoupler 2,4-dinitrophenol, fourteen naturally-occurring phenolic compounds identified as uncouplers in isolated rat liver mitochondria, and fourteen related compounds with little or no uncoupling activity were tested for enhancement of glucose uptake in differentiated C2C12 skeletal muscle cells following 18h of treatment at 25-100muM. A subset of compounds were tested for suppression of glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes following 16h at 12.5-25muM. Metformin (400muM) was used as a standard in both assays. RESULTS: Dinitrophenol and nine of eleven compounds that induced 50% or more uncoupling at 100muM in isolated mitochondria enhanced basal glucose uptake by 53 to 269%; the effect of the 4'-hydroxychalcone butein was more than 6-fold that of metformin; negative control compounds increased uptake by no more than 25%. Dinitrophenol and four 4'-hydroxychalconoids also suppressed hepatocyte G6Pase as well as, or more effectively than metformin, whereas the unsubstituted parent compound chalcone, devoid of uncoupling activity, had no effect. CONCLUSIONS: Activities key to glycemic control can be induced by a wide range of weak uncouplers, including compounds free of difficult-to-metabolize groups typically associated with uncouplers. GENERAL SIGNIFICANCE: Uncoupling represents a valid and possibly more efficient alternative to inhibition for triggering cytoprotective effects of therapeutic relevance to insulin resistance in both muscle and liver. Identification of actives of natural origin and the insights into their structure-activity relationship reported herein may lead to alternatives to metformin.

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Trichostatin A and sirtinol suppressed survivin expression through AMPK and p38MAPK in HT29 colon cancer cells.

Publication Date: 2012 Feb PMID: 22155142
Authors: Hsu, Y. F. - Sheu, J. R. - Lin, C. H. - Yang, D. S. - Hsiao, G. - Ou, G. - Chiu, P. T. - Huang, Y. H. - Kuo, W. H. - Hsu, M. J.
Journal: Biochim Biophys Acta

BACKGROUND: Elevated levels of survivin and histone deacetylases (HDACs) are often found over-expressed in human cancers, including colorectal cancer, and have been implicated in tumorigenesis. HDAC inhibition induces growth arrest and cell death in various transformed cell; however, the mechanisms by which this reduces cell viability in colorectal cancer cells remain unexplained. METHODS: We explored the actions of two HDAC inhibitors, trichostatin A (TSA) and sirtinol, in HT29 colon cancer cells. RESULTS: TSA and sirtinol induced apoptosis and inhibited cell proliferation in HT29 cells. These results are associated with the modulation of survivin. Survivin promoter luciferase activity and Sp1, a transcription factor that contributes to survivin expression, were suppressed in cells exposed to TSA or sirtinol. TSA and sirtinol also activated p38 mitogen-activated protein kinase (p38MAPK) and AMP-activated protein kinase (AMPK). Inhibitors of p38MAPK or AMPK signaling abrogated TSA and sirtinol's effects of decreasing cell viability. Survivin promoter luciferase activity in the presence of TSA or sirtinol was restored by AMPK dominant negative mutant or p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p63 binding to the promoter region increased after TSA or sirtinol exposure. CONCLUSIONS: We report a p38MAPK- and AMPK-mediated downregulation of survivin, and its functional correlation with decreased colon cancer cell viability in the presence of HDAC inhibitor. p63 and Sp1 may also contribute to TSA and sirtinol actions. GENERAL SIGNIFICANCE: This study delineates, in part, the underlying mechanisms of TSA and sirtinol in decreasing survivin expression and subsequent colon cancer cell viability.

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Structural characterization of the RNA chaperone Hfq from the nitrogen-fixing bacterium Herbaspirillum seropedicae SmR1.

Publication Date: 2012 Feb PMID: 22154803
Authors: Kadowaki, M. A. - Iulek, J. - Barbosa, J. A. - Pedrosa Fde, O. - de Souza, E. M. - Chubatsu, L. S. - Monteiro, R. A. - de Oliveira, M. A. - Steffens, M. B.
Journal: Biochim Biophys Acta

The RNA chaperone Hfq is a homohexamer protein identified as an E. coli host factor involved in phage Qbeta replication and it is an important posttranscriptional regulator of several types of RNA, affecting a plethora of bacterial functions. Although twenty Hfq crystal structures have already been reported in the Protein Data Bank (PDB), new insights into these protein structures can still be discussed. In this work, the structure of Hfq from the beta-proteobacterium Herbaspirillum seropedicae, a diazotroph associated with economically important agricultural crops, was determined by X-ray crystallography and small-angle X-ray scattering (SAXS). Biochemical assays such as exclusion chromatography and RNA-binding by the electrophoretic shift assay (EMSA) confirmed that the purified protein is homogeneous and active. The crystal structure revealed a conserved Sm topology, composed of one N-terminal alpha-helix followed by five twisted beta-strands, and a novel pi-pi stacking intra-subunit interaction of two histidine residues, absent in other Hfq proteins. Moreover, the calculated ab initio envelope based on small-angle X-ray scattering (SAXS) data agreed with the Hfq crystal structure, suggesting that the protein has the same folding structure in solution.

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Effect of insulin deprivation on metabolism and metabolism-associated gene transcript levels of in vitro cultured human Sertoli cells.

Publication Date: 2012 Feb PMID: 22146232
Authors: Oliveira, P. F. - Alves, M. G. - Rato, L. - Laurentino, S. - Silva, J. - Sa, R. - Barros, A. - Sousa, M. - Carvalho, R. A. - Cavaco, J. E. - Socorro, S.
Journal: Biochim Biophys Acta

BACKGROUND: Sertoli cells metabolize glucose producing lactate for developing germ cells. As insulin regulates glucose uptake and its disturbance/insensitivity is associated with diabetes mellitus, we aimed to determine the effect of insulin deprivation in human Sertoli cell (hSC) metabolism and metabolism-associated gene expression. METHODS: hSC-enriched primary cultures were maintained in the absence/presence of insulin and metabolite variations were determined by (1)H-NMR. mRNA expression levels of glucose transporters (GLUT1, GLUT3), lactate dehydrogenase (LDHA) and monocarboxylate transporter (MCT4) were determined by RT-PCR. RESULTS: Insulin deprivation resulted in decreased lactate production and in decrease of glucose consumption that was completely reverted after 6h. Cells of both groups consumed similar amounts of glucose. In insulin-deprived cells, transcript levels of genes associated to lactate metabolism (LDHA and MCT4) were decreased. Transcript levels of genes involved in glucose uptake exhibited a divergent variation: GLUT3 levels were decreased while GLUT1 levels increased. Insulin-deprived hSCs presented: 1) altered glucose consumption and lactate secretion; 2) altered expression of metabolism-associated genes involved in lactate production and export; 3) an adaptation of glucose uptake by modulating the expression of GLUT1 and GLUT3. GENERAL SIGNIFICANCE: This is the first report regarding the effect of insulin-deprivation on hSC metabolism.

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Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold.

Publication Date: 2012 Feb PMID: 22138634
Authors: Fernandez, F. J. - de Vries, D. - Pena-Soler, E. - Coll, M. - Christen, P. - Gehring, H. - Vega, M. C.
Journal: Biochim Biophys Acta

The joint substitution of three active-site residues in Escherichia colil-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the alpha-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for beta-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate beta-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.

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Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress.

Publication Date: 2012 Feb PMID: 22138629
Authors: Tamary, E. - Kiss, V. - Nevo, R. - Adam, Z. - Bernat, G. - Rexroth, S. - Rogner, M. - Reich, Z.
Journal: Biochim Biophys Acta

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.

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During the stationary growth phase, Yarrowia lipolytica prevents the overproduction of reactive oxygen species by activating an uncoupled mitochondrial respiratory pathway.

Publication Date: 2012 Feb PMID: 22138628
Authors: Guerrero-Castillo, S. - Cabrera-Orefice, A. - Vazquez-Acevedo, M. - Gonzalez-Halphen, D. - Uribe-Carvajal, S.
Journal: Biochim Biophys Acta

In the branched mitochondrial respiratory chain from Yarrowia lipolytica there are two alternative oxido-reductases that do not pump protons, namely an external type II NADH dehydrogenase (NDH2e) and the alternative oxidase (AOX). Direct electron transfer between these proteins is not coupled to ATP synthesis and should be avoided in most physiological conditions. However, under low energy-requiring conditions an uncoupled high rate of oxygen consumption would be beneficial, as it would prevent overproduction of reactive oxygen species (ROS). In mitochondria from high energy-requiring, logarithmic-growth phase cells, most NDH2e was associated to cytochrome c oxidase and electrons from NADH were channeled to the cytochromic pathway. In contrast, in the low energy requiring, late stationary-growth phase, complex IV concentration decreased, the cells overexpressed NDH2e and thus a large fraction of this enzyme was found in a non-associated form. Also, the NDH2e-AOX uncoupled pathway was activated and the state IV external NADH-dependent production of ROS decreased. Association/dissociation of NDH2e to/from complex IV is proposed to be the switch that channels electrons from external NADH to the coupled cytochrome pathway or allows them to reach an uncoupled, alternative, DeltaPsi-independent pathway.

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Biogenesis of the cytochrome bc(1) complex and role of assembly factors.

Publication Date: 2012 Feb PMID: 22138626
Authors: Smith, P. M. - Fox, J. L. - Winge, D. R.
Journal: Biochim Biophys Acta

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

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Microphthalmia-associated transcription factor is required for mature myotube formation.

Publication Date: 2012 Feb PMID: 22138449
Authors: Ooishi, R. - Shirai, M. - Funaba, M. - Murakami, M.
Journal: Biochim Biophys Acta

BACKGROUND: The roles of microphthalmia-associated transcription factor (Mitf) in the skeletal muscle and during myogenesis are unclear. METHODS: Expression of Mitf in mouse tissues and during myogenesis was evaluated. Effects of Mitf knockdown on myogenesis and gene expression related to myogenesis were subsequently explored. Furthermore, effects of p21, a cyclin-dependent kinase inhibitor, and integrin alpha9 (Itga9) were examined. RESULTS: Mitf was highly expressed in the skeletal muscle; Mitf-A and -J were expressed. Mitf expression increased after differentiation stimulation in C2C12 myogenic cells. Down-regulation of Mitf expression by transfection of siRNA for common Mitf inhibited myotube formation, which was reproduced by Mitf-A knockdown. Morphometric analyses indicated that both multinucleated cell number and the proportion of myotubes with more than 6 nuclei were decreased in Mitf-knockdown cells, suggesting that Mitf is required for not only the formation of nascent myotubes but also their maturation. Searching for genes positively regulated by Mitf revealed p21 and Itga9; decreasing Mitf expression inhibited up-regulation of p21 expression after differentiation stimulation and blocked the induction of Itga9 expression in response to differentiation. Knockdown of p21 decreased the number of multinucleated cells, whereas Itga9 knockdown did not affect the myotube number. Both p21 knockdown and Itga9 knockdown decreased the proportion of myotubes with more than 6 nuclei. GENERAL SIGNIFICANCE: Mitf positively regulates skeletal muscle formation; Mitf is significantly expressed during myogenesis, and is required for efficient myotube formation through expression of p21 and Itga9.

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Atomistic models of ion and solute transport by the sodium-dependent secondary active transporters.

Publication Date: 2012 Feb PMID: 22138368
Authors: Zdravkovic, I. - Zhao, C. - Lev, B. - Cuervo, J. E. - Noskov, S. Y.
Journal: Biochim Biophys Acta

The recent determination of high-resolution crystal structures of several transporters offers unprecedented insights into the structural mechanisms behind secondary transport. These proteins utilize the facilitated diffusion of the ions down their electrochemical gradients to transport the substrate against its concentration gradient. The structural studies revealed striking similarities in the structural organization of ion and solute binding sites and a well-conserved inverted-repeat topology between proteins from several gene families. In this paper we will overview recent atomistic simulations applied to study the mechanisms of selective binding of ion and substrate in LeuT, Glt, vSGLT and hSERT as well as its consequences for the transporter conformational dynamics. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Delta(12,14)-PGJ(2)-dependent mechanism.

Publication Date: 2012 Feb PMID: 22138303
Authors: Gates, D. - Dollin, K. - Connolly, R. - Young, I. - Powell, L. - McEneny, J. - Gleave, M. - McGinty, A.
Journal: Biochim Biophys Acta

Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-kappaB signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-kappaB signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), but not PGE(2), and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ(2), heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)gamma, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-alpha-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-kappaB signalling, basal levels of IkappaB were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-IkappaB was obtained. Moreover, while PCXII cells exhibited reduced NF-kappaB transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ(2)-dependent manner, and via activation of HSF-1 and PPARgamma. However, the results do not support a model whereby Cox-2/15d-PGJ(2)-dependent inhibition of NF-kappaB signalling involves clusterin.

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Preferential interactions between ApoE-containing lipoproteins and Abeta revealed by a detection method that combines size exclusion chromatography with non-reducing gel-shift.

Publication Date: 2012 Feb PMID: 22138302
Authors: Ladu, M. J. - Munson, G. W. - Jungbauer, L. - Getz, G. S. - Reardon, C. A. - Tai, L. M. - Yu, C.
Journal: Biochim Biophys Acta

The association between apolipoprotein E (apoE) and amyloid-beta peptide (Abeta) may significantly impact the function of both proteins, thus affecting the etiology of Alzheimer's disease (AD). However, apoE/Abeta interactions remain fundamentally defined by the stringency of the detection method. Here we use size exclusion chromatography (SEC) as a non-stringent approach to the detection of apoE/Abeta interactions in solution, specifically apoE and both endogenous and exogenous Abeta from plasma, CSF and astrocyte conditioned media. By SEC analysis, Abeta association with plasma and CNS lipoproteins is apoE-dependent. While endogenous Abeta elutes to specific human plasma lipoproteins distinct from those containing apoE, it is the apoE-containing lipoproteins that absorb excess amounts of exogenous Abeta40. In human CSF, apoE, endogenous Abeta and phospholipid elute in an almost identical profile, as do apoE, exogenous Abeta and phospholipid from astrocyte conditioned media. Combining SEC fractionation with subsequent analysis for SDS-stable apoE/Abeta complex reveals that apoE-containing astrocyte lipoproteins exhibit the most robust interactions with Abeta. Thus, standardization of the methods for detecting apoE/Abeta complex is necessary to determine its functional significance in the neuropathology characteristic of AD. Importantly, a systematic understanding of the role of apoE-containing plasma and CNS lipoproteins in Abeta homeostasis could potentially contribute to identifying a plasma biomarker currently over-looked because it has multiple components.

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Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes.

Publication Date: 2012 Feb PMID: 22138128
Authors: Li, T. - Hu, J. - He, G. H. - Li, Y. - Zhu, C. C. - Hou, W. G. - Zhang, S. - Li, W. - Zhang, J. S. - Wang, Z. - Liu, X. P. - Yao, L. B. - Zhang, Y. Q.
Journal: Biochim Biophys Acta

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-kappaB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-kappaB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-kappaB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.

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Localization of nerve growth factor (NGF) receptors in the mitochondrial compartment: Characterization and putative role.

Publication Date: 2012 Feb PMID: 22138126
Authors: Carito, V. - Pingitore, A. - Cione, E. - Perrotta, I. - Mancuso, D. - Russo, A. - Genchi, G. - Caroleo, M. C.
Journal: Biochim Biophys Acta

BACKGROUND: The neurotrophin NGF receptors trkA and p75NTR are expressed in the central and peripheral nervous system as well as in non-neuronal tissues; originally described to localize to the plasma membrane, recent studies have suggested other intracellular localizations for both NGF receptors. SCOPE OF REVIEW: In order to determine whether NGF receptors localize to the mitochondrial compartment mitochondria isolated from human kidney, rat tissues and a human podocyte as cell line before and after differentiation were used. MAJOR CONCLUSIONS: Our results demonstrate that NGF receptors are localized in the mitochondrial compartment of undifferentiated human podocytes and in all tissues analyzed including rat central nervous system. In mitochondria p75NTR, but not trkA, co-immunoprecipitates with the adenine nucleotide translocator (ANT) and the phosphodiesterase 4 isoform A5 (PDE4A5). Moreover, NGF, via trkA, protects isolated mitochondria of rat brain cortex from mitochondrial permeability transition induced by Ca(2+). GENERAL SIGNIFICANCE: Although NGF receptors have been described as mainly citoplasmatic so far, we proved evidence of their expression at the mitochondrial level and their interaction with specific proteins. Our results demonstrating the expression of NGF receptors in the mitochondria provide new insights into the role of NGF at subcellular level, in different areas of the organism, including CNS.

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Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416.

Publication Date: 2012 Feb PMID: 22137889
Authors: Hornig, M. - Markoutsa, S. - Hafner, A. K. - George, S. - Wisniewska, J. M. - Rodl, C. B. - Hofmann, B. - Maier, T. - Karas, M. - Werz, O. - Steinhilber, D.
Journal: Biochim Biophys Acta

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.

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Autoantibodies in primary Sjogren's syndrome patients induce internalization of muscarinic type 3 receptors.

Publication Date: 2012 Feb PMID: 22137887
Authors: Jin, M. - Hwang, S. M. - Davies, A. J. - Shin, Y. - Bae, J. S. - Lee, J. H. - Lee, E. B. - Song, Y. W. - Park, K.
Journal: Biochim Biophys Acta

Objectives. Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial. Methods. Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) was measured by microspectrofluorimetry. Results. Incubation of hSMG cells with pSS IgG (1mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca(2+)](i) transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R. Conclusion. pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.

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Electrogenic events upon photolysis of CO from fully reduced cytochrome c oxidase.

Publication Date: 2012 Feb PMID: 22133637
Authors: Rintanen, M. - Belevich, I. - Verkhovsky, M. I.
Journal: Biochim Biophys Acta

CO photolysis from fully reduced Paracoccus denitrificans aa(3)-type cytochrome c oxidase in the absence of O(2) was studied by time-resolved potential electrometry. Surprisingly, photo dissociation of the uncharged carbon monoxide results in generation of a small-amplitude electric potential with the same sign as the physiological charge separation during activity. The number of electrogenic events after CO photolysis depends on the state of the enzyme. CO photolysis following immediately after activation by an enzymatic turnover, showed a two-component potential development. A fast (~1.5mus) phase was followed by slower potential generation with a time constant varying from 8mus at pH 7 to 250mus at pH 10. The amplitude of the fast phase was independent of the time of incubation after enzyme activation, whereas the slower phase vanished with a time constant of ~25min. CO photolysis from enzyme that had not undergone a prior single turnover showed the fast phase, but the amplitude of the slow phase was reduced to 10-30%. The amplitude of the fast phase corresponds to charge movement of 0.83A perpendicular to the membrane dielectric, and is independent of the time after enzyme activation. Thus it can be used as an internal ruler for normalization of the electrogenic responses of CcO. The slow phase was absent in the K354M mutant with a blocked proton-conducting K channel. We propose that CO photolysis increases the pK of the K354 residue, which results in its partial protonation, and generation of electric potential.

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Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

Publication Date: 2012 Feb PMID: 22133636
Authors: Rexroth, S. - Poetsch, A. - Rogner, M. - Hamann, A. - Werner, A. - Osiewacz, H. D. - Schafer, E. R. - Seelert, H. - Dencher, N. A.
Journal: Biochim Biophys Acta

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimer's and Parkinson's Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

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Lysosomal vitamin E accumulation in Niemann-Pick type C disease.

Publication Date: 2012 Feb PMID: 22120593
Authors: Yevenes, L. F. - Klein, A. - Castro, J. F. - Marin, T. - Leal, N. - Leighton, F. - Alvarez, A. R. - Zanlungo, S.
Journal: Biochim Biophys Acta

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (alpha-tocopherol, alpha-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of alpha-TOH in NPC cells. We observed accumulation and entrapment of alpha-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, alpha-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the alpha-TOH transporter, alpha-Tocopherol Transfer Protein (alpha-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic alpha-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to alpha-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.

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Acetylation of alphaA-crystallin in the human lens: Effects on structure and chaperone function.

Publication Date: 2012 Feb PMID: 22120592
Authors: Nagaraj, R. H. - Nahomi, R. B. - Shanthakumar, S. - Linetsky, M. - Padmanabha, S. - Pasupuleti, N. - Wang, B. - Santhoshkumar, P. - Panda, A. K. - Biswas, A.
Journal: Biochim Biophys Acta

alpha-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including alphaA-crystallin are acetylated in vivo. We found that K70 and K99 in alphaA-crystallin and, K92 and K166 in alphaB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, alphaA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(epsilon)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated alphaA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in alphaA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against gamma-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of alphaA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.

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Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype.

Publication Date: 2012 Feb PMID: 22119597
Authors: Jandova, J. - Shi, M. - Norman, K. G. - Stricklin, G. P. - Sligh, J. E.
Journal: Biochim Biophys Acta

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse rho(0) cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.

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Impaired hepatic function and central dopaminergic denervation in a rodent model of Parkinson's disease: A self-perpetuating crosstalk?

Publication Date: 2012 Feb PMID: 22119596
Authors: Vairetti, M. - Ferrigno, A. - Rizzo, V. - Ambrosi, G. - Bianchi, A. - Richelmi, P. - Blandini, F. - Armentero, M. T.
Journal: Biochim Biophys Acta

In Parkinson's disease (PD), aside from the central lesion, involvement of visceral organs has been proposed as part of the complex clinical picture of the disease. The issue is still poorly understood and relatively unexplored. In this study we used a classic rodent model of nigrostriatal degeneration, induced by the intrastriatal injection of 6-hydroxydopamine (6-OHDA), to investigate whether and how a PD-like central dopaminergic denervation may influence hepatic functions. Rats received an intrastriatal injection of 6-OHDA or saline (sham), and blood, cerebrospinal fluid, liver and brain samples were obtained for up to 8weeks after surgery. Specimens were analyzed for changes in cytokine and thyroid hormone levels, as well as liver mitochondrial alterations. Hepatic mitochondria isolated from animals bearing extended nigrostriatal lesion displayed increased ROS production, while membrane potential (DeltaPsi) and ATP production were significantly decreased. Reduced ATP production correlated with nigral neuronal loss. Thyroid hormone levels were significantly increased in serum of PD rats compared to sham animals while steady expression of selected cytokines was detected in all groups. Hepatic enzyme functions were comparable in all animals. Our study indicates for the first time that in a rodent model of PD, hepatic mitochondria dysfunctions arise as a consequence of nigrostriatal degeneration, and that thyroid hormone represents a key interface in this CNS-liver interaction. Liver plays a fundamental detoxifying function and a better understanding of PD-related hepatic mitochondrial alterations, which might further promote neurodegeneration, may represent an important step for the development of novel therapeutic strategies.

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The use of PIB-PET as a dual pathological and functional biomarker in AD.

Publication Date: 2012 Mar PMID: 22115832
Authors: Forsberg, A. - Engler, H. - Blomquist, G. - Langstrom, B. - Nordberg, A.
Journal: Biochim Biophys Acta

Amyloid imaging with positron emission tomography (PET) is presently used in Alzheimer's disease (AD) research. In this study we investigated the possibility to use early frames (ePIB) of the PIB scans as a rough index of CBF by comparing normalised early PIB values with cerebral glucose metabolism (rCMRglc). PIB-PET and FDG-PET were performed in 37 AD patients, 21 subjects with mild cognitive impairment (MCI) and 6 healthy controls (HC). The patients were divided based on their PIB retention (amyloid load) as either PIB positive (PIB+) or PIB negative (PIB-). Data of the unidirectional influx K(1) from a subset of the subjects including 7 AD patients and 3 HC was used for correlative analysis. Data was analysed using regions of interest (ROI) analysis. A strong, positive correlation was observed across brain regions between K(1) and ePIB (r=0.70; p
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Structure-based discovery of anti-influenza virus A compounds among medicines.

Publication Date: 2012 Feb PMID: 22108550
Authors: Fukuoka, M. - Minakuchi, M. - Kawaguchi, A. - Nagata, K. - Kamatari, Y. O. - Kuwata, K.
Journal: Biochim Biophys Acta

BACKGROUND: Influenza A virus (IAV) infection is nowadays a major public health concern, in particular since the 2009 H1N1 flu pandemic. The outbreak of IAV strains resistant to currently available drugs, such as oseltamivir or zanamivir targeting the neuraminidase, is a real threat for humans as well as for animals. Thus the development of anti-IAV drugs with a novel action mechanism may be an urgent theme. METHODS: We performed a docking simulation targeting the interface of PA interacting with PB1 using a drug database including ~4000 compounds. We then conducted cell viability assay and plaque assay using IAV/WSN/33. Finally we examined their anti-IAV mechanism by surface plasmon resonance and IAV replicon assay. RESULTS: We found that benzbromarone, diclazuril, and trenbolone acetate had strong anti-IAV activities. We confirmed that benzbromarone and diclazuril bound with PA subunit, and decreased the transcriptional activity of the viral RNA polymerase. CONCLUSIONS: Benzbromarone and diclazuril had strong anti-IAV activities with novel action mechanism, i.e. inhibition of viral RNA polymerase. GENERAL SIGNIFICANCE: Since benzbromarone and diclazuril are already used in public as medicines, these could be the candidates for alternatives in case of an outbreak of IAV which is resistant to pre-existing anti-IAV drugs.

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PET amyloid-beta imaging in preclinical Alzheimer's disease.

Publication Date: 2012 Mar PMID: 22108203
Authors: Vlassenko, A. G. - Benzinger, T. L. - Morris, J. C.
Journal: Biochim Biophys Acta

Alzheimer's disease (AD) is the leading cause of dementia, accounting for 60-70% of all cases [Hebert et al., 2003, 1]. The need for effective therapies for AD is great. Current approaches, including cholinesterase inhibitors and N-methyl-d-aspartate (NMDA) receptor antagonists, are symptomatic treatments for AD but do not prevent disease progression. Many diagnostic and therapeutic approaches to AD are currently changing due to the knowledge that underlying pathology starts 10 to 20years before clinical signs of dementia appear [Holtzman et al., 2011, 2]. New therapies which focus on prevention or delay of the onset or cognitive symptoms are needed. Recent advances in the identification of AD biomarkers now make it possible to detect AD pathology in the preclinical stage of the disease, in cognitively normal (CN) individuals; this biomarker data should be used in the selection of high-risk populations for clinical trials. In vivo visualization of AD neuropathology and biological, biochemical or physiological confirmation of the effects of treatment likely will substantially improve development of novel pharmaceuticals. Positron emission tomography (PET) is the leading neuroimaging tool to detect and provide quantitative measures of AD amyloid pathology in vivo at the early stages and follow its course longitudinally. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Mechanism of structural transformations induced by antimicrobial peptides in lipid membranes.

Publication Date: 2012 Feb PMID: 22100601
Authors: Lam, K. L. - Wang, H. - Siaw, T. A. - Chapman, M. R. - Waring, A. J. - Kindt, J. T. - Lee, K. Y.
Journal: Biochim Biophys Acta

It has long been suggested that pore formation is responsible for the increase in membrane permeability by antimicrobial peptides (AMPs). To better understand the mechanism of AMP activity, the disruption of model membrane by protegrin-1 (PG-1), a cationic antimicrobial peptide, was studied using atomic force microscopy. We present here the direct visualization of the full range of structural transformations in supported lipid bilayer patches induced by PG-1 on zwitterionic 1,2-dimyristoyl-snglycero-phospho-choline (DMPC) membranes. When PG-1 is added to DMPC, the peptide first induces edge instability at low concentrations, then pore-like surface defects at intermediate concentrations, and finally wormlike structures with a specific length scale at high concentrations. The formation of these structures can be understood using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent. Atomistic molecular dynamics simulations on lipid bilayer ribbons with PG-1 molecules placed at the edge or interior positions are carried out to calculate the effect of PG-1 in reducing line tension. Further investigation of the placement of PG-1 and its association with defects in the bilayer is carried out using unbiased assembly of a PG-1 containing bilayer from a random mixture of PG-1, DMPC, and water. A generalized model of AMP induced structural transformations is also presented in this work. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Stabilization and modulation of the phycobilisome by calcium in the calciphilic freshwater red alga Bangia atropurpurea.

Publication Date: 2012 Feb PMID: 22093772
Authors: Yokono, M. - Uchida, H. - Suzawa, Y. - Akiomoto, S. - Murakami, A.
Journal: Biochim Biophys Acta

The bangiophycean filamentous red alga Bangia atropurpurea is distributed in freshwater habitats such as littoral and splash zones of lakes or rapid currents distant from the sea. In these habitats, the distribution and growth of this alga appear to be related to hard water rich in calcium ions. To characterize the eco-physiological properties of this calciphilic red alga, we examined the effects of long-term and short-term Ca(2+) depletion on photosynthetic growth of the thallus and on the phycobilisome. Long-term culture experiments suggested that higher Ca(2+) concentrations (>50mgL(-1)) were required to sustain thallus growth and pigmentation of cells. In short-term Ca(2+)-depletion treatments, fluorescence derived from phycoerythrin (PE) fluctuated, although the absorption spectra of the thalli did not change. After 30min of Ca(2+) depletion, the fluorescence lifetime of PE became markedly longer, indicating that the energy transfer from PE to phycocyanin (PC) was suppressed. The fluorescence lifetime of PE returned to its original value within a short time after 4h of Ca(2+) depletion, however, energy transfer from PE to PC was still suppressed. This suggested that the excitation energy absorbed by PE was quenched during prolonged Ca(2+) depletion. The efficient energy transfer from PC and allophycocyanin were unchanged during these treatments.

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The effects of idebenone on mitochondrial bioenergetics.

Publication Date: 2012 Feb PMID: 22086148
Authors: Giorgio, V. - Petronilli, V. - Ghelli, A. - Carelli, V. - Rugolo, M. - Lenaz, G. - Bernardi, P.
Journal: Biochim Biophys Acta

We have studied the effects of idebenone on mitochondrial function in cybrids derived from one normal donor (HQB17) and one patient harboring the G3460A/MT-ND1 mutation of Leber's Hereditary Optic Neuropathy (RJ206); and in XTC.UC1 cells bearing a premature stop codon at aminoacid 101 of MT-ND1 that hampers complex I assembly. Addition of idebenone to HQB17 cells caused mitochondrial depolarization and NADH depletion, which were inhibited by cyclosporin (Cs) A and decylubiquinone, suggesting an involvement of the permeability transition pore (PTP). On the other hand, addition of dithiothreitol together with idebenone did not cause PTP opening and allowed maintenance of the mitochondrial membrane potential even in the presence of rotenone. Addition of dithiothreitol plus idebenone, or of idebenol, to HQB17, RJ206 and XTC.UC1 cells sustained membrane potential in intact cells and ATP synthesis in permeabilized cells even in the presence of rotenone and malonate, and restored a good level of coupled respiration in complex I-deficient XTC.UC1 cells. These findings demonstrate that idebenol can feed electrons at complex III. If the quinone is maintained in the reduced state, a task that in some cell types appears to be performed by dicoumarol-sensitive NAD(P)H:quinone oxidoreductase 1 [Haefeli et al. (2011) PLoS One 6, e17963], electron transfer to complex III may allow reoxidation of NADH in complex I deficiencies.

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The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A).

Publication Date: 2012 Feb PMID: 22085909
Authors: Mason, J. M. - Bendall, D. S. - Howe, C. J. - Worrall, J. A.
Journal: Biochim Biophys Acta

Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core alpha-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.

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Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy.

Publication Date: 2012 Feb PMID: 22085605
Authors: Biancini, G. B. - Vanzin, C. S. - Rodrigues, D. B. - Deon, M. - Ribas, G. S. - Barschak, A. G. - Manfredini, V. - Netto, C. B. - Jardim, L. B. - Giugliani, R. - Vargas, C. R.
Journal: Biochim Biophys Acta

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of alpha-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-alpha were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.

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Mutant huntingtin, abnormal mitochondrial dynamics, defective axonal transport of mitochondria, and selective synaptic degeneration in Huntington's disease.

Publication Date: 2012 Feb PMID: 22080977
Authors: Reddy, P. H. - Shirendeb, U. P.
Journal: Biochim Biophys Acta

Huntington's disease (HD) is a progressive, fatal neurodegenerative disease caused by expanded polyglutamine repeats in the HD gene. HD is characterized by chorea, seizures, involuntary movements, dystonia, cognitive decline, intellectual impairment and emotional disturbances. Research into mutant huntingtin (Htt) and mitochondria has found that mutant Htt interacts with the mitochondrial protein dynamin-related protein 1 (Drp1), enhances GTPase Drp1 enzymatic activity, and causes excessive mitochondrial fragmentation and abnormal distribution, leading to defective axonal transport of mitochondria and selective synaptic degeneration. This article summarizes latest developments in HD research and focuses on the role of abnormal mitochondrial dynamics and defective axonal transport in HD neurons. This article also discusses the therapeutic strategies that decrease mitochondrial fragmentation and neuronal damage in HD.

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Cross-species investigation of the functions of the Rhodobacter PufX polypeptide and the composition of the RC-LH1 core complex.

Publication Date: 2012 Feb PMID: 22079525
Authors: Crouch, L. I. - Jones, M. R.
Journal: Biochim Biophys Acta

In well-characterised species of the Rhodobacter (Rba.) genus of purple photosynthetic bacteria it is known that the photochemical reaction centre (RC) is intimately-associated with an encircling LH1 antenna pigment protein, and this LH1 antenna is prevented from completely surrounding the RC by a single copy of the PufX protein. In Rba. veldkampii only monomeric RC-LH1 complexes are assembled in the photosynthetic membrane, whereas in Rba. sphaeroides and Rba. blasticus a dimeric form is also assembled in which two RCs are surrounded by an S-shaped LH1 antenna. The present work established that dimeric RC-LH1 complexes can also be isolated from Rba. azotoformans and Rba. changlensis, but not from Rba. capsulatus or Rba. vinaykumarii. The compositions of the monomers and dimers isolated from these four species of Rhodobacter were similar to those of the well-characterised RC-LH1 complexes present in Rba. sphaeroides. Pigment proteins were also isolated from strains of Rba. sphaeroides expressing chimeric RC-LH1 complexes. Replacement of either the Rba. sphaeroides LH1 antenna or PufX with its counterpart from Rba. capsulatus led to a loss of the dimeric form of the RC-LH1 complex, but the monomeric form had a largely unaltered composition, even in strains in which the expression level of LH1 relative to the RC was reduced. The chimeric RC-LH1 complexes were also functional, supporting bacterial growth under photosynthetic conditions. The findings help to tease apart the different functions of PufX in different species of Rhodobacter, and a specific protein structural arrangement that allows PufX to fulfil these three functions is proposed.

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The ratio of phosphatidylcholine to phosphatidylethanolamine does not predict integrity of growing MT58 Chinese hamster ovary cells.

Publication Date: 2012 Feb PMID: 22079326
Authors: Niebergall, L. J. - Vance, D. E.
Journal: Biochim Biophys Acta

Phosphatidylcholine (PC) homeostasis is important for maintaining cellular growth and survival. Cellular growth and apoptosis may also be influenced by the PC to phosphatidylethanolamine (PE) ratio as a reduction in this ratio can result in a loss of membrane integrity. To investigate whether a reduced PC:PE ratio influences cellular growth and apoptosis, we utilized the MT58 cell line, which contains a thermo-sensitive mutation in CTP:phosphocholine cytidylyltransferase-alpha, the rate-limiting enzyme for PC biosynthesis. Incubation of MT58 cells at the restrictive temperature of 41 degrees C results in a reduction of cellular PC and induces apoptosis. Furthermore, MT58 cells have a 50% reduction in the PC:PE ratio when incubated at 41 degrees C. In an attempt to normalize the PC:PE ratio, which may stabilize cellular membranes and rescue MT58 cells from apoptosis, the cells were treated with either silencing RNA to impair PE biosynthesis or lysophosphatidylcholine to increase PC mass. Impairing PE biosynthesis in MT58 cells reduced cellular PE and PC concentrations by 30% and 20%, but did not normalize the PC:PE ratio. Loss of both phospholipids enhanced the onset of apoptosis in MT58 cells. Lysophosphatidylcholine normalized cellular PC, increased PE mass by 10%, restored cellular growth and prevented apoptosis of MT58 cells without normalizing the PC:PE ratio. Furthermore, total amount of cellular PC and PE, but not the PC:PE ratio, correlated with cellular growth (R(2)=0.76), and inversely with cellular apoptosis (R(2)=0.97). These data suggest the total cellular amount of PC and PE, not the PC:PE ratio, influences growth and membrane integrity of MT58 cells.

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LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1.

Publication Date: 2012 Feb PMID: 22079202
Authors: Patsi, J. - Maliniemi, P. - Pakanen, S. - Hinttala, R. - Uusimaa, J. - Majamaa, K. - Nystrom, T. - Kervinen, M. - Hassinen, I. E.
Journal: Biochim Biophys Acta

Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.

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Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages.

Publication Date: 2012 Feb PMID: 22074701
Authors: Fournier, N. - Attia, N. - Rousseau-Ralliard, D. - Vedie, B. - Destaillats, F. - Grynberg, A. - Paul, J. L.
Journal: Biochim Biophys Acta

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34h, 70muM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (-36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA+polyunsaturated FA):(saturated FA+TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.

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Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone.

Publication Date: 2012 Feb PMID: 22064122
Authors: Andreasen, M. - Nielsen, S. B. - Mittag, T. - Bjerring, M. - Nielsen, J. T. - Zhang, S. - Nielsen, E. H. - Jeppesen, M. - Christiansen, G. - Besenbacher, F. - Dong, M. - Nielsen, N. C. - Skrydstrup, T. - Otzen, D. E.
Journal: Biochim Biophys Acta

The well-ordered cross beta-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form beta-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. beta-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.

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Binding of the plant hormone kinetin in the active site of Mistletoe Lectin I from Viscum album.

Publication Date: 2012 Feb PMID: 22064121
Authors: Malecki, P. H. - Rypniewski, W. - Szymanski, M. - Barciszewski, J. - Meyer, A.
Journal: Biochim Biophys Acta

The crystal structure of the ribosome inhibiting protein Mistletoe Lectin I (ML-I) derived from the European mistletoe, Viscum album, in complex with kinetin has been refined at 2.7A resolution. Suitably large crystals of ML-I were obtained applying the counter diffusion method using the Gel Tube R Crystallization Kit (GT-R) on board the Russian Service Module on the international space station ISS within the GCF mission No. 6, arranged by the Japanese aerospace exploration agency (JAXA). Hexagonal bi-pyramidal crystals were grown during three months under microgravity. Before data collection the crystals were soaked in a saturated solution of kinetin and diffraction data to 2.7A were collected using synchrotron radiation and cryogenic techniques. The atomic model was refined and revealed a single kinetin molecule in the ribosome inactivation site of ML-I. The complex demonstrates the feasibility of mistletoe to bind plant hormones out of the host regulation system as part of a self protection mechanism.

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The role of membrane thickness in charged protein-lipid interactions.

Publication Date: 2012 Feb PMID: 22063722
Authors: Li, L. B. - Vorobyov, I. - Allen, T. W.
Journal: Biochim Biophys Acta

Charged amino acids are known to be important in controlling the actions of integral and peripheral membrane proteins and cell disrupting peptides. Atomistic molecular dynamics studies have shed much light on the mechanisms of membrane binding and translocation of charged protein groups, yet the impact of the full diversity of membrane physico-chemical properties and topologies has yet to be explored. Here we have performed a systematic study of an arginine (Arg) side chain analog moving across saturated phosphatidylcholine (PC) bilayers of variable hydrocarbon tail length from 10 to 18 carbons. For all bilayers we observe similar ion-induced defects, where Arg draws water molecules and lipid head groups into the bilayers to avoid large dehydration energy costs. The free energy profiles all exhibit sharp climbs with increasing penetration into the hydrocarbon core, with predictable shifts between bilayers of different thickness, leading to barrier reduction from 26kcal/mol for 18 carbons to 6kcal/mol for 10 carbons. For lipids of 10 and 12 carbons we observe narrow transmembrane pores and corresponding plateaus in the free energy profiles. Allowing for movements of the protein and side chain snorkeling, we argue that the energetic cost for burying Arg inside a thin bilayer will be small, consistent with recent experiments, also leading to a dramatic reduction in pK(a) shifts for Arg. We provide evidence that Arg translocation occurs via an ion-induced defect mechanism, except in thick bilayers (of at least 18 carbons) where solubility-diffusion becomes energetically favored. Our findings shed light on the mechanisms of ion movement through membranes of varying composition, with implications for a range of charged protein-lipid interactions and the actions of cell-perturbing peptides. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Distinct cellular pools of perilipin 5 point to roles in lipid trafficking.

Publication Date: 2012 Feb PMID: 22063271
Authors: Bartholomew, S. R. - Bell, E. H. - Summerfield, T. - Newman, L. C. - Miller, E. L. - Patterson, B. - Niday, Z. P. - Ackerman, W. E. 4th - Tansey, J. T.
Journal: Biochim Biophys Acta

The PAT family of lipid storage droplet proteins comprised five members, each of which has become an established regulator of cellular neutral lipid metabolism. Perilipin 5 (also known as lsdp-5, MLDP, PAT-1, and OXPAT), the most recently discovered member of the family, has been shown to localize to two distinct intracellular pools: the lipid storage droplet (LD), and a poorly characterized cytosolic fraction. We have characterized the denser of these intracellular pools and find that a population of perilipin 5 not associated with large LDs resides in complexes with a discrete density (~1.15g/ml) and size (~575kDa). Using immunofluorescence, western blotting of isolated sucrose density fractions, native gradient gel electrophoresis, and co-immunoprecipitation, we have shown that these small (~15nm), perilipin 5-encoated structures do not contain the PAT protein perilipin 2 (ADRP), but do contain perilipin 3 and several other as of yet uncharacterized proteins. The size and density of these particles as well as their susceptibility to degradation by lipases suggest that like larger LDs, they have a neutral lipid rich core. When treated with oleic acid to promote neutral lipid deposition, cells ectopically expressing perilipin 5 experienced a reorganization of LDs in the cell, resulting in fewer, larger droplets at the expense of smaller ones. Collectively, these data demonstrate that a portion of cytosolic perilipin 5 resides in high density lipid droplet complexes that participate in cellular neutral lipid accumulation.

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Endoplasmic reticulum stress-mediated inhibition of NSMase2 elevates plasma membrane cholesterol and attenuates NO production in endothelial cells.

Publication Date: 2012 Feb PMID: 22063270
Authors: Chaube, R. - Kallakunta, V. M. - Espey, M. G. - McLarty, R. - Faccenda, A. - Ananvoranich, S. - Mutus, B.
Journal: Biochim Biophys Acta

Chronic exposure of blood vessels to cardiovascular risk factors such as free fatty acids, LDL-cholesterol, homocysteine and hyperglycemia can give rise to endothelial dysfunction, partially due to decreased synthesis and bioavailability of nitric oxide (NO). Many of these same risk factors have been shown to induce endoplasmic reticulum (ER) stress in endothelial cells. The objective of this study was to examine the mechanisms responsible for endothelial dysfunction mediated by ER stress. ER stress elevated both intracellular and plasma membrane (PM) cholesterols in BAEC by ~3-fold, indicated by epifluorescence and cholesterol oxidase methods. Increases in cholesterol levels inversely correlated with neutral sphingomyelinase 2 (NSMase2) activity, endothelial nitric oxide synthase (eNOS) phospho-activation and NO-production. To confirm that ER stress-induced effects on PM cholesterol were a direct consequence of decreased NSMase2 activity, enzyme expression was either enhanced or knocked down in BAEC. NSMase2 over-expression did not significantly affect cholesterol levels or NO-production, but increased eNOS phosphorylation by ~1.7-fold. Molecular knock down of NSMase2 decreased eNOS phosphorylation and NO-production by 50% and 40%, respectively while increasing PM cholesterol by 1.7-fold and intracellular cholesterol by 2.7-fold. Furthermore, over-expression of NSMase2 in ER-stressed BAEC lowered cholesterol levels to within control levels as well as nearly doubled the NO production, restoring it to ~74% and 68% of controls using tunicamycin and palmitate, respectively. This study establishes NSMase2 as a pivotal enzyme in the onset of endothelial ER stress-mediated vascular dysfunction as its inactivation leads to the attenuation of NO production and the elevation of cellular cholesterol.

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Suppression of MIP-2 or IL-8 production by annexins A1 and A4 during coculturing of macrophages with late apoptotic human peripheral blood neutrophils.

Publication Date: 2012 Feb PMID: 22056994
Authors: Iwasa, T. - Takahashi, R. - Nagata, K. - Kobayashi, Y.
Journal: Biochim Biophys Acta

Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils.

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Protective effects of allopurinol against acute liver damage and cirrhosis induced by carbon tetrachloride: Modulation of NF-kappaB, cytokine production and oxidative stress.

Publication Date: 2012 Feb PMID: 22056511
Authors: Aldaba-Muruato, L. R. - Moreno, M. G. - Shibayama, M. - Tsutsumi, V. - Muriel, P.
Journal: Biochim Biophys Acta

BACKGROUND: The aim of this work was to evaluate the hepatoprotective ability of allopurinol to prevent the liver injury induced by carbon tetrachloride (CCl(4)). METHODS: Acute liver damage was induced with CCl(4) (4g/kg, by gavage); allopurinol (50mg/kg, by gavage) was given 1h before and 1h after CCl(4) intoxication and two daily doses for the previous three days. Cirrhosis was established by CCl(4) administration (0.4g/kg, i. p. three times a week, eight weeks); allopurinol was administered (100mg/kg, by gavage, daily) during the long-term of CCl(4) treatment. Alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (gamma-GTP), xanthine oxidase (XO), lipid peroxidation, reduced and oxidized glutathione (GSH, GSSG, respectively), hydroxyproline and histopathologycal analysis were performed. Nuclear factor-kappaB (NF-kappaB), pro-inflammatory and anti-inflammatory cytokines, transforming growth factor-beta (TGF-beta) and metalloproteinase-13 (MMP-13) were analyzed by Western blots. RESULTS: Acute injury increased ALT and gamma-GTP activities, additionally enhanced NF-kappaB nuclear translocation and cytokines production such as tumor necrosis factor-alpha, interleukine-1beta, and interleukine-6. Allopurinol partially prevented these effects, while increased interleukine-10. Acute and chronic CCl(4) treatments altered the levels of XO activity, lipid peroxidation, and GSH/GSSG ratio, while these remained within normal range with allopurinol administration. Necrosis, fibrosis and TGF-beta production induced in chronic injury were partially prevented by allopurinol, interestingly, this drug induced MMP-13 activity. CONCLUSIONS: Allopurinol possesses antioxidant, anti-inflammatory and antifibrotic properties, probably by its capacity to reduce NF-kappaB nuclear translocation and TGF-beta expression, as well as to induce MMP-13. General significance Allopurinol might be effective treatment of liver diseases.

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Mitochondrial DNA deletions and differential mitochondrial DNA content in Rhesus monkeys: Implications for aging.

Publication Date: 2012 Feb PMID: 22056405
Authors: Mao, P. - Gallagher, P. - Nedungadi, S. - Manczak, M. - Shirendeb, U. P. - Kohama, S. G. - Ferguson, B. - Park, B. S. - Reddy, P. H.
Journal: Biochim Biophys Acta

The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8kb mtDNA fragment covering a common 5.7kb deletion and the entire 16.5kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function.

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Substrate kringle-mediated catalysis by the streptokinase-plasmin activator complex: Critical contribution of kringle-4 revealed by the mutagenesis approaches.

Publication Date: 2012 Feb PMID: 22056293
Authors: Joshi, K. K. - Nanda, J. S. - Kumar, P. - Sahni, G.
Journal: Biochim Biophys Acta

Streptokinase (SK) is a protein co-factor with a potent capability for human plasminogen (HPG) activation. Our previous studies [1] have indicated a major role of long-range protein-protein contacts between the three domains (alpha, beta, and gamma) of SK and the multi-domain HPG substrate (K1-K5CD). To further explore this phenomenon, we prepared truncated derivatives of HPG with progressive removal of kringle domains, like K5CD, K4K5CD, K3-K5CD (K3K4K5CD), K2-K5CD (K2K3K4K5CD) and K1-K5CD (K1K2K3K4K5CD). While urokinase (uPA) cleaved the scissile peptide in the isolated catalytic domain (muPG) with nearly the same rate as with full-length HPG, SK-plasmin showed only 1-2% activity, revealing mutually distinct mechanisms of HPG catalysis between the eukaryotic and prokaryotic activators. Remarkably, with SK.HPN (plasmin), the 'addition' of both kringles 4 and 5 onto the catalytic domain showed catalytic rates comparable to full length HPG, thus identifying the dependency of the "long-range" enzyme-substrate interactions onto these two CD-proximal domains. Further, chimeric variants of K5CD were generated by swapping the kringle domains of HPG with those of uPA and TPA (tissue plasminogen activator), separately. Surprisingly, although native-like catalytic turnover rates were retained when either K1, K2 or K4 of HPG was substituted at the K5 position in K5CD, these were invariably lost once substituted with the evolutionarily more distant TPA- and uPA-derived kringles. The present results unveil a novel mechanism of SK.HPN action in which augmented catalysis occurs through enzyme-substrate interactions centered on regions in substrate HPG (kringles 4 and 5) that are spatially distant from the scissile peptide bond.

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Physiology and global gene expression of a Corynebacterium glutamicum DeltaF(1)F(O)-ATP synthase mutant devoid of oxidative phosphorylation.

Publication Date: 2012 Feb PMID: 22050934
Authors: Koch-Koerfges, A. - Kabus, A. - Ochrombel, I. - Marin, K. - Bott, M.
Journal: Biochim Biophys Acta

A mutant of Corynebacterium glutamicum ATCC 13032 with a deletion of the atpBEFHAGDC genes encoding F(1)F(O)-ATP synthase was characterized. Whereas no growth was observed with acetate as sole carbon source, the DeltaF(1)F(O) mutant reached 47% of the growth rate and 65% of the biomass of the wild type during shake-flask cultivation in glucose minimal medium. Initially, the mutant strain showed a strongly increased glucose uptake rate accompanied by a high oxygen consumption rate and pyruvate secretion into the medium. When oxygen became limiting, the glucose consumption rate was reduced below that of the wild type and pyruvate was consumed again. The DeltaF(1)F(O) mutant had increased levels of b- and d-type cytochromes and a significantly increased proton motive force. Transcription of genes involved in central carbon metabolism was essentially unchanged, whereas genes for cytochrome bd oxidase, pyruvate:quinone oxidoreductase, oxidative stress response, and others showed increased mRNA levels. On the other hand, genes for amino acid biosynthesis and ribosomal proteins as well as many genes involved in transport displayed decreased mRNA levels. Several of the transcriptional changes were reflected at the protein level, but there were also discrepancies between the mRNA and protein levels suggesting some kind of posttranscriptional regulation. The results prove for the first time that F(1)F(O)-ATP synthase and oxidative phosphorylation are in general not essential for growth of C. glutamicum.

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Generation, characterization and crystallization of a cytochrome c(1)-subunit IV fused cytochrome bc(1) complex from Rhodobacter sphaeroides.

Publication Date: 2012 Feb PMID: 22050933
Authors: Su, T. - Esser, L. - Xia, D. - Yu, C. A. - Yu, L.
Journal: Biochim Biophys Acta

Cytochrome bc(1) complex catalyzes the reaction of electron transfer from ubiquinol to cytochrome c (or cytochrome c(2)) and couples this reaction to proton translocation across the membrane. Crystallization of the Rhodobacter sphaeroides bc(1) complex resulted in crystals containing only three core subunits. To mitigate the problem of subunit IV being dissociated from the three-subunit core complex during crystallization, we recently engineered an R. sphaeroides mutant in which the N-terminus of subunit IV was fused to the C-terminus of cytochrome c(1) with a 14-glycine linker between the two fusing subunits, and a 6-histidine tag at the C-terminus of subunit IV (c(1)-14Gly-IV-6His). The purified fusion mutant complex shows higher electron transfer activity, more structural stability, and less superoxide generation as compared to the wild-type enzyme. Preliminary crystallization attempts with this mutant complex yielded crystals containing four subunits and diffracting X-rays to 5.5A resolution.

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Acidity and lipolysis by group V secreted phospholipase A(2) strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans.

Publication Date: 2012 Feb PMID: 22041135
Authors: Lahdesmaki, K. - Oorni, K. - Alanne-Kinnunen, M. - Jauhiainen, M. - Hurt-Camejo, E. - Kovanen, P. T.
Journal: Biochim Biophys Acta

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A(2) (sPLA(2)-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA(2)-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased >60-fold and sPLA(2)-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA(2)-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA(2)-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.

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Proteomic profiling of hempseed proteins from Cheungsam.

Publication Date: 2012 Feb PMID: 22040604
Authors: Park, S. K. - Seo, J. B. - Lee, M. Y.
Journal: Biochim Biophys Acta

Proteomic profiling of hempseed proteins from a non-drug type of industrial hemp (Cannabis sativa L.), Cheungsam, was conducted using two-dimensional gel electrophoresis and mass spectrometry. A total of 1102 protein spots were resolved on pH 3-10 immobilized pH gradient strips, and 168 unique protein spots were identified. The proteins were categorized based on function, including involvement in energy regulation (23%), metabolism (18%), stress response (16%), unclassified (12%), cytoskeleton (11%), binding function (5%), and protein synthesis (3%). These proteins might have important biological functions in hempseed, such as germination, storage, or development.

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Compromised mitochondrial complex II in models of Machado-Joseph disease.

Publication Date: 2012 Feb PMID: 22037589
Authors: Laco, M. N. - Oliveira, C. R. - Paulson, H. L. - Rego, A. C.
Journal: Biochim Biophys Acta

Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.

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Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II.

Publication Date: 2012 Feb PMID: 22037395
Authors: Itoh, S. - Kozuki, T. - Nishida, K. - Fukushima, Y. - Yamakawa, H. - Domonkos, I. - Laczko-Dobos, H. - Kis, M. - Ughy, B. - Gombos, Z.
Journal: Biochim Biophys Acta

Functional roles of an anionic lipid phosphatidylglycerol (PG) were studied in pgsA-gene-inactivated and cdsA-gene-inactivated/phycobilisome-less mutant cells of a cyanobacterium Synechocystis sp. PCC 6803, which can grow only in PG-supplemented media. 1) A few days of PG depletion suppressed oxygen evolution of mutant cells supported by p-benzoquinone (BQ). The suppression was recovered slowly in a week after PG re-addition. Measurements of fluorescence yield indicated the enhanced sensitivity of Q(B) to the inactivation by BQ. It is assumed that the loss of low-affinity PG (PG(L)) enhances the affinity for BQ that inactivates Q(B). 2) Oxygen evolution without BQ, supported by the endogenous electron acceptors, was slowly suppressed due to the direct inactivation of Q(B) during 10days of PG depletion, and was recovered rapidly within 10h upon the PG re-addition. It is concluded that the loss of high-affinity PG (PG(H)) displaces Q(B) directly. 3) Electron microscopy images of PG-depleted cells showed the specific suppression of division of mutant cells, which had developed thylakoid membranes attaching phycobilisomes (PBS). 4) Although the PG-depletion for 14days decreased the chlorophyll/PBS ratio to about 1/4, florescence spectra/lifetimes were not modified indicating the flexible energy transfer from PBS to different numbers of PSII. Longer PG-depletion enhanced allophycocyanin fluorescence at 683nm with a long 1.2ns lifetime indicating the suppression of energy transfer from PBS to PSII. 5) Action sites of PG(H), PG(L) and other PG molecules on PSII structure are discussed.

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Kinetics of phyllosemiquinone oxidation in the Photosystem I reaction centre of Acaryochloris marina.

Publication Date: 2012 Feb PMID: 22037394
Authors: Santabarbara, S. - Bailleul, B. - Redding, K. - Barber, J. - Rappaport, F. - Telfer, A.
Journal: Biochim Biophys Acta

Light-induced electron transfer reactions in the chlorophyll a/d-binding Photosystem I reaction centre of Acaryochloris marina were investigated in whole cells by pump-probe optical spectroscopy with a temporal resolution of ~5ns at room temperature. It is shown that phyllosemiquinone, the secondary electron transfer acceptor anion, is oxidised with bi-phasic kinetics characterised by lifetimes of 88+/-6ns and 345+/-10ns. These lifetimes, particularly the former, are significantly slower than those reported for chlorophyll a-binding Photosystem I, which typically range in the 5-30ns and 200-300ns intervals. The possible mechanism of electron transfer reactions in the chlorophyll a/d-binding Photosystem I and the slower oxidation kinetics of the secondary acceptors are discussed.

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Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate.

Publication Date: 2012 Feb PMID: 22037183
Authors: Luka, Z. - Pakhomova, S. - Loukachevitch, L. V. - Newcomer, M. E. - Wagner, C.
Journal: Biochim Biophys Acta

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

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Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions.

Publication Date: 2012 Feb PMID: 22037182
Authors: Ceruso, M. - Howe, N. - Malthouse, J. P.
Journal: Biochim Biophys Acta

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82ppm and 182.87ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5ppm to 182.9ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is
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Copper binding traps the folded state of the SCO protein from Bacillus subtilis.

Publication Date: 2012 Feb PMID: 22036877
Authors: Lai, M. - Yam, K. C. - Andrews, D. - Hill, B. C.
Journal: Biochim Biophys Acta

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.

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A catalytic defect in mitochondrial respiratory chain complex I due to a mutation in NDUFS2 in a patient with Leigh syndrome.

Publication Date: 2012 Feb PMID: 22036843
Authors: Ngu, L. H. - Nijtmans, L. G. - Distelmaier, F. - Venselaar, H. - van Emst-de Vries, S. E. - van den Brand, M. A. - Stoltenborg, B. J. - Wintjes, L. T. - Willems, P. H. - van den Heuvel, L. P. - Smeitink, J. A. - Rodenburg, R. J.
Journal: Biochim Biophys Acta

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.

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An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification.

Publication Date: 2012 Feb PMID: 22027266
Authors: Dittrich, A. C. - Devarenne, T. P.
Journal: Biochim Biophys Acta

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the "gatekeeper" residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3(M385G) can also specifically utilize N(6)-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.

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Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis.

Publication Date: 2012 Feb PMID: 22027215
Authors: Gomes, A. P. - Duarte, F. V. - Nunes, P. - Hubbard, B. P. - Teodoro, J. S. - Varela, A. T. - Jones, J. G. - Sinclair, D. A. - Palmeira, C. M. - Rolo, A. P.
Journal: Biochim Biophys Acta

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.

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Arabidopsis thaliana PECP1 - Enzymatic characterization and structural organization of the first plant phosphoethanolamine/phosphocholine phosphatase.

Publication Date: 2012 Feb PMID: 22024570
Authors: May, A. - Spinka, M. - Kock, M.
Journal: Biochim Biophys Acta

Maintenance of cellular phosphate homeostasis is crucial for primary and energy metabolism. In plants, low exogenous phosphate availability activates adaptive responses that include the immediate liberation of Pi from phosphorylated metabolites by yet uncharacterized intracellular phosphatases. Based on transcriptional analyses, the Arabidopsis thaliana gene At1g17710, a member of the HAD (Haloacid Dehalogenase) superfamily, was one of the most promising candidates. Here, we show by recombinant protein production and analysis of purified protein that the gene At1g17710 encodes a phosphoethanolamine/phosphocholine phosphatase (EC 3.1.3.75). Thus, the gene product was termed AtPECP1. The present study demonstrates that the Mg(2+)-dependent enzyme exhibits pronounced specificity for both substrates. The enzyme displays a broad pH optimum ranging from pH 6 to pH 8. Comparison of K(m) values indicates a slightly higher affinity for phosphocholine (0.44mM) than for phosphoethanolamine (1.16mM). The catalytic efficiency, however, is markedly higher for phosphoethanolamine than for phosphocholine being 1.06x10(4)M(-1)s(-1) and 2.34x10(3)M(-1)s(-1), respectively. Size exclusion chromatography, native gel electrophoresis and SAXS experiments with recombinant protein clearly point to a rapid monomer-dimer equilibrium of protein subunits. Given its established substrate specificity the enzyme is likely to be involved in the liberation of inorganic phosphate from intracellular sources and is especially in demand under phosphate-deprived conditions.

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alpha-Synuclein misfolding and Parkinson's disease.

Publication Date: 2012 Feb PMID: 22024360
Authors: Breydo, L. - Wu, J. W. - Uversky, V. N.
Journal: Biochim Biophys Acta

Substantial evidence links alpha-synuclein, a small highly conserved presynaptic protein with unknown function, to both familial and sporadic Parkinson's disease (PD). alpha-Synuclein has been identified as the major component of Lewy bodies and Lewy neurites, the characteristic proteinaceous deposits that are the hallmarks of PD. alpha-Synuclein is a typical intrinsically disordered protein, but can adopt a number of different conformational states depending on conditions and cofactors. These include the helical membrane-bound form, a partially-folded state that is a key intermediate in aggregation and fibrillation, various oligomeric species, and fibrillar and amorphous aggregates. The molecular basis of PD appears to be tightly coupled to the aggregation of alpha-synuclein and the factors that affect its conformation. This review examines the different aggregation states of alpha-synuclein, the molecular mechanism of its aggregation, and the influence of environmental and genetic factors on this process.

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Multi-faced neuroprotective effects of Ginsenoside Rg1 in an Alzheimer mouse model.

Publication Date: 2012 Feb PMID: 22015470
Authors: Fang, F. - Chen, X. - Huang, T. - Lue, L. F. - Luddy, J. S. - Yan, S. S.
Journal: Biochim Biophys Acta

There has been no extensive characterization of the effects of Ginsenoside Rg1, a pharmacological active component purified from the nature product ginseng, in an Alzheimer's disease mouse model. The well-characterized transgenic Alzheimer disease (AD) mice over expressing amyloid precursor protein (APP)/Abeta (Tg mAPP) and nontransgenic (nonTg) littermates at age of 6 and 9months were treated with Rg 1 for three months via intraperitoneal injection. Mice were then evaluated for changes in amyloid pathology, neuropathology and behavior. Tg mAPP treated with Rg1 showed a significant reduction of cerebral Abeta levels, reversal of certain neuropathological changes, and preservation of spatial learning and memory, as compared to vehicle-treated mice. Rg1 treatment inhibited activity of gamma-secretase in both Tg mAPP mice and B103-APP cells, indicating the involvement of Rg1 in APP regulation pathway. Furthermore, administration of Rg1 enhanced PKA/CREB pathway activation in mAPP mice and in cultured cortical neurons exposed to Abeta or glutamate-mediated synaptic stress. Most importantly, the beneficial effects on attenuation of cerebral Abeta accumulation, improvement in neuropathological and behavioral changes can be extended to the aged mAPP mice, even to 12-13months old mice that had extensive amyloid pathology and severe neuropathological and cognitive malfunction. These studies indicate that Rg1 has profound multi-faced and neuroprotective effects in an AD mouse model. Rg1 induces neuroprotection through ameliorating amyloid pathology, modulating APP process, improving cognition, and activating PKA/CREB signaling. These findings provide a new perspective for the treatment of AD and demonstrate potential for a new class of drugs for AD treatment.

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Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome.

Publication Date: 2012 Feb PMID: 22009041
Authors: Cenini, G. - Dowling, A. L. - Beckett, T. L. - Barone, E. - Mancuso, C. - Murphy, M. P. - Levine, H. 3rd - Lott, I. T. - Schmitt, F. A. - Butterfield, D. A. - Head, E.
Journal: Biochim Biophys Acta

Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n=29 control and n=41 DS). By ELISA, we quantified soluble and insoluble Abeta40 and Abeta42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Abeta) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Abeta40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.

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Constant electric field simulations of the membrane potential illustrated with simple systems.

Publication Date: 2012 Feb PMID: 22001851
Authors: Gumbart, J. - Khalili-Araghi, F. - Sotomayor, M. - Roux, B.
Journal: Biochim Biophys Acta

Advances in modern computational methods and technology make it possible to carry out extensive molecular dynamics simulations of complex membrane proteins based on detailed atomic models. The ultimate goal of such detailed simulations is to produce trajectories in which the behavior of the system is as realistic as possible. A critical aspect that requires consideration in the case of biological membrane systems is the existence of a net electric potential difference across the membrane. For meaningful computations, it is important to have well validated methodologies for incorporating the latter in molecular dynamics simulations. A widely used treatment of the membrane potential in molecular dynamics consists of applying an external uniform electric field E perpendicular to the membrane. The field acts on all charged particles throughout the simulated system, and the resulting applied membrane potential V is equal to the applied electric field times the length of the periodic cell in the direction perpendicular to the membrane. A series of test simulations based on simple membrane-slab models are carried out to clarify the consequences of the applied field. These illustrative tests demonstrate that the constant-field method is a simple and valid approach for accounting for the membrane potential in molecular dynamics studies of biomolecular systems. This article is part of a Special Issue entitled: Membrane protein structure and function.

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AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: Implication of the cell survival in mitochondrial diseases.

Publication Date: 2012 Feb PMID: 22001850
Authors: Wu, S. B. - Wei, Y. H.
Journal: Biochim Biophys Acta

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

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MiR-21 is involved in cervical squamous cell tumorigenesis and regulates CCL20.

Publication Date: 2012 Feb PMID: 22001440
Authors: Yao, T. - Lin, Z.
Journal: Biochim Biophys Acta

MicroRNA 21 (miR-21) has been implicated in various aspects of carcinogenesis. However, its function and molecular mechanism in cervical squamous carcinoma have not been studied. Using TaqMan quantitative real-time PCR and Northern blot, we confirmed that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines. Remarkably, we showed that the level of miR-21 correlates with the tumor differentiation and nodal status by ISH. Furthermore, we demonstrated that miR-21 regulates proliferation, apoptosis, and migration of HPV16-positive cervical squamous cells. In order to identify candidate target genes for miR-21, we used gene expression profiling. By luciferase reporter assays, we confirmed that CCL20 is one of its target genes, which is related to the HPV16 E6 and E7 oncogenes. Our results suggest that miR-21 may be involved in cervical squamous cell tumorigenesis.

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Alteration in intestine tight junction protein phosphorylation and apoptosis is associated with increase in IL-18 levels following alcohol intoxication and burn injury.

Publication Date: 2012 Feb PMID: 22001439
Authors: Li, X. - Akhtar, S. - Choudhry, M. A.
Journal: Biochim Biophys Acta

Intestinal mucosal barrier is the first line of defense against bacteria and their products originating from the intestinal lumen. We have shown a role for IL-18 in impaired gut barrier function following acute alcohol (EtOH) intoxication combined with burn injury. To further delineate the mechanism, this study examined whether IL-18 alters intestine tight junction proteins or induces mucosal apoptosis under these conditions. To accomplish this, rats were gavaged with EtOH (3.2g/kg) prior to ~12.5% total body surface area burn or sham injury. One day after injury, EtOH combined with burn injury resulted in a significant decrease in total occludin protein and its phosphorylation in small intestine compared to either EtOH or burn injury alone. There was no change in claudin-1 protein content but its phosphorylation on tyrosine was decreased following EtOH and burn injury. This was accompanied with an increase in mucosal apoptosis (p<0.05). The treatment of rats with anti-IL-18 antibody at the time of burn injury prevented intestine apoptosis and normalized tight junction proteins following EtOH and burn injury. Altogether, these findings suggest that IL-18 modulates tight junction proteins and cause apoptosis leading to impaired intestinal mucosal integrity following EtOH intoxication combined with burn injury.

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Functional magnetic resonance imaging of semantic memory as a presymptomatic biomarker of Alzheimer's disease risk.

Publication Date: 2012 Mar PMID: 21996618
Authors: Sugarman, M. A. - Woodard, J. L. - Nielson, K. A. - Seidenberg, M. - Smith, J. C. - Durgerian, S. - Rao, S. M.
Journal: Biochim Biophys Acta

Extensive research efforts have been directed toward strategies for predicting risk of developing Alzheimer's disease (AD) prior to the appearance of observable symptoms. Existing approaches for early detection of AD vary in terms of their efficacy, invasiveness, and ease of implementation. Several non-invasive magnetic resonance imaging strategies have been developed for predicting decline in cognitively healthy older adults. This review will survey a number of studies, beginning with the development of a famous name discrimination task used to identify neural regions that participate in semantic memory retrieval and to test predictions of several key theories of the role of the hippocampus in memory. This task has revealed medial temporal and neocortical contributions to recent and remote memory retrieval, and it has been used to demonstrate compensatory neural recruitment in older adults, apolipoprotein E epsilon4 carriers, and amnestic mild cognitive impairment patients. Recently, we have also found that the famous name discrimination task provides predictive value for forecasting episodic memory decline among asymptomatic older adults. Other studies investigating the predictive value of semantic memory tasks will also be presented. We suggest several advantages associated with the use of semantic processing tasks, particularly those based on person identification, in comparison to episodic memory tasks to study AD risk. Future directions for research and potential clinical uses of semantic memory paradigms are also discussed. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Cellular retinaldehyde-binding protein (CRALBP) is a direct downstream target of transcription factor Pax6.

Publication Date: 2012 Feb PMID: 21996446
Authors: Boppana, S. - Scheglov, A. - Geffers, R. - Tarabykin, V.
Journal: Biochim Biophys Acta

BACKGROUND: Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated. METHODS: We studied the expression of CRALBP in wt and Pax6-/- mutants through in situ hybridization and immunohistochemistry. ChIP assay and luciferase reporter assay were performed to show the regulatory role of Pax6 on CRALBP promoter. RESULTS: RNA and protein expression data show that CRALBP expression was completely abolished in Pax6 mutants. In vivo binding assays and in vitro reporter assays indicate that Pax6 not only binds the promoter of CRALBP but also positively regulates protein expression. CONCLUSIONS: This work provides evidence supporting that CRALBP is a direct downstream target of Pax6. However, the role of CRALBP in the cortex is yet to be elucidated. GENERAL SIGNIFICANCE: Pax6 is a marker expressed on neural stem cells and progenitor cells. Understanding Pax6-dependent gene regulatory mechanisms unravels signaling cascades that occur early during development.

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Exploring the neural basis of cognitive reserve in aging.

Publication Date: 2012 Mar PMID: 21982946
Authors: Steffener, J. - Stern, Y.
Journal: Biochim Biophys Acta

The concept of reserve arose from the mismatch between the extent of brain changes or pathology and the clinical manifestations of these brain changes. The cognitive reserve hypothesis posits that individual differences in the flexibility and adaptability of brain networks underlying cognitive function may allow some people to cope better with brain changes than others. Although there is ample epidemiologic evidence for cognitive reserve, the neural substrate of reserve is still a topic of ongoing research. Here we review some representative studies from our group that exemplify possibilities for the neural substrate of reserve including neural reserve, neural compensation, and generalized cognitive reserve networks. We also present a schematic overview of our ongoing research in this area. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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A gate-free pathway for substrate release from the inward-facing state of the Na(+)-galactose transporter.

Publication Date: 2012 Feb PMID: 21978597
Authors: Li, J. - Tajkhorshid, E.
Journal: Biochim Biophys Acta

Employing molecular dynamics (MD) simulations, the pathway and mechanism of substrate unbinding from the inward-facing state of the Na(+)-coupled galactose transporter, vSGLT, have been investigated. During a 200-ns equilibrium simulation, repeated spontaneous unbinding events of the substrate from its binding site have been observed. In contrast to the previously proposed gating role of a tyrosine residue (Y263), the unbinding mechanism captured in the present equilibrium simulation does not rely on the displacement and/or rotation of this side chain. Rather, the unbinding involves an initial lateral displacement of the substrate out of the binding site which allows the substrate to completely emerge from the region covered by the side chain of Y263 without any noticeable conformational changes of the latter. Starting with the snapshots taken from this equilibrium simulation with the substrate outside the binding site, steered MD (SMD) simulations were then used to probe the translocation of the substrate along the remaining of the release pathway within the protein's lumen and to characterize the nature of protein-substrate interactions involved in the process. Combining the results of the equilibrium and SMD simulations, we provide a description of the full translocation pathway for the substrate release from the binding site into the cytoplasm. Residues E68, N142, T431, and N267 facilitate the initial substrate's displacement out of the binding site, while the translocation of the substrate along the remainder of the exit pathway formed between TM6 and TM8 is facilitated by H-bond interactions between the substrate and a series of conserved, polar residues (Y138, N267, R273, S365, S368, N371, S372, and T375). The observed molecular events indicate that no gating is required for the release of the substrate from the crystallographically captured structure of the inward-facing state of SGLT, suggesting that this conformation might represent an open, rather than occluded, state of the transporter. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Charge equilibration force fields for molecular dynamics simulations of lipids, bilayers, and integral membrane protein systems.

Publication Date: 2012 Feb PMID: 21967961
Authors: Lucas, T. R. - Bauer, B. A. - Patel, S.
Journal: Biochim Biophys Acta

With the continuing advances in computational hardware and novel force fields constructed using quantum mechanics, the outlook for non-additive force fields is promising. Our work in the past several years has demonstrated the utility of polarizable force fields, those based on the charge equilibration formalism, for a broad range of physical and biophysical systems. We have constructed and applied polarizable force fields for lipids and lipid bilayers. In this review of our recent work, we discuss the formalism we have adopted for implementing the charge equilibration (CHEQ) method for lipid molecules. We discuss the methodology, related issues, and briefly discuss results from recent applications of such force fields. Application areas include DPPC-water monolayers, potassium ion permeation free energetics in the gramicidin A bacterial channel, and free energetics of permeation of charged amino acid analogs across the water-bilayer interface. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Structural correlates of selectivity and inactivation in potassium channels.

Publication Date: 2012 Feb PMID: 21958666
Authors: McCoy, J. G. - Nimigean, C. M.
Journal: Biochim Biophys Acta

Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the "selectivity filter". The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Empirical derivation of the reference region for computing diagnostic sensitive (18)fluorodeoxyglucose ratios in Alzheimer's disease based on the ADNI sample.

Publication Date: 2012 Mar PMID: 21958592
Authors: Rasmussen, J. M. - Lakatos, A. - van Erp, T. G. - Kruggel, F. - Keator, D. B. - Fallon, J. T. - Macciardi, F. - Potkin, S. G.
Journal: Biochim Biophys Acta

Careful selection of the reference region for non-quantitative positron emission tomography (PET) analyses is critically important for Region of Interest (ROI) data analyses. We introduce an empirical method of deriving the most suitable reference region for computing neurodegeneration sensitive (18)fluorodeoxyglucose (FDG) PET ratios based on the dataset collected by the Alzheimer's Disease Neuroimaging Initiative (ADNI) study. Candidate reference regions are selected based on a heat map of the difference in coefficients of variation (COVs) of FDG ratios over time for each of the Automatic Anatomical Labeling (AAL) atlas regions normalized by all other AAL regions. Visual inspection of the heat map suggests that the portion of the cerebellum and vermis superior to the horizontal fissure is the most sensitive reference region. Analyses of FDG ratio data show increases in significance on the order of ten-fold when using the superior portion of the cerebellum as compared with the traditionally used full cerebellum. The approach to reference region selection in this paper can be generalized to other radiopharmaceuticals and radioligands as well as to other disorders where brain changes over time are hypothesized and longitudinal data is available. Based on the empirical evidence presented in this study, we demonstrate the usefulness of the COV heat map method and conclude that intensity normalization based on the superior portion of the cerebellum may be most sensitive to measuring change when performing longitudinal analyses of FDG-PET ratios as well as group comparisons in Alzheimer's disease. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Leisure activities, cognition and dementia.

Publication Date: 2012 Mar PMID: 21930203
Authors: Wang, H. X. - Xu, W. - Pei, J. J.
Journal: Biochim Biophys Acta

Accumulated evidence shows that leisure activities have a positive impact on cognitive function and dementia. This review aimed to systematically summarize the current evidence on this topic taking into account the limitations of the studies and biological plausibility for the underlying mechanisms linking cognition, dementia and leisure activities, with special attention on mental, physical and social activities. We included only longitudinal studies, with a follow-up time of at least 2years, published in English from 1991 to March 2011 on leisure activities and cognition (n=29) or dementia (n=23) and provided some evidence from intervention studies on the topic. A protective effect of mental activity on cognitive function has been consistently reported in both observational and interventional studies. The association of mental activity with the risk of dementia was robust in observational studies but inconsistent in clinical trials. The protective effect of physical activity on the risk of cognitive decline and dementia has been reported in most observational studies, but has been less evident in interventional studies. Current evidence concerning the beneficial effect of other types of leisure activities on the risk of dementia is still limited and results are inconsistent. For future studies it is imperative that the assessment of leisure activities is standardized, for example, the frequency, intensity, duration and the type of activity; and also that the cognitive test batteries and the definition of cognitive decline are harmonized/standardized. Further, well designed studies with long follow-up times are necessary. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Age-related white matter microstructural differences partly mediate age-related decline in processing speed but not cognition.

Publication Date: 2012 Mar PMID: 21930202
Authors: Salami, A. - Eriksson, J. - Nilsson, L. G. - Nyberg, L.
Journal: Biochim Biophys Acta

Aging is associated with declining cognitive performance as well as structural changes in brain gray and white matter (WM). The WM deterioration contributes to a disconnection among distributed brain networks and may thus mediate age-related cognitive decline. The present diffusion tensor imaging (DTI) study investigated age-related differences in WM microstructure and their relation to cognition (episodic memory, visuospatial processing, fluency, and speed) in a large group of healthy subjects (n=287) covering 6 decades of the human life span. Age related decreases in fractional anisotropy (FA) and increases in mean diffusivity (MD) were observed across the entire WM skeleton as well as in specific WM tracts, supporting the WM degeneration hypothesis. The anterior section of the corpus callosum was more susceptible to aging compared to the posterior section, lending support to the anterior-posterior gradient of WM integrity in the corpus callosum. Finally, and of critical interest, WM integrity differences were found to mediate age-related reductions in processing speed but no significant mediation was found for episodic memory, visuospatial ability, or fluency. These findings suggest that compromised WM integrity is not a major contributing factor to declining cognitive performance in normal aging. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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QM/MM study of the mechanism of enzymatic limonene 1,2-epoxide hydrolysis.

Publication Date: 2012 Feb PMID: 21925621
Authors: Hou, Q. Q. - Sheng, X. - Wang, J. H. - Liu, Y. J. - Liu, C. B.
Journal: Biochim Biophys Acta

Limonene 1,2-epoxide hydrolase (LEH) is completely different from those of classic epoxide hydrolases (EHs) which catalyze the hydrolysis of epoxides to vicinal diols. A novel concerted general acid catalysis step involving the Asp101-Arg99-Asp132 triad is proposed to play an important role in the mechanism. Combined quantum-mechanical/molecular-mechanical (QM/MM) calculations gave activation barriers of 16.9 and 25.1kcal/mol at the B3LYP/6-31G(d,p)//CHARMM level for nucleophilic attack on the more and less substituted epoxide carbons, respectively. Furthermore, the important roles of residues Arg99, Tyr53 and Asn55 on mutated LEH were evaluated by QM/MM-scanned energy mapping. These results may provide an explanation for site-directed mutagenesis.

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Forster resonance energy transfer as a probe of membrane protein folding.

Publication Date: 2012 Feb PMID: 21925139
Authors: Kang, G. - Lopez-Pena, I. - Oklejas, V. - Gary, C. S. - Cao, W. - Kim, J. E.
Journal: Biochim Biophys Acta

The folding reaction of a beta-barrel membrane protein, outer membrane protein A (OmpA), is probed with Forster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.

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The role of the lipid matrix for structure and function of the GPCR rhodopsin.

Publication Date: 2012 Feb PMID: 21924236
Authors: Soubias, O. - Gawrisch, K.
Journal: Biochim Biophys Acta

Photoactivation of rhodopsin in lipid bilayers results within milliseconds in a metarhodopsin I (MI)-metarhodopsin II (MII) equilibrium that is very sensitive to the lipid composition. It has been well established that lipid bilayers that are under negative curvature elastic stress from incorporation of lipids like phosphatidylethanolamines (PE) favor formation of MII, the rhodopsin photointermediate that is capable of activating G protein. Furthermore, formation of the MII state is favored by negatively charged lipids like phosphatidylserine and by lipids with longer hydrocarbon chains that yield bilayers with larger membrane hydrophobic thickness. Cholesterol and rhodopsin-rhodopsin interactions from crowding of rhodopsin molecules in lipid bilayers shift the MI-MII equilibrium towards MI. A variety of mechanisms seems to be responsible for the large, lipid-induced shifts between MI and MII: adjustment of the thickness of lipid bilayers to rhodopsin and adjustment of rhodopsin helicity to the thickness of bilayers, curvature elastic deformations in the lipid matrix surrounding the protein, direct interactions of PE headgroups and polyunsaturated hydrocarbon chains with rhodopsin, and direct or lipid-mediated interactions between rhodopsin molecules. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Transmembrane helix dimerization: Beyond the search for sequence motifs.

Publication Date: 2012 Feb PMID: 21910966
Authors: Li, E. - Wimley, W. C. - Hristova, K.
Journal: Biochim Biophys Acta

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Volume of white matter hyperintensities in healthy adults: Contribution of age, vascular risk factors, and inflammation-related genetic variants.

Publication Date: 2012 Mar PMID: 21889590
Authors: Raz, N. - Yang, Y. - Dahle, C. L. - Land, S.
Journal: Biochim Biophys Acta

Aging is associated with appearance of white matter hyperintensities (WMH) on MRI scans. Vascular risk and inflammation, which increase with age, may contribute to white matter deterioration and proliferation of WMH. We investigated whether circulating biomarkers and genetic variants associated with elevated vascular risk and inflammation are associated with WMH volume in healthy adults (144 volunteers, 44-77years of age). We examined association of WMH volume with age, sex, hypertension, circulating levels of total plasma homocysteine (tHcy), cholesterol (low-density lipoprotein), and C-reactive protein (CRP), and four polymorphisms related to vascular risk and inflammation: Apolipoprotein epsilon (ApoE epsilon2,3,4), Angiotensin-Converting Enzyme insertion/deletion (ACE I/D), methylenetetrahydrofolate reductase (MTHFR) C677T, C-reactive protein (CRP)-286C>A>T, and interleukin-1beta (IL-1beta) C-511T. We found that larger WMH volume was associated with advanced age, hypertension, and elevated levels of homocysteine and CRP but not with low-density lipoprotein levels. Homozygotes for IL-1beta-511T allele and carriers of CRP-286T allele that are associated with increased inflammatory response had larger WMH than the other allelic combinations. Carriers of the APOE epsilon2 allele had larger frontal WMH than epsilon3 homozygotes and epsilon4 carriers did. Thus, in healthy adults, who are free of neurological and vascular disease, genetic variants that promote inflammation and elevated levels of vascular risk biomarkers can contribute to brain abnormalities. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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A reinterpretation of neutron scattering experiments on a lipidated Ras peptide using replica exchange molecular dynamics.

Publication Date: 2012 Feb PMID: 21872568
Authors: Vogel, A. - Roark, M. - Feller, S. E.
Journal: Biochim Biophys Acta

The Ras family of proteins plays crucial roles in a variety of cell signaling networks where they have the function of a molecular switch. Their particular medical relevance arises from mutations in these proteins that are implicated in ~30% of human cancers. The various Ras proteins exhibit a high degree of homology in their soluble domains but extremely high variability in the membrane anchoring regions that are crucial for protein function and are the focus of this study. We have employed replica exchange molecular dynamics computer simulations to study a doubly lipidated heptapeptide, corresponding to the C-terminus of the human N-Ras protein, incorporated into a dimyristoylphosphatidylcholine lipid bilayer. This same system has previously been investigated experimentally utilizing a number of techniques, including neutron scattering. Here we present results of well converged simulations that describe the subtle changes in scattering density in terms of the location of the peptide and its lipid modifications and in terms of changes in phospholipid density arising from the incorporation of the peptide into the membrane bilayer. The detailed picture that emerges from the combination of experimental and computational data exemplifies the power of combining isotopic substitution neutron scattering with atomistic molecular dynamics simulation. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Diffusion tensor imaging of cerebral white matter integrity in cognitive aging.

Publication Date: 2012 Mar PMID: 21871957
Authors: Madden, D. J. - Bennett, I. J. - Burzynska, A. - Potter, G. G. - Chen, N. K. - Song, A. W.
Journal: Biochim Biophys Acta

In this article we review recent research on diffusion tensor imaging (DTI) of white matter (WM) integrity and the implications for age-related differences in cognition. Neurobiological mechanisms defined from DTI analyses suggest that a primary dimension of age-related decline in WM is a decline in the structural integrity of myelin, particularly in brain regions that myelinate later developmentally. Research integrating behavioral measures with DTI indicates that WM integrity supports the communication among cortical networks, particularly those involving executive function, perceptual speed, and memory (i.e., fluid cognition). In the absence of significant disease, age shares a substantial portion of the variance associated with the relation between WM integrity and fluid cognition. Current data are consistent with one model in which age-related decline in WM integrity contributes to a decreased efficiency of communication among networks for fluid cognitive abilities. Neurocognitive disorders for which older adults are at risk, such as depression, further modulate the relation between WM and cognition, in ways that are not as yet entirely clear. Developments in DTI technology are providing a new insight into both the neurobiological mechanisms of aging WM and the potential contribution of DTI to understanding functional measures of brain activity. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Selective changes in white matter integrity in MCI and older adults with cognitive complaints.

Publication Date: 2012 Mar PMID: 21867750
Authors: Wang, Y. - West, J. D. - Flashman, L. A. - Wishart, H. A. - Santulli, R. B. - Rabin, L. A. - Pare, N. - Arfanakis, K. - Saykin, A. J.
Journal: Biochim Biophys Acta

BACKGROUND: White matter changes measured using diffusion tensor imaging have been reported in Alzheimer's disease and amnestic mild cognitive impairment, but changes in earlier pre-mild cognitive impairment stages have not been fully investigated. METHODS: In a cross-sectional analysis, older adults with mild cognitive impairment (n=28), older adults with cognitive complaints but without psychometric impairment (n=29) and healthy controls (n=35) were compared. Measures included whole-brain diffusion tensor imaging, T1-weighted structural magnetic resonance imaging, and neuropsychological assessment. Diffusion images were analyzed using Tract-Based Spatial Statistics. Voxel-wise fractional anisotropy and mean, axial, and radial diffusivities were assessed and compared between groups. Significant tract clusters were extracted in order to perform further region of interest comparisons. Brain volume was estimated using FreeSurfer based on T1 structural images. RESULTS: The mild cognitive impairment group showed lower fractional anisotropy and higher radial diffusivity than controls in bilateral parahippocampal white matter. When comparing extracted diffusivity measurements from bilateral parahippocampal white matter clusters, the cognitive complaint group had values that were intermediate to the mild cognitive impairment and healthy control groups. Group difference in diffusion tensor imaging measures remained significant after controlling for hippocampal atrophy. Across the entire sample, diffusion tensor imaging indices in parahippocampal white matter were correlated with memory function. CONCLUSIONS: These findings are consistent with previous results showing changes in parahippocampal white matter in Alzheimer's disease and mild cognitive impairment compared to controls. The intermediate pattern found in the cognitive complaint group suggests the potential of diffusion tensor imaging to contribute to earlier detection of neurodegenerative changes during prodromal stages. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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An ensemble dynamics approach to decipher solid-state NMR observables of membrane proteins.

Publication Date: 2012 Feb PMID: 21851810
Authors: Im, W. - Jo, S. - Kim, T.
Journal: Biochim Biophys Acta

Solid-state NMR (SSNMR) is an invaluable tool for determining orientations of membrane proteins and peptides in lipid bilayers. Such orientational descriptions provide essential information about membrane protein functions. However, when a semi-static single conformer model is used to interpret various SSNMR observables, important dynamics information can be missing, and, sometimes, even orientational information can be misinterpreted. In addition, over the last decade, molecular dynamics (MD) simulation and semi-static SSNMR interpretation have shown certain levels of discrepancies in terms of transmembrane helix orientation and dynamics. Dynamic fitting models have recently been proposed to resolve these discrepancies by taking into account transmembrane helix whole body motions using additional parameters. As an alternative approach, we have developed SSNMR ensemble dynamics (SSNMR-ED) using multiple conformer models, which generates an ensemble of structures that satisfies the experimental observables without any fitting parameters. In this review, various computational methods for determining transmembrane helix orientations are discussed, and the distributions of VpuTM (from HIV-1) and WALP23 (a synthetic peptide) orientations from SSNMR-ED simulations are compared with those from MD simulations and semi-static/dynamic fitting models. Such comparisons illustrate that SSNMR-ED can be used as a general means to extract both membrane protein structure and dynamics from the SSNMR measurements. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Molecular simulations and solid-state NMR investigate dynamical structure in rhodopsin activation.

Publication Date: 2012 Feb PMID: 21851809
Authors: Mertz, B. - Struts, A. V. - Feller, S. E. - Brown, M. F.
Journal: Biochim Biophys Acta

Rhodopsin has served as the primary model for studying G protein-coupled receptors (GPCRs)-the largest group in the human genome, and consequently a primary target for pharmaceutical development. Understanding the functions and activation mechanisms of GPCRs has proven to be extraordinarily difficult, as they are part of a complex signaling cascade and reside within the cell membrane. Although X-ray crystallography has recently solved several GPCR structures that may resemble the activated conformation, the dynamics and mechanism of rhodopsin activation continue to remain elusive. Notably solid-state ((2))H NMR spectroscopy provides key information pertinent to how local dynamics of the retinal ligand change during rhodopsin activation. When combined with molecular mechanics simulations of proteolipid membranes, a new paradigm for the rhodopsin activation process emerges. Experiment and simulation both suggest that retinal isomerization initiates the rhodopsin photocascade to yield not a single activated structure, but rather an ensemble of activated conformational states. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Artificial membrane-like environments for in vitro studies of purified G-protein coupled receptors.

Publication Date: 2012 Feb PMID: 21851807
Authors: Serebryany, E. - Zhu, G. A. - Yan, E. C.
Journal: Biochim Biophys Acta

Functional reconstitution of transmembrane proteins remains a significant barrier to their biochemical, biophysical, and structural characterization. Studies of seven-transmembrane G-protein coupled receptors (GPCRs) in vitro are particularly challenging because, ideally, they require access to the receptor on both sides of the membrane as well as within the plane of the membrane. However, understanding the structure and function of these receptors at the molecular level within a native-like environment will have a large impact both on basic knowledge of cell signaling and on pharmacological research. The goal of this article is to review the main classes of membrane mimics that have been, or could be, used for functional reconstitution of GPCRs. These include the use of micelles, bicelles, lipid vesicles, nanodiscs, lipidic cubic phases, and planar lipid membranes. Each of these approaches is evaluated with respect to its fundamental advantages and limitations and its applications in the field of GPCR research. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Water wires in atomistic models of the Hv1 proton channel.

Publication Date: 2012 Feb PMID: 21843503
Authors: Wood, M. L. - Schow, E. V. - Freites, J. A. - White, S. H. - Tombola, F. - Tobias, D. J.
Journal: Biochim Biophys Acta

The voltage-gated proton channel (Hv1) is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels but lacks a separate pore domain. The Hv1 monomer has dual functions: it gates the proton current and also serves as the proton conduction pathway. To gain insight into the structure and dynamics of the yet unresolved proton permeation pathway, we performed all-atom molecular dynamics simulations of two different Hv1 homology models in a lipid bilayer in excess water. The structure of the Kv1.2-Kv2.1 paddle-chimera VSD was used as template to generate both models, but they differ in the sequence alignment of the S4 segment. In both models, we observe a water wire that extends through the membrane, whereas the corresponding region is dry in simulations of the Kv1.2-Kv2.1 paddle-chimera. We find that the kinetic stability of the water wire is dependent upon the identity and location of the residues lining the permeation pathway, in particular, the S4 arginines. A measurement of water transport kinetics indicates that the water wire is a relatively static feature of the permeation pathway. Taken together, our results suggest that proton conduction in Hv1 may occur via Grotthuss hopping along a robust water wire, with exchange of water molecules between inner and outer ends of the permeation pathway minimized by specific water-protein interactions. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Coarse grained model for exploring voltage dependent ion channels.

Publication Date: 2012 Feb PMID: 21843502
Authors: Dryga, A. - Chakrabarty, S. - Vicatos, S. - Warshel, A.
Journal: Biochim Biophys Acta

The relationship between the membrane voltage and the gating of voltage activated ion channels and other systems have been a problem of great current interest. Unfortunately, reliable molecular simulations of external voltage effects present a major challenge, since meaningful converging microscopic simulations are not yet available and macroscopic treatments involve major uncertainties in terms of the dielectric used and other key features. This work extends our coarse grained (CG) model to simulations of membrane/protein systems under external potential. Special attention is devoted to a consistent modeling of the effect of external potential due to the electrodes, emphasizing semimacroscopic description of the electrolytes in the solution regions between the membranes and the electrodes, as well as the coupling between the combined potential from the electrodes plus the electrolytes and the protein ionized groups. We also provide a clear connection to microscopic treatment of the electrolytes and thus can explore possible conceptual problems that are hard to resolve by other current approaches. For example, we obtain a clear description of the charge distribution in the entire electrolyte system, including near the electrodes in membrane/electrodes systems (where continuum models do not seem to provide the relevant results). Furthermore, the present treatment provides an insight on the distribution of the electrolyte charges before and after equilibration across the membrane, and thus on the nature of the gating charge. The different aspects of the model have been carefully validated by considering problems ranging for the simple Debye-Huckel, and the Gouy-Chapman models to the evaluation of the electrolyte distribution between two electrodes, as well as the effect of extending the simulation system by periodic replicas. Overall the clear connection to microscopic descriptions combined with the power of the CG modeling seems to offer a powerful tool for exploring the balance between the protein conformational energy and the interaction with the external potential in voltage activated channels. To illustrate these features we present a preliminary study of the gating charge in the voltage activated Kv1.2 channel, using the actual change in the electrolyte charge distribution rather than the conventional macroscopic estimate. We also discuss other special features of the model, which include the ability to capture the effect of changes in the protonation states of the protein residues during the close to open voltage induced transition. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Structural brain imaging in frontotemporal dementia.

Publication Date: 2012 Mar PMID: 21839829
Authors: Rohrer, J. D.
Journal: Biochim Biophys Acta

Frontotemporal dementia (FTD) is the second commonest young-onset neurodegenerative dementia. The canonical clinical syndromes are a behavioural variant (bvFTD) and two language variants (progressive nonfluent aphasia, PNFA, and semantic dementia, SD) although there is overlap with motor neurone disease and the atypical parkinsonian disorders corticobasal syndrome (CBS) and progressive supranuclear palsy syndrome (PSPS). Characteristic patterns of atrophy or hypometabolism are described in each of the variants but in reality imaging studies are rather heterogeneous. This review attempts to address four key questions in the neuroimaging of FTD: 1) what are the early imaging features of the different FTD syndromes (and how do these change as the disease progresses); 2) what do studies of presymptomatic genetic cases of FTD tell us about the very early stages of the disease; 3) can neuroimaging help to differentiate the different FTD syndromes; and 4) can neuroimaging help to differentiate FTD from other neurodegenerative diseases? This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy.

Publication Date: 2012 Feb PMID: 21839724
Authors: Gustavsson, M. - Traaseth, N. J. - Veglia, G.
Journal: Biochim Biophys Acta

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Biophysics of alpha-synuclein membrane interactions.

Publication Date: 2012 Feb PMID: 21819966
Authors: Pfefferkorn, C. M. - Jiang, Z. - Lee, J. C.
Journal: Biochim Biophys Acta

Membrane proteins participate in nearly all cellular processes; however, because of experimental limitations, their characterization lags far behind that of soluble proteins. Peripheral membrane proteins are particularly challenging to study because of their inherent propensity to adopt multiple and/or transient conformations in solution and upon membrane association. In this review, we summarize useful biophysical techniques for the study of peripheral membrane proteins and their application in the characterization of the membrane interactions of the natively unfolded and Parkinson's disease (PD) related protein, alpha-synuclein (alpha-syn). We give particular focus to studies that have led to the current understanding of membrane-bound alpha-syn structure and the elucidation of specific membrane properties that affect alpha-syn-membrane binding. Finally, we discuss biophysical evidence supporting a key role for membranes and alpha-syn in PD pathogenesis. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Characterization of a potent antimicrobial lipopeptide via coarse-grained molecular dynamics.

Publication Date: 2012 Feb PMID: 21819964
Authors: Horn, J. N. - Sengillo, J. D. - Lin, D. - Romo, T. D. - Grossfield, A.
Journal: Biochim Biophys Acta

The prevalence of antibiotic-resistant pathogens is a major medical concern, prompting increased interest in the development of novel antimicrobial compounds. One such set of naturally occurring compounds, known as antimicrobial peptides (AMPs), have broad-spectrum activity, but come with many limitations for clinical use. Recent work has resulted in a set of antimicrobial lipopeptides (AMLPs) with micromolar minimum inhibitory concentrations and excellent selectivity for bacterial membranes. To characterize a potent, synthetic lipopeptide, C16-KGGK, we used multi-microsecond coarse-grained simulations with the MARTINI forcefield, with a total simulation time of nearly 46mus. These simulations show rapid binding of C16-KGGK, which forms micelles in solution, to model bacterial lipid bilayers. Furthermore, upon binding to the surface of the bilayer, these lipopeptides alter the local lipid organization by recruiting negatively charged POPG lipids to the site of binding. It is likely that this drastic reorganization of the bilayer has major effects on bilayer dynamics and cellular processes that depend on specific bilayer compositions. By contrast, the simulations revealed no association between the lipopeptides and model mammalian bilayers. These simulations provide biophysical insights into lipopeptide selectivity and suggest a possible mechanism for antimicrobial action. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Outer membrane phospholipase A in phospholipid bilayers: A model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers.

Publication Date: 2012 Feb PMID: 21816133
Authors: Fleming, P. J. - Freites, J. A. - Moon, C. P. - Tobias, D. J. - Fleming, K. G.
Journal: Biochim Biophys Acta

Understanding the forces that stabilize membrane proteins in their native states is one of the contemporary challenges of biophysics. To date, estimates of side chain partitioning free energies from water to the lipid environment show disparate values between experimental and computational measures. Resolving the disparities is particularly important for understanding the energetic contributions of polar and charged side chains to membrane protein function because of the roles these residue types play in many cellular functions. In general, computational free energy estimates of charged side chain partitioning into bilayers are much larger than experimental measurements. However, the lack of a protein-based experimental system that uses bilayers against which to vet these computational predictions has traditionally been a significant drawback. Moon & Fleming recently published a novel hydrophobicity scale that was derived experimentally by using a host-guest strategy to measure the side chain energetic perturbation due to mutation in the context of a native membrane protein inserted into a phospholipid bilayer. These values are still approximately an order of magnitude smaller than computational estimates derived from molecular dynamics calculations from several independent groups. Here we address this discrepancy by showing that the free energy differences between experiment and computation become much smaller if the appropriate comparisons are drawn, which suggests that the two fields may in fact be converging. In addition, we present an initial computational characterization of the Moon & Fleming experimental system used for the hydrophobicity scale: OmpLA in DLPC bilayers. The hydrophobicity scale used OmpLA position 210 as the guest site, and our preliminary results demonstrate that this position is buried in the center of the DLPC membrane, validating its usage in the experimental studies. We further showed that the introduction of charged Arg at position 210 is well tolerated in OmpLA and that the DLPC bilayers accommodate this perturbation by creating a water dimple that allows the Arg side chain to remain hydrated. Lipid head groups visit the dimple and can hydrogen bond with Arg, but these interactions are transient. Overall, our study demonstrates the unique advantages of this molecular system because it can be interrogated by both computational and experimental practitioners, and it sets the stage for free energy calculations in a system for which there is unambiguous experimental data. This article is part of a Special Issue entitled: Membrane protein structure and function.

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The influence of exercise on brain aging and dementia.

Publication Date: 2012 Mar PMID: 21810472
Authors: Lautenschlager, N. T. - Cox, K. - Cyarto, E. V.
Journal: Biochim Biophys Acta

Physical activity has been recognized as an important protective factor reducing disability and mortality and therefore it is focus of many health promotion activities at all ages. More recently a growing body of literature is focusing whether physical activity could also have a positive impact on brain aging with exploring healthy brain aging as well as on cognitive impairment and dementia. An increasing number of prospective studies and randomized controlled trials involving humans take place both with older adults with normal cognition as well as with mild cognitive impairment or dementia. However, the body of evidence is still sparse and many methodological issues make comparisons across studies challenging. Increasingly research into underlying mechanisms in relation to physical activity and brain aging identify biomarker candidates with especially neuroimaging measurements being more used in trials with humans. Whilst the evidence base is slowly growing more detailed research is needed to address methodological issues to finally achieve clinical relevance. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Imaging the default mode network in aging and dementia.

Publication Date: 2012 Mar PMID: 21807094
Authors: Hafkemeijer, A. - van der Grond, J. - Rombouts, S. A.
Journal: Biochim Biophys Acta

Although in the last decade brain activation in healthy aging and dementia was mainly studied using task-activation fMRI, there is increasing interest in task-induced decreases in brain activity, termed deactivations. These deactivations occur in the so-called default mode network (DMN). In parallel a growing number of studies focused on spontaneous, ongoing 'baseline' activity in the DMN. These resting state fMRI studies explored the functional connectivity in the DMN. Here we review whether normal aging and dementia affect task-induced deactivation and functional connectivity in the DMN. The majority of studies show a decreased DMN functional connectivity and task-induced DMN deactivations along a continuum from normal aging to mild cognitive impairment and to Alzheimer's disease (AD). Even subjects at risk for developing AD, either in terms of having amyloid plaques or carrying the APOE4 allele, showed disruptions in the DMN. While fMRI is a useful tool for detecting changes in DMN functional connectivity and deactivation, more work needs to be conducted to conclude whether these measures will become useful as a clinical diagnostic tool in AD. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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White matter integrity and vulnerability to Alzheimer's disease: Preliminary findings and future directions.

Publication Date: 2012 Mar PMID: 21803153
Authors: Gold, B. T. - Johnson, N. F. - Powell, D. K. - Smith, C. D.
Journal: Biochim Biophys Acta

Neuroimaging biomarkers that precede cognitive decline have the potential to aid early diagnosis of Alzheimer's disease (AD). A body of diffusion tensor imaging (DTI) work has demonstrated declines in white matter (WM) microstructure in AD and its typical prodromal state, amnestic mild cognitive impairment. The present review summarizes recent evidence suggesting that WM integrity declines are present in individuals at high AD-risk, prior to cognitive decline. The available data suggest that AD-risk is associated with WM integrity declines in a subset of tracts showing decline in symptomatic AD. Specifically, AD-risk has been associated with WM integrity declines in tracts that connect gray matter structures associated with memory function. These tracts include parahippocampal WM, the cingulum, the inferior fronto-occipital fasciculus, and the splenium of the corpus callosum. Preliminary evidence suggests that some AD-risk declines are characterized by increases of radial diffusivity, raising the possibility that a myelin-related pathology may contribute to AD onset. These findings justify future research aimed at a more complete understanding of the neurobiological bases of DTI-based declines in AD. With continued refinement of imaging methods, DTI holds promise as a method to aid identification of presymptomatic AD. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Hydrogen-bond energetics drive helix formation in membrane interfaces.

Publication Date: 2012 Feb PMID: 21802405
Authors: Almeida, P. F. - Ladokhin, A. S. - White, S. H.
Journal: Biochim Biophys Acta

The free energy cost DeltaG of partitioning many unfolded peptides into membrane interfaces is unfavorable due to the cost of partitioning backbone peptide bonds. The partitioning cost is dramatically reduced if the peptide bonds participate in hydrogen bonds. The reduced cost underlies secondary structure formation by amphiphilic peptides partitioned into membrane interfaces through a process referred to as partitioning-folding coupling. This coupling is characterized by the free energy reduction per residue, G(res) that drives folding. There is some debate about the correct value of G(res) and its dependence on the hydrophobic moment (mu(H)) of amphiphilic alpha-helical peptides. We show how to compute G(res) correctly. Using published data for two families of peptides with different hydrophobic moments and charges, we find that G(res) does not depend upon mu(H). The best estimate of G(res) is -0.37+/-0.02kcalmol(-1). This article is part of a Special Issue entitled: Membrane protein structure and function.

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Lipid composition regulates the conformation and insertion of the antimicrobial peptide maculatin 1.1.

Publication Date: 2012 Feb PMID: 21801711
Authors: Sani, M. A. - Whitwell, T. C. - Separovic, F.
Journal: Biochim Biophys Acta

Antimicrobial peptides interact with cell membranes and their selectivity is contingent on the nature of the constituent lipids. Eukaryotic and bacterial membranes are comprised of different proportions of a range of lipid species with different physical properties. Hence, characterisation of antimicrobial peptides with respect to the magnitude of their interactions with model membranes of different lipid types is needed. Maculatin 1.1 is a short antimicrobial peptide secreted from the skin of several Australian tree-frog species. Circular dichroism spectroscopy (CD) was used to explore the interaction of maculatin 1.1 with a wide range of model membrane systems of different head group and acyl chain characteristics. For neutral phosphatidylcholine (PC), unlike anionic phospholipids, the magnitude of the peptide interactions was dependent on the length and degree of saturation of the constituent acyl chains. Oriented circular dichroism (OCD) data indicated that helical structure was likely promoted by peptide insertion into the hydrophobic core of PC bilayers. The addition of cholesterol (30% mol/mol) tended to decrease the membrane interaction of maculatin 1.1. Anionic lipids locked maculatin 1.1 via electrostatic interactions onto the surface of oriented bilayers as seen in OCD spectra. Furthermore, increasing the membrane curvature by reducing the vesicle radii only slightly reduced the proportion of helical structure in all systems by approximately 10%. The peptide-lipid interaction was strongly dependent on both the lipid chain length and head group, which highlights the importance of the lipid composition used to mimic different cell types. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Neurodegeneration with brain iron accumulation - Clinical syndromes and neuroimaging.

Publication Date: 2012 Mar PMID: 21782937
Authors: Schipper, H. M.
Journal: Biochim Biophys Acta

Iron participates in a wide array of cellular functions and is essential for normal neural development and physiology. However, if inappropriately managed, the transition metal is capable of generating neurotoxic reactive oxygen species. A number of hereditary conditions perturb body iron homeostasis and some, collectively referred to as neurodegeneration with brain iron accumulation (NBIA), promote pathological deposition of the metal predominantly or exclusively within the central nervous system (CNS). In this article, we discuss seven NBIA disorders with emphasis on the clinical syndromes and neuroimaging. The latter primarily entails magnetic resonance scanning using iron-sensitive sequences. The conditions considered are Friedreich ataxia (FA), pantothenate kinase 2-associated neurodegeneration (PKAN), PLA2G6-associated neurodegeneration (PLAN), FA2H-associated neurodegeneration (FAHN), Kufor-Rakeb disease (KRD), aceruloplasminemia, and neuroferritinopathy. An approach to differential diagnosis and the status of iron chelation therapy for several of these entities are presented. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Dementia: Alzheimer pathology and vascular factors: From mutually exclusive to interaction.

Publication Date: 2012 Mar PMID: 21777675
Authors: van Norden, A. G. - van Dijk, E. J. - de Laat, K. F. - Scheltens, P. - Olderikkert, M. G. - de Leeuw, F. E.
Journal: Biochim Biophys Acta

Alzheimer's disease (AD) is the most common type of dementia. Both its incidence and prevalence are expected to increase exponentially as populations' age worldwide. Despite impressive efforts of research worldwide, neither cure nor effective preventive strategy is available for this devastating disease. Currently there are several hypotheses on what causes AD, with the amyloid hypothesis being the most investigated and accepted hypothesis over the past 20years. However the exact role of amyloid-beta in the onset and progression of AD is not yet fully understood, and even the validity of the amyloid hypothesis itself is still being discussed. This debate is fuelled by the vascular hypothesis, as increasing epidemiological, neuroimaging, pathological, pharmacotherapeutic and clinical studies suggest that vascular pathology plays a key role in the onset and progression of AD. We here will discuss arguments in favor and limitations of both hypotheses within the framework of available literature, but also provide arguments for convergence of both hypotheses. Finally we propose approaches that may aid in unraveling the etiology and treatment of AD. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Structural imaging in early pre-states of dementia.

Publication Date: 2012 Mar PMID: 21777674
Authors: Smith, C. D.
Journal: Biochim Biophys Acta

In this review focus is on structural imaging in the Alzheimer's disease (AD) pre-states, particularly cognitively normal (CN) persons at future dementia risk. Findings in mild cognitive impairment (MCI) are described here only for comparison with CN. Cited literature evidence and commentary address issues of structural imaging alterations in CN that precede MCI and AD, regional patterns of such alterations, and the time relationship between structural imaging alterations and the appearance of symptoms of AD, issues relevant to the conduct of future AD prevention trials. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Lipid-protein interactions in biological membranes: A dynamic perspective.

Publication Date: 2012 Feb PMID: 21763269
Authors: Smith, A. W.
Journal: Biochim Biophys Acta

Though an increasing number of biological functions at the membrane are attributed to direct associations between lipid head groups and protein side chains or lipid protein hydrophobic attractive forces, surprisingly limited information is available about the dynamics of these interactions. The static in vitro representation provided by membrane protein structures, including very insightful lipid-protein binding geometries, still fails to recapitulate the dynamic behavior characteristic of lipid membranes. Experimental measures of the interaction time of lipid-protein association are very rare, and have only provided order-of-magnitude estimates in an extremely limited number of systems. In this review, a brief outline of the experimental approaches taken in this area to date is given. The bulk of the review will focus on two methods that are promising techniques for measuring lipid-protein interactions: time-resolved fluorescence microscopy, and two-dimensional infrared (2D IR) spectroscopy. Time-resolved fluorescence microscopy is the name given to a sophisticated toolbox of measurements taken using pulsed laser excitation and time-correlated single photon counting (TCSPC). With this technique the dynamics of interaction can be measured on the time scale of nanoseconds to milliseconds. 2D IR is a femtosecond nonlinear spectroscopy that can resolve vibrational coupling between lipids and proteins at molecular-scale distances and at time scales from femtoseconds to picoseconds. These two methods are poised to make significant advances in our understanding of the dynamic properties of biological membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Insulin is differentially related to cognitive decline and atrophy in Alzheimer's disease and aging.

Publication Date: 2012 Mar PMID: 21745566
Authors: Burns, J. M. - Honea, R. A. - Vidoni, E. D. - Hutfles, L. J. - Brooks, W. M. - Swerdlow, R. H.
Journal: Biochim Biophys Acta

We assessed the relationship of insulin resistance with cognitive decline and brain atrophy over two years in early Alzheimer's disease (AD, n=48) and nondemented controls (n=61). Intravenous glucose tolerance tests were conducted at baseline to determine insulin area-under-the-curve (AUC). A standard battery of cognitive tasks and MRI were conducted at baseline and 2-year follow-up. In nondemented controls, higher baseline insulin AUC was associated with 2-year decline in global cognitive performance (beta=-0.36, p=0.005). In early AD, however, higher insulin AUC was associated with less decline in global cognitive performance (beta=0.26, p=0.06), slower global brain atrophy (beta=0.40, p=0.01) and less regional atrophy in the bilateral hippocampi and cingulate cortices. While insulin resistance is associated with cognitive decline in nondemented aging, higher peripheral insulin may have AD-specific benefits or insulin signaling may be affected by systemic physiologic changes associated with AD. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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Side-chain oxysterols: From cells to membranes to molecules.

Publication Date: 2012 Feb PMID: 21745458
Authors: Olsen, B. N. - Schlesinger, P. H. - Ory, D. S. - Baker, N. A.
Journal: Biochim Biophys Acta

This review discusses the application of cellular biology, molecular biophysics, and computational simulation to understand membrane-mediated mechanisms by which oxysterols regulate cholesterol homeostasis. Side-chain oxysterols, which are produced enzymatically in vivo, are physiological regulators of cholesterol homeostasis and primarily serve as cellular signals for excess cholesterol. These oxysterols regulate cholesterol homeostasis through both transcriptional and non-transcriptional pathways; however, many molecular details of their interactions in these pathways are still not well understood. Cholesterol trafficking provides one mechanism for regulation. The current model of cholesterol trafficking regulation is based on the existence of two distinct cholesterol pools in the membrane: a low and a high availability/activity pool. It is proposed that the low availability/activity pool of cholesterol is integrated into tightly packing phospholipids and relatively inaccessible to water or cellular proteins, while the high availability cholesterol pool is more mobile in the membrane and is present in membranes where the phospholipids are not as compressed. Recent results suggest that oxysterols may promote cholesterol egress from membranes by shifting cholesterol from the low to the high activity pools. Furthermore, molecular simulations suggest a potential mechanism for oxysterol "activation" of cholesterol through its displacement in the membrane. This review discusses these results as well as several other important interactions between oxysterols and cholesterol in cellular and model lipid membranes. This article is part of a Special Issue entitled: Membrane protein structure and function.

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Diffusion tensor imaging and cognition in cerebral small vessel disease The RUN DMC study.

Publication Date: 2012 Mar PMID: 21549191
Authors: van Norden, A. G. - de Laat, K. F. - van Dijk, E. J. - van Uden, I. W. - van Oudheusden, L. J. - Gons, R. A. - Norris, D. G. - Zwiers, M. P. - de Leeuw, F. E.
Journal: Biochim Biophys Acta

BACKGROUND: Cerebral small vessel disease (SVD) is very common in elderly and related to cognition, although this relation is weak. This might be because the underlying pathology of white matter lesions (WML) is diverse and cannot be properly appreciated with conventional FLAIR MRI. In addition, conventional MRI is not sensitive to early loss of microstructural integrity of the normal appearing white matter (NAWM), which might be an important factor. Diffusion tensor imaging (DTI) provides alternative information on microstructural white matter integrity and we have used this to investigate the relation between white matter integrity, in both WML and NAWM, and cognition among elderly with cerebral SVD. METHODS: The RUN DMC study is a prospective cohort study among 503 independently living, non-demented elderly with cerebral SVD aged between 50 and 85years. All subjects underwent MRI and DTI scanning. WML were segmented manually. We measured mean diffusivity (MD) and fractional anisotropy (FA), as assessed by DTI in both WML and NAWM. RESULTS: Inverse relations were found between MD in the WML and NAWM and global cognitive function (beta=-.11, p<0.05; beta=-.18, p<0.001), psychomotor speed (beta=-.15, p<0.01; beta=-.18, p<0.001), concept shifting (beta=-.11, p<0.05; beta=-.10, p<0.05) and attention (beta=-.12, p<0.05; beta=-.15, p<0.001). The relation between DTI parameters in both WML and NAWM and cognitive performance was most pronounced in subjects with severe WML. CONCLUSION: DTI parameters in both WML and NAWM correlate with cognitive performance, independent of SVD. DTI may be a promising tool in exploring the mechanisms of cognitive decline and could function as a surrogate marker for disease progression in therapeutic trials. This article is part of a Special Issue entitled: Imaging Brain Aging and Neurodegenerative disease.

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