RESUMO
Cell survival to replication stress depends on the activation of the Mec1ATR-Rad53 checkpoint response that protects the integrity of stalled forks and controls the origin firing program. Here we found that Mad2, a member of the spindle assembly checkpoint (SAC), contributes to efficient origin firing and to cell survival in response to replication stress. We show that Rad53 and Mad2 promote S-phase cyclin expression through different mechanisms: while Rad53 influences Clb5,6 degradation, Mad2 promotes their protein synthesis. We found that Mad2 co-sediments with polysomes and modulates the association of the translation inhibitor Caf204E-BP with the translation machinery and the initiation factor eIF4E. This Mad2-dependent translational regulatory process does not depend on other SAC proteins. Altogether our observations indicate that Mad2 has an additional function outside of mitosis to control DNA synthesis and collaborates with the Mec1-Rad53 regulatory axis to allow cell survival in response to replication stress.
Assuntos
Ciclinas/genética , Replicação do DNA , Proteínas Mad2/metabolismo , Mitose , Biossíntese de Proteínas , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclinas/metabolismo , Proteínas Mad2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in â¼5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.
Assuntos
ATPases Transportadoras de Arsenito/química , ATPases Transportadoras de Arsenito/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Animais , ATPases Transportadoras de Arsenito/genética , Linhagem Celular , Células Cultivadas , Síndrome de Down/genética , Síndrome de Down/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Microssomos Hepáticos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Proteicas , Transporte Proteico , Proteolipídeos/metabolismo , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Unrepaired DNA damage during embryonic development can be potentially inherited by a large population of cells. However, the quality control mechanisms that minimize the contribution of damaged cells to developing embryos remain poorly understood. Here, we uncovered an ATR- and CHK1-mediated transcriptional response to replication stress (RS) in mouse embryonic stem cells (ESCs) that induces genes expressed in totipotent two-cell (2C) stage embryos and 2C-like cells. This response is mediated by Dux, a multicopy retrogene defining the cleavage-specific transcriptional program in placental mammals. In response to RS, DUX triggers the transcription of 2C-like markers such as murine endogenous retrovirus-like elements (MERVL) and Zscan4. This response can also be elicited by ETAA1-mediated ATR activation in the absence of RS. ATR-mediated activation of DUX requires GRSF1-dependent post-transcriptional regulation of Dux mRNA. Strikingly, activation of ATR expands ESCs fate potential by extending their contribution to both embryonic and extra-embryonic tissues. These findings define a novel ATR dependent pathway involved in maintaining genome stability in developing embryos by controlling ESCs fate in response to RS.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Diferenciação Celular , Proliferação de Células/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem/genética , Quimera , Cromatografia Líquida , Clonagem Molecular , Dano ao DNA , Células-Tronco Embrionárias , Regulação da Expressão Gênica , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
Proteomics is nowadays a standard tool in life sciences for the analysis of protein abundance, modifications and interactions but has so far failed to enter the clinic for routine applications. New generation mass spectrometers and chromatographic systems are able to cover approximately an entire cell proteome in one run but sample preparation, in terms of time and sample recovery, is still a critical step. Here we describe a modification of the in-gel digestion method, called STAGE-diging, that reduces sample handling, decreases the analysis time and improves protein identification and quantification. This method is particularly useful for those research labs that manage different biological samples and have a limited access to MS instrumentation or are required to perform high-throughput analysis in short time like a clinical laboratory.
Assuntos
Espectrometria de Massas/métodos , Proteômica/métodosRESUMO
UNLABELLED: Proteomics based on high-resolution mass spectrometry has become a powerful tool for the analysis of protein abundance, modifications and interactions. New generation mass spectrometers and UPLC are able to cover approximately an entire cell proteome in one run, but sample preparation, in terms of time and sample recovery is still a critical step. Here we present a modification of the in-gel digestion method, called STAGE-diging. This approach was compared with the well-established procedures for sample preparation, both on high and low complexity samples, on quantitative SILAC-based experiments and on two different mass spectrometers. The results show that STAGE-diging reduces sample handling, decreases the analysis time and improves protein identification and quantification. Moreover, shorter instrument time allows performing multiple replicates that produce wider proteome coverage and more accurate quantitation. SIGNIFICANCE: In our work we detailed the set-up of a novel in-gel digestion processing for proteomics samples, called STAGE-diging. This new method can be applied to samples of different complexity both for qualitative and quantitative proteomics studies. We proved that STAGE-diging streamlines sample preparation as it is easy to use, reduces sample handling and improves protein identification and quantification all with a decreased analysis time. All these benefits make this new method appealing for laboratories handling a large number of samples, where time and reproducibility play a substantial role.
Assuntos
Géis/química , Proteoma/metabolismo , Proteômica/métodos , Tripsina/metabolismo , Cromatografia Líquida , Humanos , Células MCF-7 , Métodos , Proteômica/normas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Fluxo de TrabalhoRESUMO
Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.
Assuntos
Proteínas de Transporte/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Biotinilação , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/isolamento & purificação , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Linhagem Celular , Cromatografia Líquida , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Imunoprecipitação/métodos , RNA Helicases , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Espectrometria de Massas em Tandem , Transativadores/genética , Transativadores/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificaçãoRESUMO
Half of the human genome is made up of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using bacterial artificial chromosomes in Xenopus laevis egg extract. Using this approach we characterized the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication-dependent enrichment of a network of DNA repair factors including the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR-dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to the inability of the single-stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of topoisomerase I-dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications for our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Replicação do DNA , Animais , Pontos de Checagem do Ciclo Celular/genética , DNA/metabolismo , Dano ao DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismoRESUMO
Histone deacetylases (HDACs) are modification enzymes that regulate a plethora of biological processes. HDAC1, a crucial epigenetic modifier, is deregulated in cancer and subjected to a variety of post-translational modifications. Here, we describe the generation of a new monoclonal antibody that specifically recognizes a novel highly dynamic prophase phosphorylation of serine 406-HDAC1, providing a powerful tool for detecting early mitotic cells.
Assuntos
Anticorpos Monoclonais Murinos/química , Histona Desacetilase 1 , Fosfoproteínas , Prófase , Animais , Histona Desacetilase 1/química , Histona Desacetilase 1/metabolismo , Humanos , Camundongos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , FosforilaçãoRESUMO
Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VI(short) and myosin VI(long), which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VI(long) and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VI(short) in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VI(short). Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VI(long)) or migratory (myosin VI(short)) functional roles.
Assuntos
Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Movimento Celular , Clatrina/metabolismo , Feminino , Técnicas de Inativação de Genes , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Neoplasias/genética , Neoplasias/metabolismo , Ressonância Magnética Nuclear Biomolecular , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Mapas de Interação de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT(Nedd4)~Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Conformação ProteicaRESUMO
Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote.
Assuntos
Giardia lamblia/fisiologia , Ubiquitinação , Bioquímica , Sobrevivência Celular , Parede Celular/metabolismo , Imunofluorescência , Complexo de Endopeptidases do Proteassoma/metabolismo , ProteômicaRESUMO
Ubiquitination of histones plays a critical role in the regulation of several processes within the nucleus, including maintenance of genome stability and transcriptional regulation. The only known ubiquitination site on histones is represented by a conserved Lys residue located at the C terminus of the protein. Here, we describe a novel ubiquitin mark at the N-terminal tail of histone H2As consisting of two Lys residues at positions 13 and 15 (K13/K15). This "bidentate" site is a target of the DNA damage response (DDR) ubiquitin ligases RNF8 and RNF168. Histone mutants lacking the K13/K15 site impair RNF168- and DNA damage-dependent ubiquitination. Conversely, inactivation of the canonical C-terminal site prevents the constitutive monoubiquitination of histone H2As but does not abolish the ubiquitination induced by RNF168. A ubiquitination-defective mutant is obtained by inactivating both the N- and the C-terminal sites, suggesting that these are unique, non-redundant acceptors of ubiquitination on histone H2As. This unprecedented result implies that RNF168 generates a qualitatively different Ub mark on chromatin.
Assuntos
Histonas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Sequência de Aminoácidos , Montagem e Desmontagem da Cromatina , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Histonas/química , Histonas/genética , Humanos , Dados de Sequência Molecular , Mutação , Estrutura Terciária de ProteínaRESUMO
A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.
Assuntos
Citocromos b5/metabolismo , Membranas Intracelulares/metabolismo , Lipídeos de Membrana/metabolismo , Animais , Cães , Humanos , Membranas Intracelulares/enzimologia , Proteínas de Membrana/metabolismo , Chaperonas Moleculares , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteolipídeos/metabolismo , Proteínas de Ligação a RNA , SuínosRESUMO
C-tail-anchored proteins are defined by an N-terminal cytosolic domain followed by a transmembrane anchor close to the C terminus. Their extreme C-terminal polar residues are translocated across membranes by poorly understood post-translational mechanism(s). Here we have used the yeast system to study translocation of the C terminus of a tagged form of mammalian cytochrome b(5), carrying an N-glycosylation site in its C-terminal domain (b(5)-Nglyc). Utilization of this site was adopted as a rigorous criterion for translocation across the ER membrane of yeast wild-type and mutant cells. The C terminus of b(5)-Nglyc was rapidly glycosylated in mutants where Sec61p was defective and incapable of translocating carboxypeptidase Y, a well known substrate for post-translational translocation. Likewise, inactivation of several other components of the translocon machinery had no effect on b(5)-Nglyc translocation. The kinetics of translocation were faster for b(5)-Nglyc than for a signal peptide-containing reporter. Depletion of the cellular ATP pool to a level that retarded Sec61p-dependent post-translational translocation still allowed translocation of b(5)-Nglyc. Similarly, only low ATP concentrations (below 1 microm), in addition to cytosolic protein(s), were required for in vitro translocation of b(5)-Nglyc into mammalian microsomes. Thus, translocation of tail-anchored b(5)-Nglyc proceeds by a mechanism different from that of signal peptide-driven post-translational translocation.