EMBO Journal

Cell cycle coordination and regulation of bacterial chromosome segregation dynamics by polarly localized proteins.

Publication Date: 2010 Aug 27 PMID: 20802464
Authors: Schofield, W. B. - Lim, H. C. - Jacobs-Wagner, C.
Journal: EMBO J

What regulates chromosome segregation dynamics in bacteria is largely unknown. Here, we show in Caulobacter crescentus that the polarity factor TipN regulates the directional motion and overall translocation speed of the parS/ParB partition complex by interacting with ParA at the new pole. In the absence of TipN, ParA structures can regenerate behind the partition complex, leading to stalls and back-and-forth motions of parS/ParB, reminiscent of plasmid behaviour. This extrinsic regulation of the parS/ParB/ParA system directly affects not only division site selection, but also cell growth. Other mechanisms, including the pole-organizing protein PopZ, compensate for the defect in segregation regulation in DeltatipN cells. Accordingly, synthetic lethality of PopZ and TipN is caused by severe chromosome segregation and cell division defects. Our data suggest a mechanistic framework for adapting a self-organizing oscillator to create motion suitable for chromosome segregation.

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ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery.

Publication Date: 2010 Aug 27 PMID: 20802463
Authors: Ammazzalorso, F. - Pirzio, L. M. - Bignami, M. - Franchitto, A. - Pichierri, P.
Journal: EMBO J

Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM-dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C-terminal S/TQ residues. Suppression of ATR-mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co-localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM-unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.

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Disulphide production by Ero1alpha-PDI relay is rapid and effectively regulated.

Publication Date: 2010 Aug 27 PMID: 20802462
Authors: Appenzeller-Herzog, C. - Riemer, J. - Zito, E. - Chin, K. T. - Ron, D. - Spiess, M. - Ellgaard, L.
Journal: EMBO J

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1alpha and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1alpha, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

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beta-arrestin Kurtz inhibits MAPK and Toll signalling in Drosophila development.

Publication Date: 2010 Aug 27 PMID: 20802461
Authors: Tipping, M. - Kim, Y. - Kyriakakis, P. - Tong, M. - Shvartsman, S. Y. - Veraksa, A.
Journal: EMBO J

beta-Arrestins have been implicated in the regulation of multiple signalling pathways. However, their role in organism development is not well understood. In this study, we report a new in vivo function of the Drosophila beta-arrestin Kurtz (Krz) in the regulation of two distinct developmental signalling modules: MAPK ERK and NF-kappaB, which transmit signals from the activated receptor tyrosine kinases (RTKs) and the Toll receptor, respectively. Analysis of the expression of effectors and target genes of Toll and the RTK Torso in krz maternal mutants reveals that Krz limits the activity of both pathways in the early embryo. Protein interaction studies suggest a previously uncharacterized mechanism for ERK inhibition: Krz can directly bind and sequester an inactive form of ERK, thus preventing its activation by the upstream kinase, MEK. A simultaneous dysregulation of different signalling systems in krz mutants results in an abnormal patterning of the embryo and severe developmental defects. Our findings uncover a new in vivo function of beta-arrestins and present a new mechanism of ERK inhibition by the Drosophila beta-arrestin Krz.

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Oct-3/4 regulates stem cell identity and cell fate decisions by modulating Wnt/beta-catenin signalling.

Publication Date: 2010 Aug 24 PMID: 20736927
Authors: Abu-Remaileh, M. - Gerson, A. - Farago, M. - Nathan, G. - Alkalay, I. - Zins Rousso, S. - Gur, M. - Fainsod, A. - Bergman, Y.
Journal: EMBO J

Although the transcriptional regulatory events triggered by Oct-3/4 are well documented, understanding the proteomic networks that mediate the diverse functions of this POU domain homeobox protein remains a major challenge. Here, we present genetic and biochemical studies that suggest an unexpected novel strategy for Oct-3/4-dependent regulation of embryogenesis and cell lineage determination. Our data suggest that Oct-3/4 specifically interacts with nuclear beta-catenin and facilitates its proteasomal degradation, resulting in the maintenance of an undifferentiated, early embryonic phenotype both in Xenopus embryos and embryonic stem (ES) cells. Our data also show that Oct-3/4-mediated control of beta-catenin stability has an important function in regulating ES cell motility. Down-regulation of Oct-3/4 increases beta-catenin protein levels, enhancing Wnt signalling and initiating invasive cellular activity characteristic of epithelial-mesenchymal transition. Our data suggest a novel mode of regulation by which a delicate balance between beta-catenin, Tcf3 and Oct-3/4 regulates maintenance of stem cell identity. Altering the balance between these proteins can direct cell fate decisions and differentiation.

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Molecular architecture of the DNA replication origin activation checkpoint.

Publication Date: 2010 Aug 20 PMID: 20729811
Authors: Tudzarova, S. - Trotter, M. W. - Wollenschlaeger, A. - Mulvey, C. - Godovac-Zimmermann, J. - Williams, G. H. - Stoeber, K.
Journal: EMBO J

Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-mid R:Hdm2-mid R:p53 --> p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/beta-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.

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Molecular basis for activation of G protein-coupled receptor kinases.

Publication Date: 2010 Aug 20 PMID: 20729810
Authors: Boguth, C. A. - Singh, P. - Huang, C. C. - Tesmer, J. J.
Journal: EMBO J

G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

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Ku prevents Exo1 and Sgs1-dependent resection of DNA ends in the absence of a functional MRX complex or Sae2.

Publication Date: 2010 Aug 20 PMID: 20729809
Authors: Mimitou, E. P. - Symington, L. S.
Journal: EMBO J

In this study, we investigate the interplay between Ku, a central non-homologous end-joining component, and the Mre11-Rad50-Xrs2 (MRX) complex and Sae2, end-processing factors crucial for initiating 5'-3' resection of double-strand break (DSB) ends. We show that in the absence of end protection by Ku, the requirement for the MRX complex is bypassed and resection is executed by Exo1. In contrast, both the Exo1 and Sgs1 resection pathways contribute to DSB processing in the absence of Ku and Sae2 or when the MRX complex is intact, but functionally compromised by elimination of the Mre11 nuclease activity. The ionizing radiation sensitivity of a mutant defective for extensive resection (exo1Delta sgs1Delta) cannot be suppressed by the yku70Delta mutation, indicating that Ku suppression is specific to the initiation of resection. We provide evidence that replication-associated DSBs need to be processed by Sae2 for repair by homologous recombination unless Ku is absent. Finally, we show that the presence of Ku exacerbates DNA end-processing defects established in the sae2Delta sgs1Delta mutant, leading to its lethality.

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A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression.

Publication Date: 2010 Aug 20 PMID: 20729808
Authors: Bernard, D. - Prasanth, K. V. - Tripathi, V. - Colasse, S. - Nakamura, T. - Xuan, Z. - Zhang, M. Q. - Sedel, F. - Jourdren, L. - Coulpier, F. - Triller, A. - Spector, D. L. - Bessis, A.
Journal: EMBO J

A growing number of long nuclear-retained non-coding RNAs (ncRNAs) have recently been described. However, few functions have been elucidated for these ncRNAs. Here, we have characterized the function of one such ncRNA, identified as metastasis-associated lung adenocarcinoma transcript 1 (Malat1). Malat1 RNA is expressed in numerous tissues and is highly abundant in neurons. It is enriched in nuclear speckles only when RNA polymerase II-dependent transcription is active. Knock-down studies revealed that Malat1 modulates the recruitment of SR family pre-mRNA-splicing factors to the transcription site of a transgene array. DNA microarray analysis in Malat1-depleted neuroblastoma cells indicates that Malat1 controls the expression of genes involved not only in nuclear processes, but also in synapse function. In cultured hippocampal neurons, knock-down of Malat1 decreases synaptic density, whereas its over-expression results in a cell-autonomous increase in synaptic density. Our results suggest that Malat1 regulates synapse formation by modulating the expression of genes involved in synapse formation and/or maintenance.

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Neurons don't appreciate FUSsing in the cytoplasm.

Publication Date: 2010 Aug 18 PMID: 20717146
Authors: Buratti, E. - Baralle, F. E.
Journal: EMBO J

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Spatial organization of adhesion: force-dependent regulation and function in tissue morphogenesis.

Publication Date: 2010 Aug 18 PMID: 20717145
Authors: Papusheva, E. - Heisenberg, C. P.
Journal: EMBO J

Integrin- and cadherin-mediated adhesion is central for cell and tissue morphogenesis, allowing cells and tissues to change shape without loosing integrity. Studies predominantly in cell culture showed that mechanosensation through adhesion structures is achieved by force-mediated modulation of their molecular composition. The specific molecular composition of adhesion sites in turn determines their signalling activity and dynamic reorganization. Here, we will review how adhesion sites respond to mecanical stimuli, and how spatially and temporally regulated signalling from different adhesion sites controls cell migration and tissue morphogenesis.

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Protein homeostasis and synaptic plasticity.

Publication Date: 2010 Aug 18 PMID: 20717144
Authors: Cajigas, I. J. - Will, T. - Schuman, E. M.
Journal: EMBO J

It is clear that de novo protein synthesis has an important function in synaptic transmission and plasticity. A substantial amount of work has shown that mRNA translation in the hippocampus is spatially controlled and that dendritic protein synthesis is required for different forms of long-term synaptic plasticity. More recently, several studies have highlighted a function for protein degradation by the ubiquitin proteasome system in synaptic plasticity. These observations suggest that changes in synaptic transmission involve extensive regulation of the synaptic proteome. Here, we review experimental data supporting the idea that protein homeostasis is a regulatory motif for synaptic plasticity.

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Cellular responses to extracellular guidance cues.

Publication Date: 2010 Aug 18 PMID: 20717143
Authors: Berzat, A. - Hall, A.
Journal: EMBO J

Extracellular guidance cues have a key role in orchestrating cell behaviour. They can take many forms, including soluble and cell-bound ligands (proteins, lipids, peptides or small molecules) and insoluble matrix substrates, but to act as guidance cues, they must be presented to the cell in a spatially restricted manner. Cells that recognize such cues respond by activating intracellular signal transduction pathways in a spatially restricted manner and convert the extracellular information into intracellular polarity. Although extracellular cues influence a broad range of cell polarity decisions, such as mitotic spindle orientation during asymmetric cell division, or the establishment of apical-basal polarity in epithelia, this review will focus specifically on guidance cues that promote cell migration (chemotaxis), or localized cell shape changes (chemotropism).

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Spatial organization in bacterial chemotaxis.

Publication Date: 2010 Aug 18 PMID: 20717142
Authors: Sourjik, V. - Armitage, J. P.
Journal: EMBO J

Spatial organization of signalling is not an exclusive property of eukaryotic cells. Despite the fact that bacterial signalling pathways are generally simpler than those in eukaryotes, there are several well-documented examples of higher-order intracellular signalling structures in bacteria. One of the most prominent and best-characterized structures is formed by proteins that control bacterial chemotaxis. Signals in chemotaxis are processed by ordered arrays, or clusters, of receptors and associated proteins, which amplify and integrate chemotactic stimuli in a highly cooperative manner. Receptor clusters further serve to scaffold protein interactions, enhancing the efficiency and specificity of the pathway reactions and preventing the formation of signalling gradients through the cell body. Moreover, clustering can also ensure spatial separation of multiple chemotaxis systems in one bacterium. Assembly of receptor clusters appears to be a stochastic process, but bacteria evolved mechanisms to ensure optimal cluster distribution along the cell body for partitioning to daughter cells at division.

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An intimate liaison: spatial organization of the endoplasmic reticulum-mitochondria relationship.

Publication Date: 2010 Aug 18 PMID: 20717141
Authors: de Brito, O. M. - Scorrano, L.
Journal: EMBO J

Organelle localization is often crucial to properly modulate cellular functions and signalling cascades. For example, the distribution of organelles in axons is crucial for their function and is dysregulated in several diseases. Similarly, relative positioning of two or more organelles is also important to perform certain specialized processes. Perhaps, the best-known form of interorganellar organization is that between endoplasmic reticulum (ER) and mitochondria. Close communication between these two compartments has been observed for a long time. Recent evidence suggests that this is the basis for a bidirectional communication regulating a number of physiological processes ranging from mitochondrial energy and lipid metabolism to Ca(2+) signalling and cell death. The recent discovery of some of the molecular mediators of the tethering already allowed to extend the function of this paradigmatic spatial organization to previously unexpected functions, and will foster future research to explore it in cellular signalling cascades as well as in disease.

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The march of the PINs: developmental plasticity by dynamic polar targeting in plant cells.

Publication Date: 2010 Aug 18 PMID: 20717140
Authors: Grunewald, W. - Friml, J.
Journal: EMBO J

Development of plants and their adaptive capacity towards ever-changing environmental conditions largely depend on the spatial distribution of the plant hormone auxin. At the cellular level, various internal and external signals are translated into specific changes in the polar, subcellular localization of auxin transporters from the PIN family thereby directing and redirecting the intercellular fluxes of auxin. The current model of polar targeting of PIN proteins towards different plasma membrane domains encompasses apolar secretion of newly synthesized PINs followed by endocytosis and recycling back to the plasma membrane in a polarized manner. In this review, we follow the subcellular march of the PINs and highlight the cellular and molecular mechanisms behind polar foraging and subcellular trafficking pathways. Also, the entry points for different signals and regulations including by auxin itself will be discussed within the context of morphological and developmental consequences of polar targeting and subcellular trafficking.

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Spatial cycles in G-protein crowd control.

Publication Date: 2010 Aug 18 PMID: 20717139
Authors: Vartak, N. - Bastiaens, P.
Journal: EMBO J

The nature of living systems and their apparent resilience to the second law of thermodynamics has been the subject of extensive investigation and imaginative speculation. The segregation and compartmentalization of proteins is one manifestation of this departure from equilibrium conditions; the effect of which is now beginning to be elucidated. This should not come as a surprise, as even a cursory inspection of cellular processes reveals the large amount of energetic cost borne to maintain cell-scale patterns, separations and gradients of molecules. The G-proteins, kinases, calcium-responsive proteins have all been shown to contain reaction cycles that are inherently coupled to their signalling activities. G-proteins represent an important and diverse toolset used by cells to generate cellular asymmetries. Many small G-proteins in particular, are dynamically acylated to modify their membrane affinities, or localized in an activity-dependent manner, thus manipulating the mobility modes of these proteins beyond pure diffusion and leading to finely tuned steady state partitioning into cellular membranes. The rates of exchange of small G-proteins over various compartments, as well as their steady state distributions enrich and diversify the landscape of possibilities that GTPase-dependent signalling networks can display over cellular dimensions. The chemical manipulation of spatial cycles represents a new approach for the modulation of cellular signalling with potential therapeutic benefits.

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Spatial organization of transmembrane receptor signalling.

Publication Date: 2010 Aug 18 PMID: 20717138
Authors: Bethani, I. - Skanland, S. S. - Dikic, I. - Acker-Palmer, A.
Journal: EMBO J

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis-interactions as well as intercellular trans-interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand-induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid-protein interactions important for the spatial organization of signalling at the membrane.

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Focus on the spatial organization of signalling.

Publication Date: 2010 Aug 18 PMID: 20717137
Authors: Schwarz-Romond, T. - Gorski, S. A.
Journal: EMBO J

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Crystal structure of a transfer-ribonucleoprotein particle that promotes asparagine formation.

Publication Date: 2010 Aug 17 PMID: 20717102
Authors: Blaise, M. - Bailly, M. - Frechin, M. - Behrens, M. A. - Fischer, F. - Oliveira, C. L. - Becker, H. D. - Pedersen, J. S. - Thirup, S. - Kern, D.
Journal: EMBO J

Four out of the 22 aminoacyl-tRNAs (aa-tRNAs) are systematically or alternatively synthesized by an indirect, two-step route requiring an initial mischarging of the tRNA followed by tRNA-dependent conversion of the non-cognate amino acid. During tRNA-dependent asparagine formation, tRNA(Asn) promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-tRNA from non-discriminating aspartyl-tRNA synthetase active site to the GatCAB amidotransferase site. The crystal structure of the Thermus thermophilus transamidosome determined at 3 A resolution reveals a particle formed by two GatCABs, two dimeric ND-AspRSs and four tRNAs(Asn) molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl-tRNA(Asn) without dissociation of the complex. We propose that the crystal structure represents a transient state of the transamidation reaction. The transamidosome constitutes a transfer-ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine.

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The nuclear receptor PPARgamma individually responds to serotonin- and fatty acid-metabolites.

Publication Date: 2010 Aug 17 PMID: 20717101
Authors: Waku, T. - Shiraki, T. - Oyama, T. - Maebara, K. - Nakamori, R. - Morikawa, K.
Journal: EMBO J

The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPARgamma through conformational changes of the Omega loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARgamma to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARgamma antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARgamma-target gene. Collectively, these results suggest a novel agonism, in which PPARgamma functions as a multiple sensor in response to distinct metabolites.

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Growth habit determination by the balance of histone methylation activities in Arabidopsis.

Publication Date: 2010 Aug 13 PMID: 20711170
Authors: Ko, J. H. - Mitina, I. - Tamada, Y. - Hyun, Y. - Choi, Y. - Amasino, R. M. - Noh, B. - Noh, Y. S.
Journal: EMBO J

In Arabidopsis, the rapid-flowering summer-annual versus the vernalization-requiring winter-annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter-annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)-specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36- and H3K4-specific histone demethylase (HDM) activities of autonomous-pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter-annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36-specific HMT and HDM activities.

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Rif1 provides a new DNA-binding interface for the Bloom syndrome complex to maintain normal replication.

Publication Date: 2010 Aug 13 PMID: 20711169
Authors: Xu, D. - Muniandy, P. - Leo, E. - Yin, J. - Thangavel, S. - Shen, X. - Ii, M. - Agama, K. - Guo, R. - Fox, D. 3rd - Meetei, A. R. - Wilson, L. - Nguyen, H. - Weng, N. P. - Brill, S. J. - Li, L. - Vindigni, A. - Pommier, Y. - Seidman, M. - Wang, W.
Journal: EMBO J

BLM, the helicase defective in Bloom syndrome, is part of a multiprotein complex that protects genome stability. Here, we show that Rif1 is a novel component of the BLM complex and works with BLM to promote recovery of stalled replication forks. First, Rif1 physically interacts with the BLM complex through a conserved C-terminal domain, and the stability of Rif1 depends on the presence of the BLM complex. Second, Rif1 and BLM are recruited with similar kinetics to stalled replication forks, and the Rif1 recruitment is delayed in BLM-deficient cells. Third, genetic analyses in vertebrate DT40 cells suggest that BLM and Rif1 work in a common pathway to resist replication stress and promote recovery of stalled forks. Importantly, vertebrate Rif1 contains a DNA-binding domain that resembles the alphaCTD domain of bacterial RNA polymerase alpha; and this domain preferentially binds fork and Holliday junction (HJ) DNA in vitro and is required for Rif1 to resist replication stress in vivo. Our data suggest that Rif1 provides a new DNA-binding interface for the BLM complex to restart stalled replication forks.

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A Dyn2-CIN85 complex mediates degradative traffic of the EGFR by regulation of late endosomal budding.

Publication Date: 2010 Aug 13 PMID: 20711168
Authors: Schroeder, B. - Weller, S. G. - Chen, J. - Billadeau, D. - McNiven, M. A.
Journal: EMBO J

The epidermal growth factor receptor (EGFR) is over-expressed in a variety of human cancers. Downstream signalling of this receptor is tightly regulated both spatially and temporally by controlling its internalization and subsequent degradation. Internalization of the EGFR requires dynamin 2 (Dyn2), a large GTPase that deforms lipid bilayers, leading to vesicle scission. The adaptor protein CIN85 (cbl-interacting protein of 85 kDa), which has been proposed to indirectly link the EGFR to the endocytic machinery at the plasma membrane, is also thought to be involved in receptor internalization. Here, we report a novel and direct interaction between Dyn2 and CIN85 that is induced by EGFR stimulation and, most surprisingly, occurs late in the endocytic process. Importantly, disruption of the CIN85-Dyn2 interaction results in accumulation of internalized EGFR in late endosomes that become aberrantly elongated into distended tubules. Consistent with the accumulation of this receptor is a sustention of downstream signalling cascades. These findings provide novel insights into a previously unknown protein complex that can regulate EGFR traffic at very late stages of the endocytic pathway.

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An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition.

Publication Date: 2010 Aug 13 PMID: 20711167
Authors: Warzecha, C. C. - Jiang, P. - Amirikian, K. - Dittmar, K. A. - Lu, H. - Shen, S. - Guo, W. - Xing, Y. - Carstens, R. P.
Journal: EMBO J

Alternative splicing achieves coordinated changes in post-transcriptional gene expression programmes through the activities of diverse RNA-binding proteins. Epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) are cell-type-specific regulators of transcripts that switch splicing during the epithelial-mesenchymal transition (EMT). To define a comprehensive programme of alternative splicing that is regulated during the EMT, we identified an extensive ESRP-regulated splicing network of hundreds of alternative splicing events within numerous genes with functions in cell-cell adhesion, polarity, and migration. Loss of this global ESRP-regulated epithelial splicing programme induces the phenotypic changes in cell morphology that are observed during the EMT. Components of this splicing signature provide novel molecular markers that can be used to characterize the EMT. Bioinformatics and experimental approaches revealed a high-affinity ESRP-binding motif and a predictive RNA map that governs their activity. This work establishes the ESRPs as coordinators of a complex alternative splicing network that adds an important post-transcriptional layer to the changes in gene expression that underlie epithelial-mesenchymal transitions during development and disease.

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Dynamin GTPase regulation is altered by PH domain mutations found in centronuclear myopathy patients.

Publication Date: 2010 Aug 10 PMID: 20700106
Authors: Kenniston, J. A. - Lemmon, M. A.
Journal: EMBO J

The large GTPase dynamin has an important membrane scission function in receptor-mediated endocytosis and other cellular processes. Self-assembly on phosphoinositide-containing membranes stimulates dynamin GTPase activity, which is crucial for its function. Although the pleckstrin-homology (PH) domain is known to mediate phosphoinositide binding by dynamin, it remains unclear how this promotes activation. Here, we describe studies of dynamin PH domain mutations found in centronuclear myopathy (CNM) that increase dynamin's GTPase activity without altering phosphoinositide binding. CNM mutations in the PH domain C-terminal alpha-helix appear to cause conformational changes in dynamin that alter control of the GTP hydrolysis cycle. These mutations either 'sensitize' dynamin to lipid stimulation or elevate basal GTPase rates by promoting self-assembly and thus rendering dynamin no longer lipid responsive. We also describe a low-resolution structure of dimeric dynamin from small-angle X-ray scattering that reveals conformational changes induced by CNM mutations, and defines requirements for domain rearrangement upon dynamin self-assembly at membrane surfaces. Our data suggest that changes in the PH domain may couple lipid binding to dynamin GTPase activation at sites of vesicle invagination.

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alpha-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells.

Publication Date: 2010 Aug 10 PMID: 20700105
Authors: Chen, B. - Liu, P. - Wang, S. J. - Ge, Q. - Zhan, H. - Mohler, W. A. - Wang, Z. W.
Journal: EMBO J

The BK channel, a voltage- and Ca(2+)-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian alpha-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel alpha-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization.

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Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc.

Publication Date: 2010 Aug 10 PMID: 20700104
Authors: Yang, C. S. - Thomenius, M. J. - Gan, E. C. - Tang, W. - Freel, C. D. - Merritt, T. J. - Nutt, L. K. - Kornbluth, S.
Journal: EMBO J

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome-wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc-mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII-mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death-inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non-phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.

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Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4.

Publication Date: 2010 Aug 6 PMID: 20693977
Authors: Polo, S. E. - Kaidi, A. - Baskcomb, L. - Galanty, Y. - Jackson, S. P.
Journal: EMBO J

The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in the DNA-damage response (DDR) and cell-cycle control. We show that CHD4 mediates rapid poly(ADP-ribose)-dependent recruitment of the NuRD complex to DNA-damage sites, and we identify CHD4 as a phosphorylation target for the apical DDR kinase ataxia-telangiectasia mutated. Functionally, we show that CHD4 promotes repair of DNA double-strand breaks and cell survival after DNA damage. In addition, we show that CHD4 acts as an important regulator of the G1/S cell-cycle transition by controlling p53 deacetylation. These results provide new insights into how the chromatin remodelling complex NuRD contributes to maintaining genome stability.

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Nanog, Gli, and p53: a new network of stemness in development and cancer.

Publication Date: 2010 Aug 4 PMID: 20683467
Authors: Brandner, S.
Journal: EMBO J

MeSH Categories: Animals, Homeodomain Proteins/*metabolism, Humans, Mice, Neoplasms/*metabolism, Oncogene Proteins/*metabolism, Signal Transduction, Stem Cells/*metabolism, Trans-Activators/*metabolism, Tumor Suppressor Protein p53/*metabolism

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The glucose signal and metabolic p[H+]lux.

Publication Date: 2010 Aug 4 PMID: 20683466
Authors: Rubenstein, E. M. - Schmidt, M. C.
Journal: EMBO J

MeSH Categories: Animals, Cyclic AMP-Dependent Protein Kinases/metabolism, Glucose/*metabolism, Hydrogen-Ion Concentration, *Second Messenger Systems, Vacuolar Proton-Translocating ATPases/*metabolism

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Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1-E2F pathway activity.

Publication Date: 2010 Sep 1 PMID: 20683442
Authors: Henriques, R. - Magyar, Z. - Monardes, A. - Khan, S. - Zalejski, C. - Orellana, J. - Szabados, L. - de la Torre, C. - Koncz, C. - Bogre, L.
Journal: EMBO J

The 40S ribosomal protein S6 kinase (S6K) is a conserved component of signalling pathways controlling growth in eukaryotes. To study S6K function in plants, we isolated single- and double-knockout mutations and RNA-interference (RNAi)-silencing lines in the linked Arabidopsis S6K1 and S6K2 genes. Hemizygous s6k1s6k2/++ mutant and S6K1 RNAi lines show high phenotypic instability with variation in size, increased trichome branching, produce non-viable pollen and high levels of aborted seeds. Analysis of their DNA content by flow cytometry, as well as chromosome counting using DAPI staining and fluorescence in situ hybridization, revealed an increase in ploidy and aneuploidy. In agreement with this data, we found that S6K1 associates with the Retinoblastoma-related 1 (RBR1)-E2FB complex and this is partly mediated by its N-terminal LVxCxE motif. Moreover, the S6K1-RBR1 association regulates RBR1 nuclear localization, as well as E2F-dependent expression of cell cycle genes. Arabidopsis cells grown under nutrient-limiting conditions require S6K for repression of cell proliferation. The data suggest a new function for plant S6K as a repressor of cell proliferation and required for maintenance of chromosome stability and ploidy levels.

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Integrating anaerobic/aerobic sensing and the general stress response through the ArcZ small RNA.

Publication Date: 2010 Aug 3 PMID: 20683441
Authors: Mandin, P. - Gottesman, S.
Journal: EMBO J

The alternative sigma factor RpoS responds to multiple stresses and activates a large number of genes that allow bacteria to adapt to changing environments. The accumulation of RpoS is regulated at multiple levels, including the regulation of its translation by small regulatory RNAs (sRNAs). A library of plasmids expressing each of 26 Escherichia coli sRNAs that bind Hfq was created to globally and rapidly analyse regulation of an rpoS-lacZ translational fusion. The approach can be easily applied to any gene of interest. When overexpressed, four sRNAs, including OxyS, previously shown to repress rpoS, were observed to repress the expression of the rpoS-lacZ fusion. Along with DsrA and RprA, two previously defined activators of rpoS translation, a third new sRNA activator, ArcZ, was identified. The expression of arcZ is repressed by the aerobic/anaerobic-sensing ArcA-ArcB two-component system under anaerobic conditions and adds translational regulation to the ArcA-ArcB regulon. ArcZ directly represses, and is repressed by, arcB transcription, providing a negative feedback loop that may affect functioning of the ArcA-ArcB regulon.

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AGAP1/AP-3-dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release.

Publication Date: 2010 Aug 18 PMID: 20664521
Authors: Bendor, J. - Lizardi-Ortiz, J. E. - Westphalen, R. I. - Brandstetter, M. - Hemmings, H. C. Jr - Sulzer, D. - Flajolet, M. - Greengard, P.
Journal: EMBO J

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M(5) is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M(5) and the AP-3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP-3 to M(5). Interaction with AGAP1 and activity of AP-3 were required for the endocytic recycling of M(5) in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP-3 or abrogation of AGAP1-M(5) interaction in vivo decreased the magnitude of presynaptic M(5)-mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor-recycling pathway that may underlie mechanisms of G-protein-coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.

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Sub-picomolar relaxin signalling by a pre-assembled RXFP1, AKAP79, AC2, beta-arrestin 2, PDE4D3 complex.

Publication Date: 2010 Aug 18 PMID: 20664520
Authors: Halls, M. L. - Cooper, D. M.
Journal: EMBO J

Biochemical studies suggest that G-protein-coupled receptors (GPCRs) achieve exquisite signalling specificity by forming selective complexes, termed signalosomes. Here, using cAMP biosensors in single cells, we uncover a pre-assembled, constitutively active GPCR signalosome, that couples the relaxin receptor, relaxin family peptide receptor 1 (RXFP1), to cAMP following receptor stimulation with sub-picomolar concentrations of peptide. The physiological effects of relaxin, a pleiotropic hormone with therapeutic potential in cancer metastasis and heart failure, are generally attributed to local production of the peptide, that occur in response to sub-micromolar concentrations. The highly sensitive signalosome identified here provides a regulatory mechanism for the extremely low levels of relaxin that circulate. The signalosome includes requisite Galpha(s), Gbetagamma and adenylyl cyclase 2 (AC2); AC2 is functionally coupled to RXFP1 through AKAP79 binding to helix 8 of the receptor; activation of AC2 is tonically opposed by protein kinase A (PKA)-activated PDE4D3, scaffolded through a beta-arrestin 2 interaction with Ser(704) of the receptor C-terminus. This elaborate, pre-assembled, ligand-independent GPCR signalosome represents a new paradigm in GPCR signalling and provides a mechanism for the distal actions of low circulating levels of relaxin.

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14-3-3gamma mediates Cdc25A proteolysis to block premature mitotic entry after DNA damage.

Publication Date: 2010 Aug 18 PMID: 20639859
Authors: Kasahara, K. - Goto, H. - Enomoto, M. - Tomono, Y. - Kiyono, T. - Inagaki, M.
Journal: EMBO J

14-3-3 proteins control various cellular processes, including cell cycle progression and DNA damage checkpoint. At the DNA damage checkpoint, some subtypes of 14-3-3 (beta and zeta isoforms in mammalian cells and Rad24 in fission yeast) bind to Ser345-phosphorylated Chk1 and promote its nuclear retention. Here, we report that 14-3-3gamma forms a complex with Chk1 phosphorylated at Ser296, but not at ATR sites (Ser317 and Ser345). Ser296 phosphorylation is catalysed by Chk1 itself after Chk1 phosphorylation by ATR, and then ATR sites are rapidly dephosphorylated on Ser296-phosphorylated Chk1. Although Ser345 phosphorylation is observed at nuclear DNA damage foci, it occurs more diffusely in the nucleus. The replacement of endogenous Chk1 with Chk1 mutated at Ser296 to Ala induces premature mitotic entry after ultraviolet irradiation, suggesting the importance of Ser296 phosphorylation in the DNA damage response. Although Ser296 phosphorylation induces the only marginal change in Chk1 catalytic activity, 14-3-3gamma mediates the interaction between Chk1 and Cdc25A. This ternary complex formation has an essential function in Cdc25A phosphorylation and degradation to block premature mitotic entry after DNA damage.

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Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection.

Publication Date: 2010 Aug 18 PMID: 20639858
Authors: Dai, X. - Huang, C. - Bhusari, A. - Sampathi, S. - Schubert, K. - Chai, W.
Journal: EMBO J

Telomeric G-overhangs are required for the formation of the protective telomere structure and telomerase action. However, the mechanism controlling G-overhang generation at human telomeres is poorly understood. Here, we show that G-overhangs can undergo cell cycle-regulated changes independent of telomerase activity. G-overhangs at lagging telomeres are lengthened in S phase and then shortened in late S/G2 because of C-strand fill-in, whereas the sizes of G-overhangs at leading telomeres remain stable throughout S phase and are lengthened in G2/M. The final nucleotides at measurable C-strands are precisely defined throughout the cell cycle, indicating that C-strand resection is strictly regulated. We demonstrate that C-strand fill-in is mediated by DNA polymerase alpha (polalpha) and controlled by cyclin-dependent kinase 1 (CDK1). Inhibition of CDK1 leads to accumulation of lengthened G-overhangs and induces telomeric DNA damage response. Furthermore, depletion of hStn1 results in elongation of G-overhangs and an increase in telomeric DNA damage. Our results suggest that G-overhang generation at human telomeres is regulated by multiple tightly controlled processes and C-strand fill-in is under the control of polalpha and CDK1.

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Cellular pathways controlling integron cassette site folding.

Publication Date: 2010 Aug 4 PMID: 20628355
Authors: Loot, C. - Bikard, D. - Rachlin, A. - Mazel, D.
Journal: EMBO J

By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single-stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double-stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non-recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.

MeSH Categories: DNA/*chemistry, DNA Replication, *Integrons, Molecular Sequence Data, *Nucleic Acid Conformation, Recombination, Genetic

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PTEN is recruited to the postsynaptic terminal for NMDA receptor-dependent long-term depression.

Publication Date: 2010 Aug 18 PMID: 20628354
Authors: Jurado, S. - Benoist, M. - Lario, A. - Knafo, S. - Petrok, C. N. - Esteban, J. A.
Journal: EMBO J

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is an important regulator of phosphatidylinositol-(3,4,5,)-trisphosphate signalling, which controls cell growth and differentiation. However, PTEN is also highly expressed in the adult brain, in which it can be found in dendritic spines in hippocampus and other brain regions. Here, we have investigated specific functions of PTEN in the regulation of synaptic function in excitatory hippocampal synapses. We found that NMDA receptor activation triggers a PDZ-dependent association between PTEN and the synaptic scaffolding molecule PSD-95. This association is accompanied by PTEN localization at the postsynaptic density and anchoring within the spine. On the other hand, enhancement of PTEN lipid phosphatase activity is able to drive depression of AMPA receptor-mediated synaptic responses. This activity is specifically required for NMDA receptor-dependent long-term depression (LTD), but not for LTP or metabotropic glutamate receptor-dependent LTD. Therefore, these results reveal PTEN as a regulated signalling molecule at the synapse, which is recruited to the postsynaptic membrane upon NMDA receptor activation, and is required for the modulation of synaptic activity during plasticity.

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Crystal structure of the human CNOT6L nuclease domain reveals strict poly(A) substrate specificity.

Publication Date: 2010 Aug 4 PMID: 20628353
Authors: Wang, H. - Morita, M. - Yang, X. - Suzuki, T. - Yang, W. - Wang, J. - Ito, K. - Wang, Q. - Zhao, C. - Bartlam, M. - Yamamoto, T. - Rao, Z.
Journal: EMBO J

CCR4, an evolutionarily conserved member of the CCR4-NOT complex, is the main cytoplasmic deadenylase. It contains a C-terminal nuclease domain with homology to the endonuclease-exonuclease-phosphatase (EEP) family of enzymes. We have determined the high-resolution three-dimensional structure of the nuclease domain of CNOT6L, a human homologue of CCR4, by X-ray crystallography using the single-wavelength anomalous dispersion method. This first structure of a deadenylase belonging to the EEP family adopts a complete alpha/beta sandwich fold typical of hydrolases with highly conserved active site residues similar to APE1. The active site of CNOT6L should recognize the RNA substrate through its negatively charged surface. In vitro deadenylase assays confirm the critical active site residues and show that the nuclease domain of CNOT6L exhibits full Mg(2+)-dependent deadenylase activity with strict poly(A) RNA substrate specificity. To understand the structural basis for poly(A) RNA substrate binding, crystal structures of the CNOT6L nuclease domain have also been determined in complex with AMP and poly(A) DNA. The resulting structures suggest a molecular deadenylase mechanism involving a pentacovalent phosphate transition.

MeSH Categories: Biocatalysis, Crystallography, X-Ray, DNA, Single-Stranded/metabolism, Humans, Magnesium/metabolism, Models, Molecular, Nucleic Acid Conformation, Poly A/*chemistry/*metabolism, Protein Binding, Protein Structure, Tertiary, Ribonucleases/*chemistry/genetics/*metabolism, Substrate Specificity

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ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import.

Publication Date: 2010 Aug 18 PMID: 20606625
Authors: Dormann, D. - Rodde, R. - Edbauer, D. - Bentmann, E. - Fischer, I. - Hruscha, A. - Than, M. E. - Mackenzie, I. R. - Capell, A. - Schmid, B. - Neumann, M. - Haass, C.
Journal: EMBO J

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C-terminus of FUS show neuronal cytoplasmic FUS-positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD-FUS). We show that a non-classical PY nuclear localization signal (NLS) in the C-terminus of FUS is necessary for nuclear import. The majority of fALS-associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease-causing mutations within a PY-NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co-deposit with inclusions in fALS and FTLD-FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS-opathies.

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Cis interaction between Semaphorin6A and Plexin-A4 modulates the repulsive response to Sema6A.

Publication Date: 2010 Aug 4 PMID: 20606624
Authors: Haklai-Topper, L. - Mlechkovich, G. - Savariego, D. - Gokhman, I. - Yaron, A.
Journal: EMBO J

The correct navigation of axons to their targets depends on guidance molecules in the extra-cellular environment. Differential responsiveness to a particular guidance cue is largely an outcome of disparity in the expression of its receptors on the reacting axons. Here, we show that the differential responsiveness of sympathetic and sensory neurons to the transmembrane Semaphorin Sema6A is mainly determined by its co-expression in the responding neurons. Both sympathetic and sensory neurons express the Sema6A receptor Plexin-A4, but only sympathetic neurons respond to it. The expression of Sema6A counteracts this responsiveness and is detected only in sensory neurons. Remarkably, sensory neurons that lack Sema6A gain sensitivity to it in a Plexin-A4-dependent manner. Using heterologus systems, we show that the co-expression of Sema6A and Plexin-A4 hinders the binding of exogenous ligand, suggesting that a Sema6A-Plexin-A4 cis interaction serves as an inhibitory mechanism. Finally, we provide evidence for differential modes of interaction in cis versus in trans. Thus, co-expression of a transmembrane cue together with its receptor can serve as a guidance response modulator.

MeSH Categories: Animals, Cells, Cultured, Cercopithecus aethiops, Humans, Mice, Mice, Knockout, Nerve Tissue Proteins/deficiency/*metabolism, Protein Binding, Receptors, Cell Surface/deficiency/*metabolism, Semaphorins/deficiency/*metabolism

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Ubiquitin-specific proteases 7 and 11 modulate Polycomb regulation of the INK4a tumour suppressor.

Publication Date: 2010 Aug 4 PMID: 20601937
Authors: Maertens, G. N. - El Messaoudi-Aubert, S. - Elderkin, S. - Hiom, K. - Peters, G.
Journal: EMBO J

An important facet of transcriptional repression by Polycomb repressive complex 1 (PRC1) is the mono-ubiquitination of histone H2A by the combined action of the Posterior sex combs (Psc) and Sex combs extra (Sce) proteins. Here, we report that two ubiquitin-specific proteases, USP7 and USP11, co-purify with human PRC1-type complexes through direct interactions with the Psc orthologues MEL18 and BMI1, and with other PRC1 components. Ablation of either USP7 or USP11 in primary human fibroblasts results in de-repression of the INK4a tumour suppressor accompanied by loss of PRC1 binding at the locus and a senescence-like proliferative arrest. Mechanistically, USP7 and USP11 regulate the ubiquitination status of the Psc and Sce proteins themselves, thereby affecting their turnover and abundance. Our results point to a novel function for USPs in the regulation and function of Polycomb complexes.

MeSH Categories: Cell Proliferation, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p16/*metabolism, Histones/metabolism, Humans, Nuclear Proteins/metabolism, Protein Binding, Proto-Oncogene Proteins/metabolism, RNA Interference, Repressor Proteins/*metabolism, Thiolester Hydrolases/genetics/*metabolism, Ubiquitin Thiolesterase/genetics/*metabolism, Ubiquitination

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MDM2 recruitment of lysine methyltransferases regulates p53 transcriptional output.

Publication Date: 2010 Aug 4 PMID: 20588255
Authors: Chen, L. - Li, Z. - Zwolinska, A. K. - Smith, M. A. - Cross, B. - Koomen, J. - Yuan, Z. M. - Jenuwein, T. - Marine, J. C. - Wright, K. L. - Chen, J.
Journal: EMBO J

MDM2 is a key regulator of the p53 tumor suppressor acting primarily as an E3 ubiquitin ligase to promote its degradation. MDM2 also inhibits p53 transcriptional activity by recruiting histone deacetylase and corepressors to p53. Here, we show that immunopurified MDM2 complexes have significant histone H3-K9 methyltransferase activity. The histone methyltransferases SUV39H1 and EHMT1 bind specifically to MDM2 but not to its homolog MDMX. MDM2 mediates formation of p53-SUV39H1/EHMT1 complex capable of methylating H3-K9 in vitro and on p53 target promoters in vivo. Furthermore, MDM2 promotes EHMT1-mediated p53 methylation at K373. Knockdown of SUV39H1 and EHMT1 increases p53 activity during stress response without affecting p53 levels, whereas their overexpression inhibits p53 in an MDM2-dependent manner. The p53 activator ARF inhibits SUV39H1 and EHMT1 binding to MDM2 and reduces MDM2-associated methyltransferase activity. These results suggest that MDM2-dependent recruitment of methyltransferases is a novel mechanism of p53 regulation through methylation of both p53 itself and histone H3 at target promoters.

MeSH Categories: Animals, Cells, Cultured, *Gene Expression Regulation, Histone-Lysine N-Methyltransferase/genetics/*metabolism, Histones/genetics/metabolism, Humans, Lysine/genetics/metabolism, Methylation, Methyltransferases/genetics/*metabolism, Mice, Promoter Regions, Genetic, Protein Binding, Proto-Oncogene Proteins c-mdm2/deficiency/genetics/*metabolism, Repressor Proteins/genetics/*metabolism, Stress, Physiological, Transcription, Genetic, Tumor Suppressor Protein p53/deficiency/*metabolism

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Recognition of the amber UAG stop codon by release factor RF1.

Publication Date: 2010 Aug 4 PMID: 20588254
Authors: Korostelev, A. - Zhu, J. - Asahara, H. - Noller, H. F.
Journal: EMBO J

We report the crystal structure of a termination complex containing release factor RF1 bound to the 70S ribosome in response to an amber (UAG) codon at 3.6-A resolution. The amber codon is recognized in the 30S subunit-decoding centre directly by conserved elements of domain 2 of RF1, including T186 of the PVT motif. Together with earlier structures, the mechanisms of recognition of all three stop codons by release factors RF1 and RF2 can now be described. Our structure confirms that the backbone amide of Q230 of the universally conserved GGQ motif is positioned to contribute directly to the catalysis of the peptidyl-tRNA hydrolysis reaction through stabilization of the leaving group and/or transition state. We also observe synthetic-negative interactions between mutations in the switch loop of RF1 and in helix 69 of 23S rRNA, revealing that these structural features interact functionally in the termination process. These findings are consistent with our proposal that structural rearrangements of RF1 and RF2 are critical to accurate translation termination.

MeSH Categories: Amino Acid Sequence, Bacterial Proteins/*chemistry/metabolism, Biocatalysis, Codon, Terminator/*chemistry/metabolism, Models, Molecular, Nucleic Acid Conformation, Peptide Termination Factors/*chemistry/metabolism, Protein Binding, Protein Structure, Tertiary, Thermus thermophilus/*chemistry/metabolism

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CHIP-dependent termination of MEKK2 regulates temporal ERK activation required for proper hyperosmotic response.

Publication Date: 2010 Aug 4 PMID: 20588253
Authors: Maruyama, T. - Kadowaki, H. - Okamoto, N. - Nagai, A. - Naguro, I. - Matsuzawa, A. - Shibuya, H. - Tanaka, K. - Murata, S. - Takeda, K. - Nishitoh, H. - Ichijo, H.
Journal: EMBO J

The extracellular signal-regulated kinase (ERK) pathway is an important signalling pathway that regulates a large number of cellular processes, including proliferation, differentiation and gene expression. Hyperosmotic stress activates the ERK pathway, whereas little is known about the regulatory mechanisms and physiological functions of ERK activation in hyperosmotic response. Here, we show that MAPK/ERK kinase kinase 2 (MEKK2), a member of the MAPKKK family, mediated the specific and transient activation of ERK, which was required for the induction of aquaporin 1 (AQP1) and AQP5 gene expression in response to hyperosmotic stress. Moreover, we identified the E3 ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) as a binding partner of MEKK2. Depletion of CHIP by small-interference RNA or gene targeting attenuated the degradation of MEKK2 and prolonged the ERK activity. Interestingly, hyperosmolality-induced gene expression of AQP1 and AQP5 was suppressed by CHIP depletion and was reversed by inhibition of the prolonged phase of ERK activity. These findings show that transient activation of the ERK pathway, which depends not only on MEKK2 activation, but also on CHIP-dependent MEKK2 degradation, is crucial for proper gene expression in hyperosmotic stress response.

MeSH Categories: Animals, Aquaporins/metabolism, Cells, Cultured, Enzyme Activation, Extracellular Signal-Regulated MAP Kinases/*metabolism, Humans, MAP Kinase Kinase Kinase 2/*metabolism, MAP Kinase Kinase Kinases/genetics/*metabolism, Mice, Mice, Knockout, Osmotic Pressure, Protein Binding, Rats, Ubiquitin-Protein Ligases/deficiency/genetics/*metabolism, Ubiquitination

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The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres.

Publication Date: 2010 Aug 4 PMID: 20588252
Authors: Rai, R. - Zheng, H. - He, H. - Luo, Y. - Multani, A. - Carpenter, P. B. - Chang, S.
Journal: EMBO J

Repair of DNA double-stranded breaks (DSBs) is crucial for the maintenance of genome stability. DSBs are repaired by either error prone non-homologous end-joining (NHEJ) or error-free homologous recombination. NHEJ precedes either by a classic, Lig4-dependent process (C-NHEJ) or an alternative, Lig4-independent one (A-NHEJ). Dysfunctional telomeres arising either through natural attrition due to telomerase deficiency or by removal of telomere-binding proteins are recognized as DSBs. In this report, we studied which end-joining pathways are required to join dysfunctional telomeres. In agreement with earlier studies, depletion of Trf2 resulted in end-to-end chromosome fusions mediated by the C-NHEJ pathway. In contrast, removal of Tpp1-Pot1a/b initiated robust chromosome fusions that are mediated by A-NHEJ. C-NHEJ is also dispensable for the fusion of naturally shortened telomeres. Our results reveal that telomeres engage distinct DNA repair pathways depending on how they are rendered dysfunctional, and that A-NHEJ is a major pathway to process dysfunctional telomeres.

MeSH Categories: Animals, Antigens, Nuclear/metabolism, Cells, Cultured, *DNA Repair, DNA-Binding Proteins/deficiency/metabolism, Humans, Intracellular Signaling Peptides and, Proteins/deficiency/genetics/metabolism, Mice, Mice, Knockout, *Telomere, Telomeric Repeat Binding Protein 2/metabolism

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Hedgehog controls neural stem cells through p53-independent regulation of Nanog.

Publication Date: 2010 Aug 4 PMID: 20581804
Authors: Po, A. - Ferretti, E. - Miele, E. - De Smaele, E. - Paganelli, A. - Canettieri, G. - Coni, S. - Di Marcotullio, L. - Biffoni, M. - Massimi, L. - Di Rocco, C. - Screpanti, I. - Gulino, A.
Journal: EMBO J

Hedgehog (Hh) pathway has a pivotal function in development and tumorigenesis, processes sustained by stem cells (SCs). The transcription factor Nanog controls stemness acting as a key determinant of both embryonic SC self-renewal and differentiated somatic cells reprogramming to pluripotency, in concert with the loss of the oncosuppressor p53. How Nanog is regulated by microenvironmental signals in postnatal SC niches has been poorly investigated. Here, we show that Nanog is highly expressed in SCs from postnatal cerebellum and medulloblastoma, and acts as a critical mediator of Hh-driven self-renewal. Indeed, the downstream effectors of Hh activity, Gli1 and Gli2, bind to Nanog-specific cis-regulatory sequences both in mouse and human SCs. Loss of p53, a key event promoting cell stemness, activates Hh signalling, thereby contributing to Nanog upregulation. Conversely, Hh downregulates p53 but does not require p53 to control Nanog. Our data reveal a mechanism for the function of Hh in the control of stemness that represents a crucial component of an integrated circuitry determining cell fate decision and involved in the maintenance of cancer SCs.

MeSH Categories: Animals, Base Sequence, Cell Proliferation, Cells, Cultured, Gene Expression Profiling, Hedgehog Proteins/*metabolism, Homeodomain Proteins/genetics/*metabolism, Humans, Medulloblastoma/metabolism, Mice, Molecular Sequence Data, Neoplastic Stem Cells/metabolism, Neurons/cytology/*metabolism, Oncogene Proteins/genetics/metabolism, Sequence Alignment, Stem Cells/cytology/*metabolism, Trans-Activators/genetics/metabolism, Transcription, Genetic, Tumor Suppressor Protein p53/genetics/*metabolism

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Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.

Publication Date: 2010 Aug 4 PMID: 20581803
Authors: Dechant, R. - Binda, M. - Lee, S. S. - Pelet, S. - Winderickx, J. - Peter, M.
Journal: EMBO J

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 beta-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.

MeSH Categories: Animals, Cell Line, Cyclic AMP-Dependent Protein Kinases/metabolism, Cytosol/*metabolism, Glucose/*metabolism, Hydrogen-Ion Concentration, Insulin/secretion, Insulin-Secreting Cells/secretion, Mice, *Second Messenger Systems, Vacuolar Proton-Translocating ATPases/*metabolism

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NANOG regulates glioma stem cells and is essential in vivo acting in a cross-functional network with GLI1 and p53.

Publication Date: 2010 Aug 4 PMID: 20581802
Authors: Zbinden, M. - Duquet, A. - Lorente-Trigos, A. - Ngwabyt, S. N. - Borges, I. - Ruiz i Altaba, A.
Journal: EMBO J

A cohort of genes associated with embryonic stem (ES) cell behaviour, including NANOG, are expressed in a number of human cancers. They form an ES-like signature we first described in glioblastoma multiforme (GBM), a highly invasive and incurable brain tumour. We have also shown that HEDGEHOG-GLI (HH-GLI) signalling is required for GBM growth, stem cell expansion and the expression of this (ES)-like stemness signature. Here, we address the function of NANOG in human GBMs and its relationship with HH-GLI activity. We find that NANOG modulates gliomasphere clonogenicity, CD133(+) stem cell cell behavior and proliferation, and is regulated by HH-GLI signalling. However, GLI1 also requires NANOG activity forming a positive loop, which is negatively controlled by p53 and vice versa. NANOG is essential for GBM tumourigenicity in orthotopic xenografts and it is epistatic to HH-GLI activity. Our data establish NANOG as a novel HH-GLI mediator essential for GBMs. We propose that this function is conserved and that tumour growth and stem cell behaviour rely on the status of a functional GLI1-NANOG-p53 network.

MeSH Categories: Aged, Aged, 80 and over, Animals, Cell Proliferation, Female, Gene Expression Regulation, Neoplastic, Glioma/*metabolism/pathology, Homeodomain Proteins/genetics/*metabolism, Humans, Male, Middle Aged, Neoplastic Stem Cells/cytology/*metabolism, Signal Transduction, Transcription Factors/*metabolism, Tumor Cells, Cultured, Tumor Suppressor Protein p53/*metabolism

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Overlapping functions of Hdac1 and Hdac2 in cell cycle regulation and haematopoiesis.

Publication Date: 2010 Aug 4 PMID: 20571512
Authors: Wilting, R. H. - Yanover, E. - Heideman, M. R. - Jacobs, H. - Horner, J. - van der Torre, J. - DePinho, R. A. - Dannenberg, J. H.
Journal: EMBO J

Histone deacetylases (HDACs) counterbalance acetylation of lysine residues, a protein modification involved in numerous biological processes. Here, Hdac1 and Hdac2 conditional knock-out alleles were used to study the function of class I Hdac1 and Hdac2 in cell cycle progression and haematopoietic differentiation. Combined deletion of Hdac1 and Hdac2, or inactivation of their deacetylase activity in primary or oncogenic-transformed fibroblasts, results in a senescence-like G(1) cell cycle arrest, accompanied by up-regulation of the cyclin-dependent kinase inhibitor p21(Cip). Notably, concomitant genetic inactivation of p53 or p21(Cip) indicates that Hdac1 and Hdac2 regulate p53-p21(Cip)-independent pathways critical for maintaining cell cycle progression. In vivo, we show that Hdac1 and Hdac2 are not essential for liver homeostasis. In contrast, total levels of Hdac1 and Hdac2 in the haematopoietic system are critical for erythrocyte-megakaryocyte differentiation. Dual inactivation of Hdac1 and Hdac2 results in apoptosis of megakaryocytes and thrombocytopenia. Together, these data indicate that Hdac1 and Hdac2 have overlapping functions in cell cycle regulation and haematopoiesis. In addition, this work provides insights into mechanism-based toxicities observed in patients treated with HDAC inhibitors.

MeSH Categories: Anemia/enzymology, Animals, Apoptosis, Biocatalysis, *Cell Cycle, Cells, Cultured, Cyclin-Dependent Kinase Inhibitor p21/deficiency/metabolism, *Hematopoiesis, Histone Deacetylase 1/deficiency/*metabolism, Histone Deacetylase 2/deficiency/*metabolism, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Thrombocytopenia/enzymology/pathology, Tumor Suppressor Protein p53/metabolism

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Elg1, an alternative subunit of the RFC clamp loader, preferentially interacts with SUMOylated PCNA.

Publication Date: 2010 Aug 4 PMID: 20571511
Authors: Parnas, O. - Zipin-Roitman, A. - Pfander, B. - Liefshitz, B. - Mazor, Y. - Ben-Aroya, S. - Jentsch, S. - Kupiec, M.
Journal: EMBO J

Replication-factor C (RFC) is a protein complex that loads the processivity clamp PCNA onto DNA. Elg1 is a conserved protein with homology to the largest subunit of RFC, but its function remained enigmatic. Here, we show that yeast Elg1 interacts physically and genetically with PCNA, in a manner that depends on PCNA modification, and exhibits preferential affinity for SUMOylated PCNA. This interaction is mediated by three small ubiquitin-like modifier (SUMO)-interacting motifs and a PCNA-interacting protein box close to the N-terminus of Elg1. These motifs are important for the ability of Elg1 to maintain genomic stability. SUMOylated PCNA is known to recruit the helicase Srs2, and in the absence of Elg1, Srs2 and SUMOylated PCNA accumulate on chromatin. Strains carrying mutations in both ELG1 and SRS2 exhibit a synthetic fitness defect that depends on PCNA modification. Our results underscore the importance of Elg1, Srs2 and SUMOylated PCNA in the maintenance of genomic stability.

MeSH Categories: Amino Acid Sequence, Antigens, Nuclear/chemistry/genetics/*metabolism, Carrier Proteins/genetics/*metabolism, DNA Helicases/genetics/metabolism, Gene Deletion, *Genomic Instability, Molecular Sequence Data, Protein Binding, Saccharomyces cerevisiae/genetics/*metabolism, Saccharomyces cerevisiae Proteins/chemistry/genetics/*metabolism, Sequence Alignment, Small Ubiquitin-Related Modifier Proteins/chemistry/*metabolism, Ubiquitination

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Opposing functions of two sub-domains of the SNARE-complex in neurotransmission.

Publication Date: 2010 Aug 4 PMID: 20562829
Authors: Weber, J. P. - Reim, K. - Sorensen, J. B.
Journal: EMBO J

The SNARE-complex consisting of synaptobrevin-2/VAMP-2, SNAP-25 and syntaxin-1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap-25 null mice rescued with mutants challenging the C-terminal, N-terminal and middle domains of the SNARE-bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub-domains of the SNARE-bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C-terminal end of the SNARE-bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C-terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N-terminal end of the SNARE-bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N-terminal deletion delays vesicle priming after a high-frequency train. We propose that the stability of N-terminal two-thirds of the SNARE-bundle has a function for vesicle priming and limiting spontaneous release.

MeSH Categories: Amino Acid Sequence, Animals, Cells, Cultured, Gene Deletion, Hippocampus/metabolism, Mice, Mice, Knockout, Molecular Sequence Data, Mutation, Sequence Alignment, *Synaptic Transmission, Synaptosomal-Associated Protein, 25/chemistry/deficiency/genetics/*metabolism

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The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site.

Publication Date: 2010 Aug 4 PMID: 20562828
Authors: Tupin, A. - Gualtieri, M. - Leonetti, J. P. - Brodolin, K.
Journal: EMBO J

Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the sigma(70) subunit region 3.2 and the RNAP beta' subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks sigma(70)-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the beta' subunit switch-2 element and the sigma(70) subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.

MeSH Categories: Aminoglycosides/*chemistry/pharmacology, Base Sequence, *Catalytic Domain, DNA/*chemistry, DNA-Directed RNA Polymerases/*chemistry/metabolism, Gene Deletion, Models, Molecular, Nucleic Acid Denaturation, Promoter Regions, Genetic, RNA/metabolism, Transcription, Genetic/*drug effects

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The peroxisomal receptor Pex19p forms a helical mPTS recognition domain.

Publication Date: 2010 Aug 4 PMID: 20531392
Authors: Schueller, N. - Holton, S. J. - Fodor, K. - Milewski, M. - Konarev, P. - Stanley, W. A. - Wolf, J. - Erdmann, R. - Schliebs, W. - Song, Y. H. - Wilmanns, M.
Journal: EMBO J

The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured alpha-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 muM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions.

MeSH Categories: Amino Acid Sequence, Animals, Binding Sites, Crystallography, X-Ray, Humans, Membrane Proteins/*chemistry/genetics/metabolism, Models, Molecular, Molecular Sequence Data, Mutation, Peroxisomes/*chemistry/metabolism, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment

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