RESUMO
Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase.
RESUMO
Prolonged mitosis often results in apoptosis1. Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery2. Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death3,4. Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.
Assuntos
Apoptose , Mitose , Separase/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Segregação de Cromossomos , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Fosforilação , Especificidade por Substrato , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína bcl-X/metabolismoRESUMO
Separation of eukaryotic sister chromatids during the cell cycle is timed by the spindle assembly checkpoint (SAC) and ultimately triggered when separase cleaves cohesion-mediating cohesin1-3. Silencing of the SAC during metaphase activates the ubiquitin ligase APC/C (anaphase-promoting complex, also known as the cyclosome) and results in the proteasomal destruction of the separase inhibitor securin1. In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions4,5. Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma6,7, is turned into a separase inhibitor upon association with SAC-activated MAD2. SGO2-MAD2 can functionally replace securin and sequesters most separase in securin-knockout cells. Acute loss of securin and SGO2, but not of either protein individually, resulted in separase deregulation associated with premature cohesin cleavage and cytotoxicity. Similar to securin8,9, SGO2 is a competitive inhibitor that uses a pseudo-substrate sequence to block the active site of separase. APC/C-dependent ubiquitylation and action of the AAA-ATPase TRIP13 in conjunction with the MAD2-specific adaptor p31comet liberate separase from SGO2-MAD2 in vitro. The latter mechanism facilitates a considerable degree of sister chromatid separation in securin-knockout cells that lack APC/C activity. Thus, our results identify an unexpected function of SGO2 in mitotically dividing cells and a mechanism of separase regulation that is independent of securin but still supervised by the SAC.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Mad2/metabolismo , Securina , Separase/antagonistas & inibidores , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdc20/metabolismo , Linhagem Celular , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Ligação Proteica , Securina/metabolismo , Separase/metabolismo , CoesinasRESUMO
Ring-shaped cohesin keeps sister chromatids paired until cleavage of its Scc1/Rad21 subunit by separase triggers chromosome segregation in anaphase. Vertebrate separase is held inactive by mutually exclusive binding to securin or Cdk1-cyclin B1 and becomes unleashed only upon ubiquitin-dependent degradation of these regulators. Although most separase is usually found in association with securin, this anaphase inhibitor is dispensable for murine life while Cdk1-cyclin B1-dependent control of separase is essential. Here, we show that securin-independent inhibition of separase by Cdk1-cyclin B1 in early mitosis requires the phosphorylation-specific peptidyl-prolyl cis/trans isomerase Pin1. Furthermore, isomerization of previously securin-bound separase at the metaphase-to-anaphase transition renders it resistant to re-inhibition by residual securin. At the same time, isomerization also limits the half-life of separase's proteolytic activity, explaining how cohesin can be reloaded onto telophase chromatin in the absence of securin and cyclin B1 without being cleaved.
Assuntos
Segregação de Cromossomos/genética , Regulação Enzimológica da Expressão Gênica , Peptidilprolil Isomerase/genética , Separase/genética , Anáfase/genética , Proteína Quinase CDC2 , Cromátides/genética , Ciclina B1/química , Ciclina B1/genética , Ciclina B1/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Metáfase/genética , Microscopia de Fluorescência , Mitose/genética , Modelos Genéticos , Modelos Moleculares , Mutação , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/metabolismo , Ligação Proteica , Conformação Proteica , Interferência de RNA , Securina/genética , Securina/metabolismo , Separase/química , Separase/metabolismoRESUMO
Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Quebras de DNA de Cadeia Dupla , Interfase , Proteínas de Neoplasias/metabolismo , Reparo de DNA por Recombinação , Separase/metabolismo , Neoplasias Cutâneas/enzimologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Ativação Enzimática , Células HEK293 , Humanos , Ligases/genética , Ligases/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Separase/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/prevenção & controleRESUMO
The DNA-embracing, ring-shaped multiprotein complex cohesin mediates sister chromatid cohesion and is stepwise displaced in mitosis by Wapl and separase (also known as ESPL1) to facilitate anaphase. Proper regulation of chromosome cohesion throughout meiosis is critical for preventing formation of aneuploid gametes, which are associated with trisomies and infertility in humans. Studying cohesion in meiocytes is complicated by their difficult experimental amenability and the absence of cohesin turnover. Here, we use cultured somatic cells to unravel fundamental aspects of meiotic cohesin. When expressed in Hek293 cells, the kleisin Rec8 displays no affinity for the peripheral cohesin subunits Stag1 or Stag2 and remains cytoplasmic. However, co-expression of Stag3 is sufficient for Rec8 to enter the nucleus, load onto chromatin, and functionally replace its mitotic counterpart Scc1 (also known as RAD21) during sister chromatid cohesion and dissolution. Rec8-Stag3 cohesin physically interacts with Pds5, Wapl and sororin (also known as CDCA5). Importantly, Rec8-Stag3 cohesin is shown to be susceptible to Wapl-dependent ring opening and sororin-mediated protection. These findings exemplify that our model system is suitable to rapidly generate testable predictions for important unresolved issues of meiotic cohesion regulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Meiose , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Cromátides/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos , Proteínas de Ligação a DNA , Células HEK293 , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , CoesinasRESUMO
Faithful transmission of genomic information requires tight spatiotemporal regulation of DNA replication factors. In the licensing step of DNA replication, CDT-1 is loaded onto chromatin to subsequently promote the recruitment of additional replication factors, including CDC-45 and GINS. During the elongation step, the CDC-45/GINS complex moves with the replication fork; however, it is largely unknown how its chromatin association is regulated. Here, we show that the chaperone-like ATPase CDC-48/p97 coordinates degradation of CDT-1 with release of the CDC-45/GINS complex. C. elegans embryos lacking CDC-48 or its cofactors UFD-1/NPL-4 accumulate CDT-1 on mitotic chromatin, indicating a critical role of CDC-48 in CDT-1 turnover. Strikingly, CDC-48(UFD-1/NPL-4)-deficient embryos show persistent chromatin association of CDC-45/GINS, which is a consequence of CDT-1 stabilization. Moreover, our data confirmed a similar regulation in Xenopus egg extracts, emphasizing a conserved coordination of licensing and elongation events during eukaryotic DNA replication by CDC-48/p97.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Caenorhabditis elegans , Masculino , Mitose , Interferência de RNA , Espermatozoides/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/química , Ubiquitina/metabolismo , Proteína com Valosina , Xenopus laevisRESUMO
The universal triggering event of eukaryotic chromosome segregation is cleavage of centromeric cohesin by separase. Prior to anaphase, most separase is kept inactive by association with securin. Protein phosphatase 2A (PP2A) constitutes another binding partner of human separase, but the functional relevance of this interaction has remained enigmatic. We demonstrate that PP2A stabilizes separase-associated securin by dephosphorylation, while phosphorylation of free securin enhances its polyubiquitylation by the ubiquitin ligase APC/C and proteasomal degradation. Changing PP2A substrate phosphorylation sites to alanines slows degradation of free securin, delays separase activation, lengthens early anaphase, and results in anaphase bridges and DNA damage. In contrast, separase-associated securin is destabilized by introduction of phosphorylation-mimetic aspartates or extinction of separase-associated PP2A activity. G2- or prometaphase-arrested cells suffer from unscheduled activation of separase when endogenous securin is replaced by aspartate-mutant securin. Thus, PP2A-dependent stabilization of separase-associated securin prevents precocious activation of separase during checkpoint-mediated arrests with basal APC/C activity and increases the abruptness and fidelity of sister chromatid separation in anaphase.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteína Fosfatase 2/metabolismo , Securina/metabolismo , Separase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Células HeLa , Humanos , Imunoprecipitação , Proteína Fosfatase 2/genética , Securina/genética , Separase/genética , UbiquitinaçãoRESUMO
Organelles in eukaryotic cells often have complex shapes that deviate significantly from simple spheres. A prime example is the endoplasmic reticulum (ER) that forms an extensive network of membrane tubules in many mammalian cell types and in reconstitution assays in vitro. Despite the successful hunt for molecular determinants of ER shape we are still far from having a comprehensive understanding of ER network morphogenesis. Here, we have studied the hitherto neglected influence of the host substrate when reconstituting ER networks in vitro as compared to ER networks in vivo. In culture cells we observed cytoplasm-spanning ER networks with tubules being connected almost exclusively by three-way junctions and segment lengths being narrowly distributed around a mean length of about 1µm. In contrast, networks reconstituted from purified ER microsomes on flat glass or gel substrates of varying stiffness showed significantly broader length distributions with an up to fourfold larger mean length. Self-assembly of ER microsomes on small oil droplets, however, yielded networks that resembled more closely the native ER network of mammalian cells. We conclude from these observations that the ER microsomes' inherent self-assembly capacity is sufficient to support network formation with a native geometry if the influence of the host substrate's surface chemistry becomes negligible. We hypothesize that under these conditions the networks' preference for three-way junctions follows from creating 'starfish-shaped' vesicles when ER microsomes with a protein-induced spontaneous curvature undergo fusion.
Assuntos
Citoplasma/química , Retículo Endoplasmático/química , Células HeLa , HumanosRESUMO
Faithful transmission of chromosomes during eukaryotic cell division requires sister chromatids to be paired from their generation in S phase until their separation in M phase. Cohesion is mediated by the cohesin complex, whose Smc1, Smc3 and Scc1 subunits form a tripartite ring that entraps both DNA double strands. Whereas centromeric cohesin is removed in late metaphase by Scc1 cleavage, metazoan cohesin at chromosome arms is displaced already in prophase by proteolysis-independent signalling. Which of the three gates is triggered by the prophase pathway to open has remained enigmatic. Here, we show that displacement of human cohesin from early mitotic chromosomes requires dissociation of Smc3 from Scc1 but no opening of the other two gates. In contrast, loading of human cohesin onto chromatin in telophase occurs through the Smc1-Smc3 hinge. We propose that the use of differently regulated gates for loading and release facilitates unidirectionality of DNA's entry into and exit from the cohesin ring.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Prófase/fisiologia , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos/metabolismo , DNA/genética , Proteínas de Ligação a DNA , Humanos , Imunoprecipitação , Imunossupressores/farmacologia , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Microscopia de Fluorescência , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , CoesinasRESUMO
Sister chromatid cohesion is established during replication by entrapment of both dsDNAs within the cohesin ring complex. It is dissolved in anaphase when separase, a giant cysteine endopeptidase, cleaves the Scc1/Rad21 subunit of cohesin, thereby triggering chromosome segregation. Separase is held inactive by association with securin until this anaphase inhibitor is destroyed at the metaphase-to-anaphase transition by ubiquitin-dependent degradation. The relevant ubiquitin ligase, the anaphase-promoting complex/cyclosome, also targets cyclin B1, thereby causing inactivation of Cdk1 and mitotic exit. Although separase is essential, securin knock-out mice are surprisingly viable and fertile. Capitalizing on our previous finding that Cdk1-cyclin B1 can also bind and inhibit separase, we investigated whether this kinase might be suitable to maintain faithful timing and execution of anaphase in the absence of securin. We found that, similar to securin, Cdk1-cyclin B1 regulates separase in both a positive and negative manner. Although securin associates with nascent separase to co-translationally assist proper folding, Cdk1-cyclin B1 acts on native state separase. Upon entry into mitosis, Cdk1-cyclin B1-dependent phosphorylation of Ser-1126 renders separase prone to inactivation by aggregation/precipitation. Stable association of Cdk1-cyclin B1 with phosphorylated separase counteracts this tendency and stabilizes separase in an inhibited yet activatable state. These opposing effects are suited to prevent premature cleavage of cohesin in early mitosis while ensuring timely activation of separase by anaphase-promoting complex/cyclosome-dependent degradation of cyclin B1. Coupling sister chromatid separation with subsequent exit from mitosis by this simplified mode might have been the common scheme of mitotic control prior to the evolution of securin.
Assuntos
Ciclina B1/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Securina/fisiologia , Separase/fisiologia , Sequência de Bases , Proteína Quinase CDC2 , Primers do DNA , Citometria de Fluxo , Células HEK293 , Humanos , Interferência de RNARESUMO
Protein kinases are pivotal regulators of cell signaling that modulate each other's functions and activities through site-specific phosphorylation events. These key regulatory modifications have not been studied comprehensively, because low cellular abundance of kinases has resulted in their underrepresentation in previous phosphoproteome studies. Here, we combine kinase-selective affinity purification with quantitative mass spectrometry to analyze the cell-cycle regulation of protein kinases. This proteomics approach enabled us to quantify 219 protein kinases from S and M phase-arrested human cancer cells. We identified more than 1000 phosphorylation sites on protein kinases. Intriguingly, half of all kinase phosphopeptides were upregulated in mitosis. Our data reveal numerous unknown M phase-induced phosphorylation sites on kinases with established mitotic functions. We also find potential phosphorylation networks involving many protein kinases not previously implicated in mitotic progression. These results provide a vastly extended knowledge base for functional studies on kinases and their regulation through site-specific phosphorylation.
Assuntos
Ciclo Celular , Fosfoproteínas/análise , Fosfotransferases/metabolismo , Proteômica , Sequência de Aminoácidos , Ativação Enzimática , Células HeLa , Humanos , Mitose , Dados de Sequência Molecular , Fosfopeptídeos/análise , Fosforilação , Fosfotransferases/química , Fase S , Especificidade por SubstratoRESUMO
Mammalian centromeric cohesin is protected from phosphorylation-dependent displacement in mitotic prophase by shugoshin-1 (Sgo1), while shugoshin-2 (Sgo2) protects cohesin from separase-dependent cleavage in meiosis I. In higher eukaryotes, progression and faithful execution of both mitosis and meiosis are controlled by the spindle assembly checkpoint, which delays anaphase onset until chromosomes have achieved proper attachment to microtubules. According to the so-called template model, Mad1-Mad2 complexes at unattached kinetochores instruct conformational change of soluble Mad2, thus catalysing Mad2 binding to its target Cdc20. Here, we show that human Sgo2, but not Sgo1, specifically interacts with Mad2 in a manner that strongly resembles the interactions of Mad2 with Mad1 or Cdc20. Sgo2 contains a Mad1/Cdc20-like Mad2-interaction motif and competes with Mad1 and Cdc20 for binding to Mad2. NMR and biochemical analyses show that shugoshin binding induces similar conformational changes in Mad2 as do Mad1 or Cdc20. Mad2 binding regulates fine-tuning of Sgo2's sub-centromeric localization. Mad2 binding is conserved in the only known Xenopus laevis shugoshin homologue and, compatible with a putative meiotic function, the interaction occurs in oocytes.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Células Cultivadas , Centrômero/fisiologia , Feminino , Humanos , Cinetocoros/fisiologia , Proteínas Mad2 , Dados de Sequência Molecular , Proteínas Nucleares/genética , Oócitos/citologia , Oócitos/metabolismo , Conformação Proteica , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Fuso Acromático , Xenopus laevis/metabolismoRESUMO
Separase not only triggers anaphase of meiosis I by proteolytic cleavage of cohesin on chromosome arms, but in vitro vertebrate separase also acts as a direct inhibitor of cyclin-dependent kinase 1 (Cdk1) on liberation from the inhibitory protein, securin. Blocking separase-Cdk1 complex formation by microinjection of anti-separase antibodies prevents polar-body extrusion in vertebrate oocytes. Importantly, proper meiotic maturation is rescued by chemical inhibition of Cdk1 or expression of Cdk1-binding separase fragments lacking cohesin-cleaving activity.
Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Endopeptidases/metabolismo , Meiose/fisiologia , Oócitos/fisiologia , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Endopeptidases/genética , Feminino , Histonas/metabolismo , Humanos , Camundongos , Proteínas de Neoplasias/metabolismo , Oócitos/metabolismo , Securina , Separase , XenopusRESUMO
Mature Xenopus oocytes are arrested in meiosis by the activity of XErp1/Emi2, an inhibitor of the ubiquitin-ligase anaphase-promoting complex/cyclosome (APC/C). On fertilization, XErp1 is degraded, resulting in APC/C activation and the consequent degradation of cell-cycle regulators and exit from meiosis. In this study, we show that a modest increase in the activity of the ubiquitin-conjugating enzyme UbcX overrides the meiotic arrest in an APC/C-dependent reaction. Intriguingly, XErp1 remains stable in these conditions. We found that UbcX causes the ubiquitylation of XErp1, followed by its dissociation from the APC/C. Our data support the idea that ubiquitylation regulates the APC/C-inhibitory activity of XErp1.
Assuntos
Proteínas F-Box/metabolismo , Meiose/fisiologia , Oócitos/metabolismo , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Western Blotting , Plasmídeos/genética , Ubiquitinação , Xenopus/fisiologiaRESUMO
MAD2L1BP-encoded p31comet mediates Trip13-dependent disassembly of Mad2- and Rev7-containing complexes and, through this antagonism, promotes timely spindle assembly checkpoint (SAC) silencing, faithful chromosome segregation, insulin signaling, and homology-directed repair (HDR) of DNA double-strand breaks. We identified a homozygous MAD2L1BP nonsense variant, R253*, in 2 siblings with microcephaly, epileptic encephalopathy, and juvenile granulosa cell tumors of ovary and testis. Patient-derived cells exhibited high-grade mosaic variegated aneuploidy, slowed-down proliferation, and instability of truncated p31comet mRNA and protein. Corresponding recombinant p31comet was defective in Trip13, Mad2, and Rev7 binding and unable to support SAC silencing or HDR. Furthermore, C-terminal truncation abrogated an identified interaction of p31comet with tp53. Another homozygous truncation, R227*, detected in an early-deceased patient with low-level aneuploidy, severe epileptic encephalopathy, and frequent blood glucose elevations, likely corresponds to complete loss of function, as in Mad2l1bp-/- mice. Thus, human mutations of p31comet are linked to aneuploidy and tumor predisposition.
Assuntos
Encefalopatias , Tumor de Células da Granulosa , Neoplasias Ovarianas , Feminino , Humanos , Animais , Camundongos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Tumor de Células da Granulosa/genética , Mutação , AneuploidiaRESUMO
Sister chromatid cohesion is mediated by DNA catenation and proteinaceous cohesin complexes. The recent visualization of PICH (Plk1-interacting checkpoint helicase)-coated DNA threads in anaphase cells raises new questions as to the role of DNA catenation and its regulation in time and space. In the present study we show that persistent DNA catenation induced by inhibition of Topoisomerase-IIalpha can contribute to sister chromatid cohesion in the absence of cohesin complexes and that resolution of catenation is essential for abscission. Furthermore, we use an in vitro chromatid separation assay to investigate the temporal and functional relationship between cohesin removal and Topoisomerase-IIalpha-mediated decatenation. Our data suggest that centromere decatenation can occur only after separase activation and cohesin removal, providing a plausible explanation for the persistence of centromere threads after anaphase onset.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/fisiologia , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Catenado/metabolismo , DNA/metabolismo , Anáfase/genética , Anáfase/fisiologia , Divisão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Centrômero/genética , Cromátides/genética , Cromátides/metabolismo , DNA/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Catenado/genética , Humanos , CoesinasRESUMO
After the degradation of its inhibitor securin, separase initiates chromosome segregation during the metaphase-to-anaphase transition by cleaving cohesin. Here we present a density map at a resolution of 25 A of negatively stained separase-securin complex. Based on labeling data and sequence analysis, we propose a model for the structure of separase, consisting of 26 ARM repeats, an unstructured region of 280 residues and two caspase-like domains, with securin binding to the ARM repeats.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Endopeptidases/ultraestrutura , Humanos , Microscopia Eletrônica , Modelos Moleculares , Proteínas de Neoplasias/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Securina , SeparaseRESUMO
Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1.
Assuntos
Proteínas de Transporte/metabolismo , Cromátides/metabolismo , Meiose/fisiologia , Óvulo/citologia , Animais , Western Blotting , Proteínas de Ciclo Celular/metabolismo , Análise Citogenética , Endopeptidases/metabolismo , Camundongos , Oligonucleotídeos , Securina , SeparaseRESUMO
Separase, a cysteine protease of the CD clan, triggers chromosome segregation during mitosis by cleaving the cohesin ring entrapping the two sister chromatids. Deregulated separase activity is associated with aneuploidy, a hallmark of most human cancers. In fact, separase is highly overexpressed in many solid cancers, making it an attractive chemotherapeutic target. To identify small molecules capable of inhibiting separase in its complex cellular environment, we established a highly sensitive assay to quantify separase activity in cells and screened a 51â¯009-member library for separase inhibitors. In vitro assays confirmed that the identified compounds efficiently inhibited separase, while not affecting caspase-1, another CD-clan protease structurally related to separase. Importantly, HeLa cells with compromised separase activity displayed severe chromosome segregation defects upon compound treatment, confirming that the identified inhibitors are bioactive in tumor tissue culture cells. Structure-activity relationship studies succeeded in the optimization of the most promising inhibitor. Overall, this study demonstrates the feasibility of identifying separase-specific inhibitors, which serve as promising lead compounds for the development of clinically relevant separase inhibiting drugs.