EMBO Journal

A structural road map to unveil basal body composition and assembly.

Publication Date: 2012 Feb 1 PMID: 22293831
Authors: Jana, S. C. - Machado, P. - Bettencourt-Dias, M.
Journal: EMBO J

post to: CiteULike

Rac1 gets fattier.

Publication Date: 2012 Feb 1 PMID: 22293830
Authors: Tsai, F. D. - Philips, M. R.
Journal: EMBO J

post to: CiteULike

RNA driving the epigenetic bus.

Publication Date: 2012 Feb 1 PMID: 22293829
Authors: Mattick, J. S.
Journal: EMBO J

post to: CiteULike

DNA methylation profiling identifies epigenetic dysregulation in pancreatic islets from type 2 diabetic patients.

Publication Date: 2012 Jan 31 PMID: 22293752
Authors: Volkmar, M. - Dedeurwaerder, S. - Cunha, D. A. - Ndlovu, M. N. - Defrance, M. - Deplus, R. - Calonne, E. - Volkmar, U. - Igoillo-Esteve, M. - Naamane, N. - Del Guerra, S. - Masini, M. - Bugliani, M. - Marchetti, P. - Cnop, M. - Eizirik, D. L. - Fuks, F.
Journal: EMBO J

In addition to genetic predisposition, environmental and lifestyle factors contribute to the pathogenesis of type 2 diabetes (T2D). Epigenetic changes may provide the link for translating environmental exposures into pathological mechanisms. In this study, we performed the first comprehensive DNA methylation profiling in pancreatic islets from T2D and non-diabetic donors. We uncovered 276 CpG loci affiliated to promoters of 254 genes displaying significant differential DNA methylation in diabetic islets. These methylation changes were not present in blood cells from T2D individuals nor were they experimentally induced in non-diabetic islets by exposure to high glucose. For a subgroup of the differentially methylated genes, concordant transcriptional changes were present. Functional annotation of the aberrantly methylated genes and RNAi experiments highlighted pathways implicated in beta-cell survival and function; some are implicated in cellular dysfunction while others facilitate adaptation to stressors. Together, our findings offer new insights into the intricate mechanisms of T2D pathogenesis, underscore the important involvement of epigenetic dysregulation in diabetic islets and may advance our understanding of T2D aetiology.

post to: CiteULike

APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer.

Publication Date: 2012 Jan 31 PMID: 22293751
Authors: Brough, R. - Bajrami, I. - Vatcheva, R. - Natrajan, R. - Reis-Filho, J. S. - Lord, C. J. - Ashworth, A.
Journal: EMBO J

Mutations in BRCA2 confer an increased risk of cancer development, at least in part because the BRCA2 protein is required for the maintenance of genomic integrity. Here, we use proteomic profiling to identify APRIN (PDS5B), a cohesion-associated protein, as a BRCA2-associated protein. After exposure of cells to hydroxyurea or aphidicolin, APRIN and other cohesin components associate with BRCA2 in early S-phase. We demonstrate that APRIN expression is required for the normal response to DNA-damaging agents, the nuclear localisation of RAD51 and BRCA2 and efficient homologous recombination. The clinical significance of these findings is indicated by the observation that the BRCA2/APRIN interaction is compromised by BRCA2 missense variants of previously unknown significance and that APRIN expression levels are associated with histological grade in breast cancer and the outcome of breast cancer patients treated with DNA-damaging chemotherapy.

post to: CiteULike

Soj/ParA stalls DNA replication by inhibiting helix formation of the initiator protein DnaA.

Publication Date: 2012 Jan 27 PMID: 22286949
Authors: Scholefield, G. - Errington, J. - Murray, H.
Journal: EMBO J

Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria.

post to: CiteULike

The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.

Publication Date: 2012 Jan 27 PMID: 22286948
Authors: Akopian, T. - Kandror, O. - Raju, R. M. - Unnikrishnan, M. - Rubin, E. J. - Goldberg, A. L.
Journal: EMBO J

Mycobacterium tuberculosis (Mtb) contains two clpP genes, both of which are essential for viability. We expressed and purified Mtb ClpP1 and ClpP2 separately. Although each formed a tetradecameric structure and was processed, they lacked proteolytic activity. We could, however, reconstitute an active, mixed ClpP1P2 complex after identifying N-blocked dipeptides that stimulate dramatically (>1000-fold) ClpP1P2 activity against certain peptides and proteins. These activators function cooperatively to induce the dissociation of ClpP1 and ClpP2 tetradecamers into heptameric rings, which then re-associate to form the active ClpP1P2 2-ring mixed complex. No analogous small molecule-induced enzyme activation mechanism involving dissociation and re-association of multimeric rings has been described. ClpP1P2 possesses chymotrypsin and caspase-like activities, and ClpP1 and ClpP2 differ in cleavage preferences. The regulatory ATPase ClpC1 was purified and shown to increase hydrolysis of proteins by ClpP1P2, but not peptides. ClpC1 did not activate ClpP1 or ClpP2 homotetradecamers and stimulated ClpP1P2 only when both ATP and a dipeptide activator were present. ClpP1P2 activity, its unusual activation mechanism and ClpC1 ATPase represent attractive drug targets to combat tuberculosis.

post to: CiteULike

PML regulates PER2 nuclear localization and circadian function.

Publication Date: 2012 Jan 24 PMID: 22274616
Authors: Miki, T. - Xu, Z. - Chen-Goodspeed, M. - Liu, M. - Van Oort-Jansen, A. - Rea, M. A. - Zhao, Z. - Lee, C. C. - Chang, K. S.
Journal: EMBO J

Studies have suggested that the clock regulator PER2 is a tumour suppressor. A cancer network involving PER2 raises the possibility that some tumour suppressors are directly involved in the mammalian clock. Here, we show that the tumour suppressor promyelocytic leukaemia (PML) protein is a circadian clock regulator and can physically interact with PER2. In the suprachiasmatic nucleus (SCN), PML expression and PML-PER2 interaction are under clock control. Loss of PML disrupts and dampens the expression of clock regulators Per2, Per1, Cry1, Bmal1 and Npas2. In the presence of PML and PER2, BMAL1/CLOCK-mediated transcription is enhanced. In Pml(-/-) SCN and mouse embryo fibroblast cells, the cellular distribution of PER2 is primarily perinuclear/cytoplasmic. PML is acetylated at K487 and its deacetylation by SIRT1 promotes PML control of PER2 nuclear localization. The circadian period of Pml(-/-) mice displays reduced precision and stability consistent with PML having a role in the mammalian clock mechanism.

post to: CiteULike

Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex.

Publication Date: 2012 Jan 24 PMID: 22274615
Authors: Rivera, T. - Gheniou, C. - Rodriguez-Corsino, M. - Mochida, S. - Funabiki, H. - Losada, A.
Journal: EMBO J

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore-microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex.

post to: CiteULike

Myeloid translocation gene 16 is required for maintenance of haematopoietic stem cell quiescence.

Publication Date: 2012 Jan 20 PMID: 22266796
Authors: Fischer, M. A. - Moreno-Miralles, I. - Hunt, A. - Chyla, B. J. - Hiebert, S. W.
Journal: EMBO J

The t(8;21) and t(16;21) that are associated with acute myeloid leukaemia disrupt two closely related genes termed Myeloid Translocation Genes 8 (MTG8) and 16 (MTG16), respectively. Many of the transcription factors that recruit Mtg16 regulate haematopoietic stem and progenitor cell functions and are required to maintain stem cell self-renewal potential. Accordingly, we found that Mtg16-null bone marrow (BM) failed in BM transplant assays. Moreover, when removed from the animal, Mtg16-deficient stem cells continued to show defects in stem cell self-renewal assays, suggesting a requirement for Mtg16 in this process. Gene expression analysis indicated that Mtg16 was required to suppress the expression of several key cell-cycle regulators including E2F2, and chromatin immunoprecipitation assays detected Mtg16 near an E2A binding site within the first intron of E2F2. BrdU incorporation assays indicated that in the absence of Mtg16 more long-term stem cells were in the S phase, even after competitive BM transplantation where normal stem and progenitor cells are present, suggesting that Mtg16 plays a role in the maintenance of stem cell quiescence.

post to: CiteULike

ATR maintains select progenitors during nervous system development.

Publication Date: 2012 Jan 20 PMID: 22266795
Authors: Lee, Y. - Shull, E. R. - Frappart, P. O. - Katyal, S. - Enriquez-Rios, V. - Zhao, J. - Russell, H. R. - Brown, E. J. - McKinnon, P. J.
Journal: EMBO J

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.

post to: CiteULike

The nuclear receptor signalling scaffold: insights from full-length structures.

Publication Date: 2012 PMID: 22252143
Authors: Nwachukwu, J. C. - Nettles, K. W.
Journal: EMBO J

post to: CiteULike

Raising cytosolic Cl(-) in cerebellar granule cells affects their excitability and vestibulo-ocular learning.

Publication Date: 2012 Jan 17 PMID: 22252133
Authors: Seja, P. - Schonewille, M. - Spitzmaul, G. - Badura, A. - Klein, I. - Rudhard, Y. - Wisden, W. - Hubner, C. A. - De Zeeuw, C. I. - Jentsch, T. J.
Journal: EMBO J

Cerebellar cortical throughput involved in motor control comprises granule cells (GCs) and Purkinje cells (PCs), both of which receive inhibitory GABAergic input from interneurons. The GABAergic input to PCs is essential for learning and consolidation of the vestibulo-ocular reflex, but the role of GC excitability remains unclear. We now disrupted the Kcc2 K-Cl cotransporter specifically in either cell type to manipulate their excitability and inhibition by GABA(A)-receptor Cl(-) channels. Although Kcc2 may have a morphogenic role in synapse development, Kcc2 disruption neither changed synapse density nor spine morphology. In both GCs and PCs, disruption of Kcc2, but not Kcc3, increased [Cl(-)](i) roughly two-fold. The reduced Cl(-) gradient nearly abolished GABA-induced hyperpolarization in PCs, but in GCs it merely affected excitability by membrane depolarization. Ablation of Kcc2 from GCs impaired consolidation of long-term phase learning of the vestibulo-ocular reflex, whereas baseline performance, short-term gain-decrease learning and gain consolidation remained intact. These functions, however, were affected by disruption of Kcc2 in PCs. GC excitability plays a previously unknown, but specific role in consolidation of phase learning.

post to: CiteULike

Enlightening the impact of immunogenic cell death in photodynamic cancer therapy.

Publication Date: 2012 Jan 17 PMID: 22252132
Authors: Galluzzi, L. - Kepp, O. - Kroemer, G.
Journal: EMBO J

post to: CiteULike

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin.

Publication Date: 2012 Jan 17 PMID: 22252131
Authors: Mukherjee, M. - Chow, S. Y. - Yusoff, P. - Seetharaman, J. - Ng, C. - Sinniah, S. - Koh, X. W. - Asgar, N. F. - Li, D. - Yim, D. - Jackson, R. A. - Yew, J. - Qian, J. - Iyu, A. - Lim, Y. P. - Zhou, X. - Sze, S. K. - Guy, G. R. - Sivaraman, J.
Journal: EMBO J

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.

post to: CiteULike

Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly.

Publication Date: 2012 Jan 17 PMID: 22252130
Authors: Hornig-Do, H. T. - Tatsuta, T. - Buckermann, A. - Bust, M. - Kollberg, G. - Rotig, A. - Hellmich, M. - Nijtmans, L. - Wiesner, R. J.
Journal: EMBO J

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly.

post to: CiteULike

Hypoxia regulates glutamate receptor trafficking through an HIF-independent mechanism.

Publication Date: 2012 Jan 17 PMID: 22252129
Authors: Park, E. C. - Ghose, P. - Shao, Z. - Ye, Q. - Kang, L. - Xu, X. Z. - Powell-Coffman, J. A. - Rongo, C.
Journal: EMBO J

Oxygen influences behaviour in many organisms, with low levels (hypoxia) having devastating consequences for neuron survival. How neurons respond physiologically to counter the effects of hypoxia is not fully understood. Here, we show that hypoxia regulates the trafficking of the glutamate receptor GLR-1 in C. elegans neurons. Either hypoxia or mutations in egl-9, a prolyl hydroxylase cellular oxygen sensor, result in the internalization of GLR-1, the reduction of glutamate-activated currents, and the depression of GLR-1-mediated behaviours. Surprisingly, hypoxia-inducible factor (HIF)-1, the canonical substrate of EGL-9, is not required for this effect. Instead, EGL-9 interacts with the Mint orthologue LIN-10, a mediator of GLR-1 membrane recycling, to promote LIN-10 subcellular localization in an oxygen-dependent manner. The observed effects of hypoxia and egl-9 mutations require the activity of the proline-directed CDK-5 kinase and the CDK-5 phosphorylation sites on LIN-10, suggesting that EGL-9 and CDK-5 compete in an oxygen-dependent manner to regulate LIN-10 activity and thus GLR-1 trafficking. Our findings demonstrate a novel mechanism by which neurons sense and respond to hypoxia.

post to: CiteULike

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death.

Publication Date: 2012 Jan 17 PMID: 22252128
Authors: Garg, A. D. - Krysko, D. V. - Verfaillie, T. - Kaczmarek, A. - Ferreira, G. B. - Marysael, T. - Rubio, N. - Firczuk, M. - Mathieu, C. - Roebroek, A. J. - Annaert, W. - Golab, J. - de Witte, P. - Vandenabeele, P. - Agostinis, P.
Journal: EMBO J

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1beta(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2alpha phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110alpha and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.

post to: CiteULike

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation.

Publication Date: 2012 Jan 13 PMID: 22246186
Authors: Barascu, A. - Le Chalony, C. - Pennarun, G. - Genet, D. - Imam, N. - Lopez, B. - Bertrand, P.
Journal: EMBO J

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.

post to: CiteULike

Rsk-mediated phosphorylation and 14-3-3varepsilon binding of Apaf-1 suppresses cytochrome c-induced apoptosis.

Publication Date: 2012 Jan 13 PMID: 22246185
Authors: Kim, J. - Parrish, A. B. - Kurokawa, M. - Matsuura, K. - Freel, C. D. - Andersen, J. L. - Johnson, C. E. - Kornbluth, S.
Journal: EMBO J

Many pro-apoptotic signals trigger mitochondrial cytochrome c release, leading to caspase activation and ultimate cellular breakdown. Cell survival pathways, including the mitogen-activated protein kinase (MAPK) cascade, promote cell viability by impeding mitochondrial cytochrome c release and by inhibiting subsequent caspase activation. Here, we describe a mechanism for the inhibition of cytochrome c-induced caspase activation by MAPK signalling, identifying a novel mode of apoptotic regulation exerted through Apaf-1 phosphorylation by the 90-kDa ribosomal S6 kinase (Rsk). Recruitment of 14-3-3varepsilon to phosphorylated Ser268 impedes the ability of cytochrome c to nucleate apoptosome formation and activate downstream caspases. High endogenous levels of Rsk in PC3 prostate cancer cells or Rsk activation in other cell types promoted 14-3-3varepsilon binding to Apaf-1 and rendered the cells insensitive to cytochrome c, suggesting a potential role for Rsk signalling in apoptotic resistance of prostate cancers and other cancers with elevated Rsk activity. Collectively, these results identify a novel locus of apoptosomal regulation wherein MAPK signalling promotes Rsk-catalysed Apaf-1 phosphorylation and consequent binding of 14-3-3varepsilon, resulting in decreased cellular responsiveness to cytochrome c.

post to: CiteULike

Microvesicles released from microglia stimulate synaptic activity via enhanced sphingolipid metabolism.

Publication Date: 2012 Jan 13 PMID: 22246184
Authors: Antonucci, F. - Turola, E. - Riganti, L. - Caleo, M. - Gabrielli, M. - Perrotta, C. - Novellino, L. - Clementi, E. - Giussani, P. - Viani, P. - Matteoli, M. - Verderio, C.
Journal: EMBO J

Microvesicles (MVs) released into the brain microenvironment are emerging as a novel way of cell-to-cell communication. We have recently shown that microglia, the immune cells of the brain, shed MVs upon activation but their possible role in microglia-to-neuron communication has never been explored. To investigate whether MVs affect neurotransmission, we analysed spontaneous release of glutamate in neurons exposed to MVs and found a dose-dependent increase in miniature excitatory postsynaptic current (mEPSC) frequency without changes in mEPSC amplitude. Paired-pulse recording analysis of evoked neurotransmission showed that MVs mainly act at the presynaptic site, by increasing release probability. In line with the enhancement of excitatory transmission in vitro, injection of MVs into the rat visual cortex caused an acute increase in the amplitude of field potentials evoked by visual stimuli. Stimulation of synaptic activity occurred via enhanced sphingolipid metabolism. Indeed, MVs promoted ceramide and sphingosine production in neurons, while the increase of excitatory transmission induced by MVs was prevented by pharmacological or genetic inhibition of sphingosine synthesis. These data identify microglia-derived MVs as a new mechanism by which microglia influence synaptic activity and highlight the involvement of neuronal sphingosine in this microglia-to-neuron signalling pathway.

post to: CiteULike

Axonal transcription factors signal retrogradely in lesioned peripheral nerve.

Publication Date: 2012 Jan 13 PMID: 22246183
Authors: Ben-Yaakov, K. - Dagan, S. Y. - Segal-Ruder, Y. - Shalem, O. - Vuppalanchi, D. - Willis, D. E. - Yudin, D. - Rishal, I. - Rother, F. - Bader, M. - Blesch, A. - Pilpel, Y. - Twiss, J. L. - Fainzilber, M.
Journal: EMBO J

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin alpha5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

post to: CiteULike

STIM1 is required for attenuation of PMCA-mediated Ca(2+) clearance during T-cell activation.

Publication Date: 2012 Jan 13 PMID: 22246182
Authors: Ritchie, M. F. - Samakai, E. - Soboloff, J.
Journal: EMBO J

T-cell activation involves a complex signalling cascade uniquely dependent on elevated cytosolic Ca(2+) levels. Further, the spatiotemporal characteristics of this Ca(2+) signal play a critical role in this process via selective activation of transcription factors. In T cells, store-operated Ca(2+) entry (SOCe) is the primary Ca(2+) influx pathway; however, cytosolic Ca(2+) concentration depends upon the balance between Ca(2+) influx and extrusion. The plasma membrane Ca(2+) ATPase (PMCA) has previously been identified as a critical player in Ca(2+) clearance in T cells. Here, we provide data revealing both functional and physical links between the activation of stromal interacting molecule 1 (STIM1) and PMCA-mediated Ca(2+) clearance. Due to the ubiquitous expression of both STIM1 and PMCA, these findings have wide-ranging implications for Ca(2+) signalling in multiple cell types.

post to: CiteULike

A DR4:tBID axis drives the p53 apoptotic response by promoting oligomerization of poised BAX.

Publication Date: 2012 Jan 13 PMID: 22246181
Authors: Henry, R. E. - Andrysik, Z. - Paris, R. - Galbraith, M. D. - Espinosa, J. M.
Journal: EMBO J

The cellular response to p53 activation varies greatly in a stimulus- and cell type-specific manner. Dissecting the molecular mechanisms defining these cell fate choices will assist the development of effective p53-based cancer therapies and also illuminate fundamental processes by which gene networks control cellular behaviour. Using an experimental system wherein stimulus-specific p53 responses are elicited by non-genotoxic versus genotoxic agents, we discovered a novel mechanism that determines whether cells undergo proliferation arrest or cell death. Strikingly, we observe that key mediators of cell-cycle arrest (p21, 14-3-3sigma) and apoptosis (PUMA, BAX) are equally activated regardless of outcome. In fact, arresting cells display strong translocation of PUMA and BAX to the mitochondria, yet fail to release cytochrome C or activate caspases. Surprisingly, the key differential events in apoptotic cells are p53-dependent activation of the DR4 death receptor pathway, caspase 8-mediated cleavage of BID, and BID-dependent activation of poised BAX at the mitochondria. These results reveal a previously unappreciated role for DR4 and the extrinsic apoptotic pathway in cell fate choice following p53 activation.

post to: CiteULike

Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre.

Publication Date: 2012 Jan 13 PMID: 22246180
Authors: Rasche, N. - Dybkov, O. - Schmitzova, J. - Akyildiz, B. - Fabrizio, P. - Luhrmann, R.
Journal: EMBO J

RNA-structural elements play key roles in pre-mRNA splicing catalysis; yet, the formation of catalytically competent RNA structures requires the assistance of spliceosomal proteins. We show that the S. cerevisiae Cwc2 protein functions prior to step 1 of splicing, and it is not required for the Prp2-mediated spliceosome remodelling that generates the catalytically active B(*) complex, suggesting that Cwc2 plays a more sophisticated role in the generation of a functional catalytic centre. In active spliceosomes, Cwc2 contacts catalytically important RNA elements, including the U6 internal stem-loop (ISL), and regions of U6 and the pre-mRNA intron near the 5' splice site, placing Cwc2 at/near the spliceosome's catalytic centre. These interactions are evolutionarily conserved, as shown by studies with Cwc2's human counterpart RBM22, indicating that Cwc2/RBM22-RNA contacts are functionally important. We propose that Cwc2 induces an active conformation of the spliceosome's catalytic RNA elements. Thus, the function of RNA-RNA tertiary interactions within group II introns, namely to induce an active conformation of domain V, may be fulfilled by proteins that contact the functionally analogous U6-ISL, within the spliceosome.

post to: CiteULike

Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase.

Publication Date: 2012 Jan 13 PMID: 22246179
Authors: Morin, B. - Rahmeh, A. A. - Whelan, S. P.
Journal: EMBO J

The minimal RNA synthesis machinery of non-segmented negative-strand RNA viruses comprises a genomic RNA encased within a nucleocapsid protein (N-RNA), and associated with the RNA-dependent RNA polymerase (RdRP). The RdRP is contained within a viral large (L) protein, which associates with N-RNA through a phosphoprotein (P). Here, we define that vesicular stomatitis virus L initiates synthesis via a de-novo mechanism that does not require N or P, but depends on a high concentration of the first two nucleotides and specific template requirements. Purified L copies a template devoid of N, and P stimulates L initiation and processivity. Full processivity of the polymerase requires the template-associated N protein. This work provides new mechanistic insights into the workings of a minimal RNA synthesis machine shared by a broad group of important human, animal and plant pathogens, and defines a mechanism by which specific inhibitors of RNA synthesis function.

post to: CiteULike

Endogenous DNA replication stress results in expansion of dNTP pools and a mutator phenotype.

Publication Date: 2012 Jan 10 PMID: 22234187
Authors: Davidson, M. B. - Katou, Y. - Keszthelyi, A. - Sing, T. L. - Xia, T. - Ou, J. - Vaisica, J. A. - Thevakumaran, N. - Marjavaara, L. - Myers, C. L. - Chabes, A. - Shirahige, K. - Brown, G. W.
Journal: EMBO J

The integrity of the genome depends on diverse pathways that regulate DNA metabolism. Defects in these pathways result in genome instability, a hallmark of cancer. Deletion of ELG1 in budding yeast, when combined with hypomorphic alleles of PCNA results in spontaneous DNA damage during S phase that elicits upregulation of ribonucleotide reductase (RNR) activity. Increased RNR activity leads to a dramatic expansion of deoxyribonucleotide (dNTP) pools in G1 that allows cells to synthesize significant fractions of the genome in the presence of hydroxyurea in the subsequent S phase. Consistent with the recognized correlation between dNTP levels and spontaneous mutation, compromising ELG1 and PCNA results in a significant increase in mutation rates. Deletion of distinct genome stability genes RAD54, RAD55, and TSA1 also results in increased dNTP levels and mutagenesis, suggesting that this is a general phenomenon. Together, our data point to a vicious circle in which mutations in gatekeeper genes give rise to genomic instability during S phase, inducing expansion of the dNTP pool, which in turn results in high levels of spontaneous mutagenesis.

post to: CiteULike

RP58 controls neuron and astrocyte differentiation by downregulating the expression of Id1-4 genes in the developing cortex.

Publication Date: 2012 Jan 10 PMID: 22234186
Authors: Hirai, S. - Miwa, A. - Ohtaka-Maruyama, C. - Kasai, M. - Okabe, S. - Hata, Y. - Okado, H.
Journal: EMBO J

Appropriate number of neurons and glial cells is generated from neural stem cells (NSCs) by the regulation of cell cycle exit and subsequent differentiation. Although the regulatory mechanism remains obscure, Id (inhibitor of differentiation) proteins are known to contribute critically to NSC proliferation by controlling cell cycle. Here, we report that a transcriptional factor, RP58, negatively regulates all four Id genes (Id1-Id4) in developing cerebral cortex. Consistently, Rp58 knockout (KO) mice demonstrated enhanced astrogenesis accompanied with an excess of NSCs. These phenotypes were mimicked by the overexpression of all Id genes in wild-type cortical progenitors. Furthermore, Rp58 KO phenotypes were rescued by the knockdown of all Id genes in mutant cortical progenitors but not by the knockdown of each single Id gene. Finally, we determined p57 as an effector gene of RP58-Id-mediated cell fate control. These findings establish RP58 as a novel key regulator that controls the self-renewal and differentiation of NSCs and restriction of astrogenesis by repressing all Id genes during corticogenesis.

post to: CiteULike

dNTP pools determine fork progression and origin usage under replication stress.

Publication Date: 2012 Jan 10 PMID: 22234185
Authors: Poli, J. - Tsaponina, O. - Crabbe, L. - Keszthelyi, A. - Pantesco, V. - Chabes, A. - Lengronne, A. - Pasero, P.
Journal: EMBO J

Intracellular deoxyribonucleoside triphosphate (dNTP) pools must be tightly regulated to preserve genome integrity. Indeed, alterations in dNTP pools are associated with increased mutagenesis, genomic instability and tumourigenesis. However, the mechanisms by which altered or imbalanced dNTP pools affect DNA synthesis remain poorly understood. Here, we show that changes in intracellular dNTP levels affect replication dynamics in budding yeast in different ways. Upregulation of the activity of ribonucleotide reductase (RNR) increases elongation, indicating that dNTP pools are limiting for normal DNA replication. In contrast, inhibition of RNR activity with hydroxyurea (HU) induces a sharp transition to a slow-replication mode within minutes after S-phase entry. Upregulation of RNR activity delays this transition and modulates both fork speed and origin usage under replication stress. Interestingly, we also observed that chromosomal instability (CIN) mutants have increased dNTP pools and show enhanced DNA synthesis in the presence of HU. Since upregulation of RNR promotes fork progression in the presence of DNA lesions, we propose that CIN mutants adapt to chronic replication stress by upregulating dNTP pools.

post to: CiteULike

A molecular mechanism that links Hippo signalling to the inhibition of Wnt/beta-catenin signalling.

Publication Date: 2012 Jan 10 PMID: 22234184
Authors: Imajo, M. - Miyatake, K. - Iimura, A. - Miyamoto, A. - Nishida, E.
Journal: EMBO J

The Hippo signalling pathway has emerged as a key regulator of organ size, tissue homeostasis, and patterning. Recent studies have shown that two effectors in this pathway, YAP/TAZ, modulate Wnt/beta-catenin signalling through their interaction with beta-catenin or Dishevelled, depending on biological contexts. Here, we identify a novel mechanism through which Hippo signalling inhibits Wnt/beta-catenin signalling. We show that YAP and TAZ, the transcriptional co-activators in the Hippo pathway, suppress Wnt signalling without suppressing the stability of beta-catenin but through preventing its nuclear translocation. Our results show that YAP/TAZ binds to beta-catenin, thereby suppressing Wnt-target gene expression, and that the Hippo pathway-stimulated phosphorylation of YAP, which induces cytoplasmic translocation of YAP, is required for the YAP-mediated inhibition of Wnt/beta-catenin signalling. We also find that downregulation of Hippo signalling correlates with upregulation of beta-catenin signalling in colorectal cancers. Remarkably, our analysis demonstrates that phosphorylated YAP suppresses nuclear translocation of beta-catenin by directly binding to it in the cytoplasm. These results provide a novel mechanism, in which Hippo signalling antagonizes Wnt signalling by regulating nuclear translocation of beta-catenin.

post to: CiteULike

CaMKII binding to GluN2B is critical during memory consolidation.

Publication Date: 2012 Jan 10 PMID: 22234183
Authors: Halt, A. R. - Dallapiazza, R. F. - Zhou, Y. - Stein, I. S. - Qian, H. - Juntti, S. - Wojcik, S. - Brose, N. - Silva, A. J. - Hell, J. W.
Journal: EMBO J

Memory is essential for our normal daily lives and our sense of self. Ca(2+) influx through the NMDA-type glutamate receptor (NMDAR) and the ensuing activation of the Ca(2+) and calmodulin-dependent protein kinase (CaMKII) are required for memory formation and its physiological correlate, long-term potentiation (LTP). The Ca(2+) influx induces CaMKII binding to the NMDAR to strategically recruit CaMKII to synapses that are undergoing potentiation. We generated mice with two point mutations that impair CaMKII binding to the NMDAR GluN2B subunit. Ca(2+)-triggered postsynaptic accumulation is largely abrogated for CaMKII and destabilized for TARPs, which anchor AMPA-type glutamate receptors (AMPAR). LTP is reduced by 50% and phosphorylation of the AMPAR GluA1 subunit by CaMKII, which enhances AMPAR conductance, impaired. The mutant mice learn the Morris water maze (MWM) as well as WT but show deficiency in recall during the period of early memory consolidation. Accordingly, the activity-driven interaction of CaMKII with the NMDAR is important for recall of MWM memory as early as 24 h, but not 1-2 h, after training potentially due to impaired consolidation.

post to: CiteULike

A microRNA that limits metastatic colonisation and endothelial recruitment.

Publication Date: 2012 Jan 10 PMID: 22234182
Authors: Khew-Goodall, Y. - Goodall, G. J.
Journal: EMBO J

post to: CiteULike

Genetic evidence of a redox-dependent systemic wound response via Hayan Protease-Phenoloxidase system in Drosophila.

Publication Date: 2012 Jan 6 PMID: 22227521
Authors: Nam, H. J. - Jang, I. H. - You, H. - Lee, K. A. - Lee, W. J.
Journal: EMBO J

Systemic wound response (SWR) through intertissue communication in response to local wounds is an essential biological phenomenon that occurs in all multicellular organisms from plants to animals. However, our understanding of SWR has been greatly hampered by the complexity of wound signalling communication operating within the context of an entire organism. Here, we show genetic evidence of a redox-dependent SWR from the wound site to remote tissues by identifying critical genetic determinants of SWR. Local wounds in the integument rapidly induce activation of a novel circulating haemolymph serine protease, Hayan, which in turn converts pro-phenoloxidase (PPO) to phenoloxidase (PO), an active form of melanin-forming enzyme. The Haemolymph Hayan-PO cascade is required for redox-dependent activation of the c-Jun N-terminal kinase (JNK)-dependent cytoprotective program in neuronal tissues, thereby achieving organism level of homeostasis to resist local physical trauma. These results imply that the PO-activating enzyme cascade, which is a prominent defense system in humoral innate immunity, also mediates redox-dependent SWR, providing a novel link between wound response and the nervous system.

post to: CiteULike

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop.

Publication Date: 2012 Jan 6 PMID: 22227520
Authors: Schmid, A. B. - Lagleder, S. - Grawert, M. A. - Rohl, A. - Hagn, F. - Wandinger, S. K. - Cox, M. B. - Demmer, O. - Richter, K. - Groll, M. - Kessler, H. - Buchner, J.
Journal: EMBO J

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous alpha-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.

post to: CiteULike

Ablation of Rassf2 induces bone defects and subsequent haematopoietic anomalies in mice.

Publication Date: 2012 Jan 6 PMID: 22227519
Authors: Song, H. - Kim, H. - Lee, K. - Lee, D. H. - Kim, T. S. - Song, J. Y. - Lee, D. - Choi, D. - Ko, C. Y. - Kim, H. S. - Shin, H. I. - Choi, J. - Park, H. - Park, C. - Jeong, D. - Lim, D. S.
Journal: EMBO J

RASSF2 belongs to the Ras-association domain family (RASSF) of proteins, which may be involved in the Hippo signalling pathway. However, the role of RASSF2 in vivo is unknown. Here, we show that Rassf2 knockout mice manifest a multisystemic phenotype including haematopoietic anomalies and defects in bone remodelling. Bone marrow (BM) transplantation showed that Rassf2(-/-) BM cells had a normal haematopoietic reconstitution activity, indicating no intrinsic haematopoietic defects. Notably, in vitro differentiation studies revealed that ablation of Rassf2 suppressed osteoblastogenesis but promoted osteoclastogenesis. Co-culture experiments showed that an intrinsic defect in osteoblast differentiation from Rassf2(-/-) osteoblast precursors likely leads to both haematopoiesis and osteoclast defects in Rassf2(-/-) mice. Moreover, Rassf2 deficiency resulted in hyperactivation of nuclear factor (NF)-kappaB during both osteoclast and osteoblast differentiation. RASSF2 associated with IkappaB kinase (IKK) alpha and beta forms, and suppressed IKK activity. Introduction of either RASSF2 or a dominant-negative form of IKK into Rassf2(-/-) osteoclast or osteoblast precursors inhibited NF-kappaB hyperactivation and normalized osteoclast and osteoblast differentiation. These observations indicate that RASSF2 regulates osteoblast and osteoclast differentiation by inhibiting NF-kappaB signalling.

post to: CiteULike