EMBO Journal

The mycobacterial Mpa-proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus.

Publication Date: 2010 Mar 4 PMID: 20203624
Authors: Striebel, F. - Hunkeler, M. - Summer, H. - Weber-Ban, E.
Journal: EMBO J

Mycobacterium tuberculosis, along with other actinobacteria, harbours proteasomes in addition to members of the general bacterial repertoire of degradation complexes. In analogy to ubiquitination in eukaryotes, substrates are tagged for proteasomal degradation with prokaryotic ubiquitin-like protein (Pup) that is recognized by the N-terminal coiled-coil domain of the ATPase Mpa (also called ARC). Here, we reconstitute the entire mycobacterial proteasome degradation system for pupylated substrates and establish its mechanistic features with respect to substrate recruitment, unfolding and degradation. We show that the Mpa-proteasome complex unfolds and degrades Pup-tagged proteins and that this activity requires physical interaction of the ATPase with the proteasome. Furthermore, we establish the N-terminal region of Pup as the structural element required for engagement of pupylated substrates into the Mpa pore. In this process, Mpa pulls on Pup to initiate unfolding of substrate proteins and to drag them toward the proteasome chamber. Unlike the eukaryotic ubiquitin, Pup is not recycled but degraded with the substrate. This assigns a dual function to Pup as both the Mpa recognition element as well as the threading determinant.

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AP-1/sigma1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory.

Publication Date: 2010 Mar 4 PMID: 20203623
Authors: Glyvuk, N. - Tsytsyura, Y. - Geumann, C. - D'Hooge, R. - Huve, J. - Kratzke, M. - Baltes, J. - Boning, D. - Klingauf, J. - Schu, P.
Journal: EMBO J

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1-sigma1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1-sigma1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three sigma1 subunit isoforms: A, B and C. The expressions of sigma1A and sigma1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from sigma1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The sigma1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation.

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Dispose to the pole-protein aggregation control in bacteria.

Publication Date: 2010 Mar 3 PMID: 20197791
Authors: Zietkiewicz, S. - Liberek, K.
Journal: EMBO J

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Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos.

Publication Date: 2010 Feb 25 PMID: 20186124
Authors: Vitale, I. - Senovilla, L. - Jemaa, M. - Michaud, M. - Galluzzi, L. - Kepp, O. - Nanty, L. - Criollo, A. - Rello-Varona, S. - Manic, G. - Metivier, D. - Vivet, S. - Tajeddine, N. - Joza, N. - Valent, A. - Castedo, M. - Kroemer, G.
Journal: EMBO J

Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells.

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The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy.

Publication Date: 2010 Feb 25 PMID: 20186123
Authors: Pedrotti, S. - Bielli, P. - Paronetto, M. P. - Ciccosanti, F. - Fimia, G. M. - Stamm, S. - Manley, J. L. - Sette, C.
Journal: EMBO J

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C-to-T transition at position +6 in exon-7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C-to-T transition in SMN2 creates a putative binding site for the RNA-binding protein Sam68. RNA pull-down assays and UV-crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon-7 skipping. Moreover, mutations in the Sam68-binding site of SMN2 or in the RNA-binding domain of Sam68 completely abrogated its effect on exon-7 skipping. Retroviral infection of dominant-negative mutants of Sam68 that interfere with its RNA-binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon-7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.

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Sam68 sequestration and partial loss of function are associated with splicing alterations in FXTAS patients.

Publication Date: 2010 Feb 25 PMID: 20186122
Authors: Sellier, C. - Rau, F. - Liu, Y. - Tassone, F. - Hukema, R. K. - Gattoni, R. - Schneider, A. - Richard, S. - Willemsen, R. - Elliott, D. J. - Hagerman, P. J. - Charlet-Berguerand, N.
Journal: EMBO J

Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) is a neurodegenerative disorder caused by expansion of 55-200 CGG repeats in the 5'-UTR of the FMR1 gene. FXTAS is characterized by action tremor, gait ataxia and impaired executive cognitive functioning. It has been proposed that FXTAS is caused by titration of RNA-binding proteins by the expanded CGG repeats. Sam68 is an RNA-binding protein involved in alternative splicing regulation and its ablation in mouse leads to motor coordination defects. Here, we report that mRNAs containing expanded CGG repeats form large and dynamic intranuclear RNA aggregates that recruit several RNA-binding proteins sequentially, first Sam68, then hnRNP-G and MBNL1. Importantly, Sam68 is sequestered by expanded CGG repeats and thereby loses its splicing-regulatory function. Consequently, Sam68-responsive splicing is altered in FXTAS patients. Finally, we found that regulation of Sam68 tyrosine phosphorylation modulates its localization within CGG aggregates and that tautomycin prevents both Sam68 and CGG RNA aggregate formation. Overall, these data support an RNA gain-of-function mechanism for FXTAS neuropathology, and suggest possible target routes for treatment options.

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Unravelling the mechanism of dual-specificity GAPs.

Publication Date: 2010 Feb 25 PMID: 20186121
Authors: Sot, B. - Kotting, C. - Deaconescu, D. - Suveyzdis, Y. - Gerwert, K. - Wittinghofer, A.
Journal: EMBO J

The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1(IP4BP) and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-alpha6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different.

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A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome.

Publication Date: 2010 Feb 25 PMID: 20186120
Authors: Richter, R. - Rorbach, J. - Pajak, A. - Smith, P. M. - Wessels, H. J. - Huynen, M. A. - Smeitink, J. A. - Lightowlers, R. N. - Chrzanowska-Lightowlers, Z. M.
Journal: EMBO J

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.

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Architecture of a nascent viral fusion pore.

Publication Date: 2010 Feb 18 PMID: 20168302
Authors: Lee, K. K.
Journal: EMBO J

Enveloped viruses use specialized protein machinery to fuse the viral membrane with that of the host cell during cell invasion. In influenza virus, hundreds of copies of the haemagglutinin (HA) fusion glycoprotein project from the virus surface. Despite intensive study of HA and its fusion activity, the protein's modus operandi in manipulating viral and target membranes to catalyse their fusion is poorly understood. Here, the three-dimensional architecture of influenza virus-liposome complexes at pH 5.5 was investigated by electron cryo-tomography. Tomographic reconstructions show that early stages of membrane remodeling take place in a target membrane-centric manner, progressing from punctate dimples, to the formation of a pinched liposomal funnel that may impinge on the apparently unperturbed viral envelope. The results suggest that the M1 matrix layer serves as an endoskeleton for the virus and a foundation for HA during membrane fusion. Fluorescence spectroscopy monitoring fusion between liposomes and virions shows that leakage of liposome contents takes place more rapidly than lipid mixing at pH 5.5. The relation of 'leaky' fusion to the observed prefusion structures is discussed.

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Chromatin association and regulation of rDNA transcription by the Ras-family protein RasL11a.

Publication Date: 2010 Feb 18 PMID: 20168301
Authors: Pistoni, M. - Verrecchia, A. - Doni, M. - Guccione, E. - Amati, B.
Journal: EMBO J

RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.

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Bacterial actin MreB assembles in complex with cell shape protein RodZ.

Publication Date: 2010 Feb 18 PMID: 20168300
Authors: van den Ent, F. - Johnson, C. M. - Persons, L. - de Boer, P. - Lowe, J.
Journal: EMBO J

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

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The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats.

Publication Date: 2010 Feb 18 PMID: 20168299
Authors: Guetg, C. - Lienemann, P. - Sirri, V. - Fussenegger, M. - Hernandez-Verdun, D. - Hottiger, M. O. - Santoro, R.
Journal: EMBO J

Maintenance of specific heterochromatic domains is crucial for genome stability. In eukaryotic cells, a fraction of the tandem-repeated ribosomal RNA (rRNA) genes is organized in the heterochromatic structures. The principal determinant of rDNA silencing is the nucleolar remodelling complex, NoRC, that consists of TIP5 (TTF-1-interacting protein-5) and the ATPase SNF2h. Here we showed that TIP5 not only mediates the establishment of rDNA silencing but also the formation of perinucleolar heterochromatin that contains centric and pericentric repeats. Our data indicated that the TIP5-mediated heterochromatin is indispensable for stability of silent rRNA genes and of major and minor satellite repeats. Moreover, depletion of TIP5 impairs rDNA silencing, upregulates rDNA transcription levels and induces cell transformation. These findings point to a role of TIP5 in protecting genome stability and suggest that it can play a role in the cellular transformation process.

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A polycystin-2 (TRPP2) dimerization domain essential for the function of heteromeric polycystin complexes.

Publication Date: 2010 Feb 18 PMID: 20168298
Authors: Giamarchi, A. - Feng, S. - Rodat-Despoix, L. - Xu, Y. - Bubenshchikova, E. - Newby, L. J. - Hao, J. - Gaudioso, C. - Crest, M. - Lupas, A. N. - Honore, E. - Williamson, M. P. - Obara, T. - Ong, A. C. - Delmas, P.
Journal: EMBO J

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in two genes, PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Earlier work has shown that PC1 and PC2 assemble into a polycystin complex implicated in kidney morphogenesis. PC2 also assembles into homomers of uncertain functional significance. However, little is known about the molecular mechanisms that direct polycystin complex assembly and specify its functions. We have identified a coiled coil in the C-terminus of PC2 that functions as a homodimerization domain essential for PC1 binding but not for its self-oligomerization. Dimerization-defective PC2 mutants were unable to reconstitute PC1/PC2 complexes either at the plasma membrane (PM) or at PM-endoplasmic reticulum (ER) junctions but could still function as ER Ca(2+)-release channels. Expression of dimerization-defective PC2 mutants in zebrafish resulted in a cystic phenotype but had lesser effects on organ laterality. We conclude that C-terminal dimerization of PC2 specifies the formation of polycystin complexes but not formation of ER-localized PC2 channels. Mutations that affect PC2 C-terminal homo- and heteromerization are the likely molecular basis of cyst formation in ADPKD.

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FANCM: fork pause, rewind and play.

Publication Date: 2010 Feb 17 PMID: 20160754
Authors: Collis, S. J. - Boulton, S. J.
Journal: EMBO J

DNA lesions or genomic regions that are difficult to traverse frequently hinder or block DNA replication. In response to replication fork stalling, the cell activates the replication stress response pathway, which acts to protect the fork from collapse, promotes the repair or bypass of the blockage and facilitates the resumption of DNA synthesis. In this issue of the EMBO Journal, two studies conducted by the Constantinou and Niedzwiedz laboratories shed light on how the DNA translocase FANCM acts to regulate the replication stress response (Luke-Glaser et al, 2009; Schwab et al, 2009). These studies help to explain how FANCM (mutated in the human cancer predisposition syndrome, Fanconi's anaemia (FA)) co-ordinately regulates checkpoint signalling and replication fork progression.

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Regulation of clathrin adaptor function in endocytosis: novel role for the SAM domain.

Publication Date: 2010 Feb 11 PMID: 20150898
Authors: Di Pietro, S. M. - Cascio, D. - Feliciano, D. - Bowie, J. U. - Payne, G. S.
Journal: EMBO J

During clathrin-mediated endocytosis, adaptor proteins play central roles in coordinating the assembly of clathrin coats and cargo selection. Here we characterize the binding of the yeast endocytic adaptor Sla1p to clathrin through a variant clathrin-binding motif that is negatively regulated by the Sla1p SHD2 domain. The crystal structure of SHD2 identifies the domain as a sterile alpha-motif (SAM) domain and shows a propensity to oligomerize. By co-immunoprecipitation, Sla1p binds to clathrin and self-associates in vivo. Mutations in the clathrin-binding motif that abolish clathrin binding and structure-based mutations in SHD2 that impede self-association result in endocytosis defects and altered dynamics of Sla1p assembly at the sites of endocytosis. These results define a novel mechanism for negative regulation of clathrin binding by an adaptor and suggest a role for SAM domains in clathrin-mediated endocytosis.

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New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation.

Publication Date: 2010 Feb 11 PMID: 20150897
Authors: Pitzschke, A. - Hirt, H.
Journal: EMBO J

Agrobacterium tumefaciens causes tumour formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plant-Agrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells.

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Structure of a double ubiquitin-like domain in the talin head: a role in integrin activation.

Publication Date: 2010 Feb 11 PMID: 20150896
Authors: Goult, B. T. - Bouaouina, M. - Elliott, P. R. - Bate, N. - Patel, B. - Gingras, A. R. - Grossmann, J. G. - Roberts, G. C. - Calderwood, D. A. - Critchley, D. R. - Barsukov, I. L.
Journal: EMBO J

Talin is a 270-kDa protein that activates integrins and couples them to cytoskeletal actin. Talin contains an N-terminal FERM domain comprised of F1, F2 and F3 domains, but it is atypical in that F1 contains a large insert and is preceded by an extra domain F0. Although F3 contains the binding site for beta-integrin tails, F0 and F1 are also required for activation of beta1-integrins. Here, we report the solution structures of F0, F1 and of the F0F1 double domain. Both F0 and F1 have ubiquitin-like folds joined in a novel fixed orientation by an extensive charged interface. The F1 insert forms a loop with helical propensity, and basic residues predicted to reside on one surface of the helix are required for binding to acidic phospholipids and for talin-mediated activation of beta1-integrins. This and the fact that basic residues on F2 and F3 are also essential for integrin activation suggest that extensive interactions between the talin FERM domain and acidic membrane phospholipids are required to orientate the FERM domain such that it can activate integrins.

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Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

Publication Date: 2010 Feb 11 PMID: 20150895
Authors: Wang, H. - Holst, J. - Woo, S. R. - Guy, C. - Bettini, M. - Wang, Y. - Shafer, A. - Naramura, M. - Mingueneau, M. - Dragone, L. L. - Hayes, S. M. - Malissen, B. - Band, H. - Vignali, D. A.
Journal: EMBO J

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

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Force generated by actomyosin contraction builds bridges between adhesive contacts.

Publication Date: 2010 Feb 11 PMID: 20150894
Authors: Rossier, O. M. - Gauthier, N. - Biais, N. - Vonnegut, W. - Fardin, M. A. - Avigan, P. - Heller, E. R. - Mathur, A. - Ghassemi, S. - Koeckert, M. S. - Hone, J. C. - Sheetz, M. P.
Journal: EMBO J

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)-coated stripes separated by non-adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non-adhesive regions in a treadmilling network. Inhibition of myosin-II (MII) or Rho-kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin-A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross-links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

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VHS domains of ESCRT-0 cooperate in high-avidity binding to polyubiquitinated cargo.

Publication Date: 2010 Feb 11 PMID: 20150893
Authors: Ren, X. - Hurley, J. H.
Journal: EMBO J

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT-0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain-ubiquitin complex was solved at 2.6 A resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS-domain containing proteins in yeast and humans, including both subunits of ESCRT-0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63-linked polyubiquitinated cargo. Intact human ESCRT-0 binds Lys63-linked tetraubiquitin 50-fold more tightly than monoubiquitin, though only 2-fold more tightly than Lys48-linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT-0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin-binding sites in yeast ESCRT-0 shows that cooperation between them is required for the sorting of the Lys63-linked polyubiquitinated cargo Cps1 to the vacuole.

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Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival.

Publication Date: 2010 Feb 11 PMID: 20150892
Authors: Drosten, M. - Dhawahir, A. - Sum, E. Y. - Urosevic, J. - Lechuga, C. G. - Esteban, L. M. - Castellano, E. - Guerra, C. - Santos, E. - Barbacid, M.
Journal: EMBO J

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras-like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D-type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.

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ESCRT-II coordinates the assembly of ESCRT-III filaments for cargo sorting and multivesicular body vesicle formation.

Publication Date: 2010 Mar 3 PMID: 20134403
Authors: Teis, D. - Saksena, S. - Judson, B. L. - Emr, S. D.
Journal: EMBO J

The sequential action of five distinct endosomal-sorting complex required for transport (ESCRT) complexes is required for the lysosomal downregulation of cell surface receptors through the multivesicular body (MVB) pathway. On endosomes, the assembly of ESCRT-III is a highly ordered process. We show that the length of ESCRT-III (Snf7) oligomers controls the size of MVB vesicles and addresses how ESCRT-II regulates ESCRT-III assembly. The first step of ESCRT-III assembly is mediated by Vps20, which nucleates Snf7/Vps32 oligomerization, and serves as the link to ESCRT-II. The ESCRT-II subunit Vps25 induces an essential conformational switch that converts inactive monomeric Vps20 into the active nucleator for Snf7 oligomerization. Each ESCRT-II complex contains two Vps25 molecules (arms) that generate a characteristic Y-shaped structure. Mutant 'one-armed' ESCRT-II complexes with a single Vps25 arm are sufficient to nucleate Snf7 oligomerization. However, these oligomers cannot execute ESCRT-III function. Both Vps25 arms provide essential geometry for the assembly of a functional ESCRT-III complex. We propose that ESCRT-II serves as a scaffold that nucleates the assembly of two Snf7 oligomers, which together are required for cargo sequestration and vesicle formation during MVB sorting.

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Bicaudal-D binds clathrin heavy chain to promote its transport and augments synaptic vesicle recycling.

Publication Date: 2010 Mar 3 PMID: 20111007
Authors: Li, X. - Kuromi, H. - Briggs, L. - Green, D. B. - Rocha, J. J. - Sweeney, S. T. - Bullock, S. L.
Journal: EMBO J

Cargo transport by microtubule-based motors is essential for cell organisation and function. The Bicaudal-D (BicD) protein participates in the transport of a subset of cargoes by the minus-end-directed motor dynein, although the full extent of its functions is unclear. In this study, we report that in Drosophila zygotic BicD function is only obligatory in the nervous system. Clathrin heavy chain (Chc), a major constituent of coated pits and vesicles, is the most abundant protein co-precipitated with BicD from head extracts. BicD binds Chc directly and interacts genetically with components of the pathway for clathrin-mediated membrane trafficking. Directed transport and subcellular localisation of Chc is strongly perturbed in BicD mutant presynaptic boutons. Functional assays show that BicD and dynein are essential for the maintenance of normal levels of neurotransmission specifically during high-frequency electrical stimulation and that this is associated with a reduced rate of recycling of internalised synaptic membrane. Our results implicate BicD as a new player in clathrin-associated trafficking processes and show a novel requirement for microtubule-based motor transport in the synaptic vesicle cycle.

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Kinesin-1/Hsc70-dependent mechanism of slow axonal transport and its relation to fast axonal transport.

Publication Date: 2010 Feb 17 PMID: 20111006
Authors: Terada, S. - Kinjo, M. - Aihara, M. - Takei, Y. - Hirokawa, N.
Journal: EMBO J

Cytoplasmic protein transport in axons ('slow axonal transport') is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport ('fast axonal transport'). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.

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The intronic splicing code: multiple factors involved in ATM pseudoexon definition.

Publication Date: 2010 Feb 17 PMID: 20094034
Authors: Dhir, A. - Buratti, E. - van Santen, M. A. - Luhrmann, R. - Baralle, F. E.
Journal: EMBO J

Abundance of pseudo splice sites in introns can potentially give rise to innumerable pseudoexons, outnumbering the real ones. Nonetheless, these are efficiently ignored by the splicing machinery, a process yet to be understood completely. Although numerous 5' splice site-like sequences functioning as splicing silencers have been found to be enriched in predicted human pseudoexons, the lack of active pseudoexons pose a fundamental challenge to how these U1snRNP-binding sites function in splicing inhibition. Here, we address this issue by focusing on a previously described pathological ATM pseudoexon whose inhibition is mediated by U1snRNP binding at intronic splicing processing element (ISPE), composed of a consensus donor splice site. Spliceosomal complex assembly demonstrates inefficient A complex formation when ISPE is intact, implying U1snRNP-mediated unproductive U2snRNP recruitment. Furthermore, interaction of SF2/ASF with its motif seems to be dependent on RNA structure and U1snRNP interaction. Our results suggest a complex combinatorial interplay of RNA structure and trans-acting factors in determining the splicing outcome and contribute to understanding the intronic splicing code for the ATM pseudoexon.

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POT1-TPP1 enhances telomerase processivity by slowing primer dissociation and aiding translocation.

Publication Date: 2010 Mar 3 PMID: 20094033
Authors: Latrick, C. M. - Cech, T. R.
Journal: EMBO J

Telomerase contributes to chromosome end replication by synthesizing repeats of telomeric DNA, and the telomeric DNA-binding proteins protection of telomeres (POT1) and TPP1 synergistically increase its repeat addition processivity. To understand the mechanism of increased processivity, we measured the effect of POT1-TPP1 on individual steps in the telomerase reaction cycle. Under conditions where telomerase was actively synthesizing DNA, POT1-TPP1 bound to the primer decreased primer dissociation rate. In addition, POT1-TPP1 increased the translocation efficiency. A template-mutant telomerase that synthesizes DNA that cannot be bound by POT1-TPP1 exhibited increased processivity only when the primer contained at least one POT1-TPP1-binding site, so a single POT1-TPP1-DNA interaction is necessary and sufficient for stimulating processivity. The POT1-TPP1 effect is specific, as another single-stranded DNA-binding protein, gp32, cannot substitute. POT1-TPP1 increased processivity even when substoichiometric relative to the DNA, providing evidence for a recruitment function. These results support a model in which POT1-TPP1 enhances telomerase processivity in a manner markedly different from the sliding clamps used by DNA polymerases.

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Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing.

Publication Date: 2010 Mar 3 PMID: 20094032
Authors: Winkler, J. - Seybert, A. - Konig, L. - Pruggnaller, S. - Haselmann, U. - Sourjik, V. - Weiss, M. - Frangakis, A. S. - Mogk, A. - Bukau, B.
Journal: EMBO J

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

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Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.

Publication Date: 2010 Feb 17 PMID: 20094031
Authors: Chen, Z. A. - Jawhari, A. - Fischer, L. - Buchen, C. - Tahir, S. - Kamenski, T. - Rasmussen, M. - Lariviere, L. - Bukowski-Wills, J. C. - Nilges, M. - Cramer, P. - Rappsilber, J.
Journal: EMBO J

Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.

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Structural diversity and dynamics of genomic replication origins in Schizosaccharomyces pombe.

Publication Date: 2010 Mar 3 PMID: 20094030
Authors: Cotobal, C. - Segurado, M. - Antequera, F.
Journal: EMBO J

DNA replication origins (ORI) in Schizosaccharomyces pombe colocalize with adenine and thymine (A+T)-rich regions, and earlier analyses have established a size from 0.5 to over 3 kb for a DNA fragment to drive replication in plasmid assays. We have asked what are the requirements for ORI function in the chromosomal context. By designing artificial ORIs, we have found that A+T-rich fragments as short as 100 bp without homology to S. pombe DNA are able to initiate replication in the genome. On the other hand, functional dissection of endogenous ORIs has revealed that some of them span a few kilobases and include several modules that may be as short as 25-30 contiguous A+Ts capable of initiating replication from ectopic chromosome positions. The search for elements with these characteristics across the genome has uncovered an earlier unnoticed class of low-efficiency ORIs that fire late during S phase. These results indicate that ORI specification and dynamics varies widely in S. pombe, ranging from very short elements to large regions reminiscent of replication initiation zones in mammals.

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Nucleolar retention of a translational C/EBPalpha isoform stimulates rDNA transcription and cell size.

Publication Date: 2010 Mar 3 PMID: 20075868
Authors: Muller, C. - Bremer, A. - Schreiber, S. - Eichwald, S. - Calkhoven, C. F.
Journal: EMBO J

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N-terminal sequences. Here, we describe the function of an N-terminally extended protein isoform of CCAAT enhancer-binding protein alpha (C/EBPalpha) that is translated from an alternative non-AUG initiation codon. We show that a basic amino-acid motif within its N-terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended-C/EBPalpha occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I-specific factors upstream-binding factor 1 (UBF-1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL-60 cells, endogenous expression of extended-C/EBPalpha is lost concomitantly with nucleolar C/EBPalpha immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended-C/EBPalpha induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPalpha adding to its role as key regulator of cell growth and proliferation.

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Evidence for a protein tether involved in somatic touch.

Publication Date: 2010 Feb 17 PMID: 20075867
Authors: Hu, J. - Chiang, L. Y. - Koch, M. - Lewin, G. R.
Journal: EMBO J

The gating of ion channels by mechanical force underlies the sense of touch and pain. The mode of gating of mechanosensitive ion channels in vertebrate touch receptors is unknown. Here we show that the presence of a protein link is necessary for the gating of mechanosensitive currents in all low-threshold mechanoreceptors and some nociceptors of the dorsal root ganglia (DRG). Using TEM, we demonstrate that a protein filament with of length approximately 100 nm is synthesized by sensory neurons and may link mechanosensitive ion channels in sensory neurons to the extracellular matrix. Brief treatment of sensory neurons with non-specific and site-specific endopeptidases destroys the protein tether and abolishes mechanosensitive currents in sensory neurons without affecting electrical excitability. Protease-sensitive tethers are also required for touch-receptor function in vivo. Thus, unlike the majority of nociceptors, cutaneous mechanoreceptors require a distinct protein tether to transduce mechanical stimuli.

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PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

Publication Date: 2010 Mar 3 PMID: 20075866
Authors: Chan, C. B. - Liu, X. - Ensslin, M. A. - Dillehay, D. L. - Ormandy, C. J. - Sohn, P. - Serra, R. - Ye, K.
Journal: EMBO J

PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.

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HDAC6 controls autophagosome maturation essential for ubiquitin-selective quality-control autophagy.

Publication Date: 2010 Mar 3 PMID: 20075865
Authors: Lee, J. Y. - Koga, H. - Kawaguchi, Y. - Tang, W. - Wong, E. - Gao, Y. S. - Pandey, U. B. - Kaushik, S. - Tresse, E. - Lu, J. - Taylor, J. P. - Cuervo, A. M. - Yao, T. P.
Journal: EMBO J

Autophagy is primarily considered a non-selective degradation process induced by starvation. Nutrient-independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin-binding deacetylase, histone deacetylase-6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin-dependent, actin-remodelling machinery, which in turn assembles an F-actin network that stimulates autophagosome-lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build-up, and neurodegeneration. Remarkably, HDAC6 and F-actin assembly are completely dispensable for starvation-induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome-lysosome fusion.

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Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element.

Publication Date: 2010 Feb 17 PMID: 20075864
Authors: Sinha, S. - Malonia, S. K. - Mittal, S. P. - Singh, K. - Kadreppa, S. - Kamat, R. - Mukhopadhyaya, R. - Pal, J. K. - Chattopadhyay, S.
Journal: EMBO J

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

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A viral E3 ligase targets RNF8 and RNF168 to control histone ubiquitination and DNA damage responses.

Publication Date: 2010 Mar 3 PMID: 20075863
Authors: Lilley, C. E. - Chaurushiya, M. S. - Boutell, C. - Landry, S. - Suh, J. - Panier, S. - Everett, R. D. - Stewart, G. S. - Durocher, D. - Weitzman, M. D.
Journal: EMBO J

The ICP0 protein of herpes simplex virus type 1 is an E3 ubiquitin ligase and transactivator required for the efficient switch between latent and lytic infection. As DNA damaging treatments are known to reactivate latent virus, we wished to explore whether ICP0 modulates the cellular response to DNA damage. We report that ICP0 prevents accumulation of repair factors at cellular damage sites, acting between recruitment of the mediator proteins Mdc1 and 53BP1. We identify RNF8 and RNF168, cellular histone ubiquitin ligases responsible for anchoring repair factors at sites of damage, as new targets for ICP0-mediated degradation. By targeting these ligases, ICP0 expression results in loss of ubiquitinated forms of H2A, mobilization of DNA repair proteins and enhanced viral fitness. Our study raises the possibility that the ICP0-mediated control of histone ubiquitination may link DNA repair, relief of transcriptional repression, and activation of latent viral genomes.

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Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway.

Publication Date: 2010 Mar 3 PMID: 20075862
Authors: Zuin, A. - Carmona, M. - Morales-Ivorra, I. - Gabrielli, N. - Vivancos, A. P. - Ayte, J. - Hidalgo, E.
Journal: EMBO J

Either calorie restriction, loss-of-function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient-dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 mitogen-activated protein (MAP) kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.

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The PTB domain of ShcA couples receptor activation to the cytoskeletal regulator IQGAP1.

Publication Date: 2010 Mar 3 PMID: 20075861
Authors: Smith, M. J. - Hardy, W. R. - Li, G. Y. - Goudreault, M. - Hersch, S. - Metalnikov, P. - Starostine, A. - Pawson, T. - Ikura, M.
Journal: EMBO J

Adaptor proteins respond to stimuli and recruit downstream complexes using interactions conferred by associated protein domains and linear motifs. The ShcA adaptor contains two phosphotyrosine recognition modules responsible for binding activated receptors, resulting in the subsequent recruitment of Grb2 and activation of Ras/MAPK. However, there is evidence that Grb2-independent signalling from ShcA has an important role in development. Using mass spectrometry, we identified the multidomain scaffold IQGAP1 as a ShcA-interacting protein. IQGAP1 and ShcA co-precipitate and are co-recruited to membrane ruffles induced by activated receptors of the ErbB family, and a reduction in ShcA protein levels inhibits the formation of lamellipodia. We used NMR to characterize a direct, non-canonical ShcA PTB domain interaction with a helical fragment from the IQGAP1 N-terminal region that is pTyr-independent. This interaction is mutually exclusive with binding to a more conventional PTB domain peptide ligand from PTP-PEST. ShcA-mediated recruitment of IQGAP1 may have an important role in cytoskeletal reorganization downstream of activated receptors at the cell surface.

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Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain.

Publication Date: 2010 Feb 17 PMID: 20075859
Authors: Perederina, A. - Esakova, O. - Quan, C. - Khanova, E. - Krasilnikov, A. S.
Journal: EMBO J

Ribonuclease (RNase) P is a site-specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix-loop-helix protein-binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 A. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.

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Function of endoplasmic reticulum calcium ATPase in innate immunity-mediated programmed cell death.

Publication Date: 2010 Mar 3 PMID: 20075858
Authors: Zhu, X. - Caplan, J. - Mamillapalli, P. - Czymmek, K. - Dinesh-Kumar, S. P.
Journal: EMBO J

Programmed cell death (PCD) initiated at the pathogen-infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER-localized type IIB Ca(2+)-ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N- and fungal-immune receptor Cf9-mediated PCD, as well as non-host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein-induced cell death. The accelerated PCD rescues loss-of-resistance phenotype of Rar1, HSP90-silenced plants, but not SGT1-silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N-immune receptor-mediated PCD. Our results indicate that ER-Ca(2+)-ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.

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SLR-2 and JMJC-1 regulate an evolutionarily conserved stress-response network.

Publication Date: 2010 Feb 17 PMID: 20057358
Authors: Kirienko, N. V. - Fay, D. S.
Journal: EMBO J

Maintaining a homeostatic interaction with the environment is crucial for the growth, survival, and propagation of all living organisms. Reestablishment of equilibrium after stress is achieved by the activation of complex transcriptional-response networks, many of which remain poorly understood. Here, we report that the zinc-finger protein, SLR-2, is a master stress regulator and is required for the normal response to pleiotropic stress conditions in Caenorhabditis elegans. Using bioinformatical tools, we identified an evolutionarily conserved nucleotide motif present in slr-2 stress-responsive genes and show that this motif is sufficient for stress induction under a variety of conditions. We also demonstrate that JMJC-1, a conserved Jumonji C domain protein, acts downstream of SLR-2 to mediate stress response in C. elegans. Moreover, the role of JMJC-1 in stress response is conserved in Drosophila and mammals. Finally, we provide evidence that the SLR-2-JMJC-1 pathway functions independently of the well-studied DAF-16/FOXO1 network. These findings point to a previously unrecognized phylogenetically conserved master stress-response pathway in metazoa.

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GPI anchoring facilitates propagation and spread of misfolded Sup35 aggregates in mammalian cells.

Publication Date: 2010 Feb 17 PMID: 20057357
Authors: Speare, J. O. - Offerdahl, D. K. - Hasenkrug, A. - Carmody, A. B. - Baron, G. S.
Journal: EMBO J

Prion diseases differ from other amyloid-associated protein misfolding diseases (e.g. Alzheimer's) because they are naturally transmitted between individuals and involve spread of protein aggregation between tissues. Factors underlying these features of prion diseases are poorly understood. Of all protein misfolding disorders, only prion diseases involve the misfolding of a glycosylphosphatidylinositol (GPI)-anchored protein. To test whether GPI anchoring can modulate the propagation and spread of protein aggregates, a GPI-anchored version of the amyloidogenic yeast protein Sup35NM (Sup35GPI) was expressed in neuronal cells. Treatment of cells with Sup35NM fibrils induced the GPI anchor-dependent formation of self-propagating, detergent-insoluble, protease-resistant, prion-like aggregates of Sup35GPI. Live-cell imaging showed intercellular spread of Sup35GPI aggregation to involve contact between aggregate-positive and aggregate-negative cells and transfer of Sup35GPI from aggregate-positive cells. These data demonstrate GPI anchoring facilitates the propagation and spread of protein aggregation and thus may enhance the transmissibility and pathogenesis of prion diseases relative to other protein misfolding diseases.

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ATR activation and replication fork restart are defective in FANCM-deficient cells.

Publication Date: 2010 Feb 17 PMID: 20057355
Authors: Schwab, R. A. - Blackford, A. N. - Niedzwiedz, W.
Journal: EMBO J

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA-ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR-mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.

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Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.

Publication Date: 2010 Feb 17 PMID: 20057354
Authors: Joshi, R. S. - Pina, B. - Roca, J.
Journal: EMBO J

How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin.

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HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner.

Publication Date: 2010 Feb 17 PMID: 20057353
Authors: Gao, G. - Walser, J. C. - Beaucher, M. L. - Morciano, P. - Wesolowska, N. - Chen, J. - Rong, Y. S.
Journal: EMBO J

Telomeres prevent chromosome ends from being repaired as double-strand breaks (DSBs). Telomere identity in Drosophila is determined epigenetically with no sequence either necessary or sufficient. To better understand this sequence-independent capping mechanism, we isolated proteins that interact with the HP1/ORC-associated protein (HOAP) capping protein, and identified HipHop as a subunit of the complex. Loss of one protein destabilizes the other and renders telomeres susceptible to fusion. Both HipHop and HOAP are enriched at telomeres, where they also interact with the conserved HP1 protein. We developed a model telomere lacking repetitive sequences to study the distribution of HipHop, HOAP and HP1 using chromatin immunoprecipitation (ChIP). We discovered that they occupy a broad region >10 kb from the chromosome end and their binding is independent of the underlying DNA sequence. HipHop and HOAP are both rapidly evolving proteins yet their telomeric deposition is under the control of the conserved ATM and Mre11-Rad50-Nbs (MRN) proteins that modulate DNA structures at telomeres and at DSBs. Our characterization of HipHop and HOAP reveals functional analogies between the Drosophila proteins and subunits of the yeast and mammalian capping complexes, implicating conservation in epigenetic capping mechanisms.

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Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex.

Publication Date: 2010 Feb 17 PMID: 20033062
Authors: Eichner, J. - Chen, H. T. - Warfield, L. - Hahn, S.
Journal: EMBO J

The RNA polymerase (pol) II general transcription factor TFIIF functions at several steps in transcription initiation including preinitiation complex (PIC) formation and start site selection. We find that two structured TFIIF domains bind Pol II at separate locations far from the active site with the TFIIF dimerization domain on the Pol II lobe and the winged helix domain of the TFIIF small subunit Tfg2 above the Pol II protrusion where it may interact with upstream promoter DNA. Binding of the winged helix to the protrusion is PIC specific. Anchoring of these two structured TFIIF domains at separate sites locates an essential and unstructured region of Tfg2 near the Pol II active site cleft where it may interact with flexible regions of Pol II and the general factor TFIIB to promote initiation and start site selection. Consistent with this mechanism, mutations far from the enzyme active site, which alter the binding of either structured TFIIF domains to Pol II, have similar defects in transcription start site usage.

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A fast dynamic mode of the EF-G-bound ribosome.

Publication Date: 2010 Feb 17 PMID: 20033061
Authors: Munro, J. B. - Altman, R. B. - Tung, C. S. - Sanbonmatsu, K. Y. - Blanchard, S. C.
Journal: EMBO J

A key intermediate in translocation is an 'unlocked state' of the pre-translocation ribosome in which the P-site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two- and three-colour smFRET imaging from multiple structural perspectives, EF-G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF-G-bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.

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FANCM regulates DNA chain elongation and is stabilized by S-phase checkpoint signalling.

Publication Date: 2010 Feb 17 PMID: 20010692
Authors: Luke-Glaser, S. - Luke, B. - Grossi, S. - Constantinou, A.
Journal: EMBO J

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase-dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S-phase checkpoint signalling.

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