The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
A Mesmasson - One of the best experts on this subject based on the ideXlab platform.
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tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer
International Journal of Cancer, 2006Co-Authors: Veronique Ouellet, Marieclaude Guyot, Cecile Le Page, Abdelali Filalimouhim, Christian Lussier, Patricia N Tonin, Diane Provencher, A MesmassonAbstract:Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0-3) and stages (I-IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p<0.01). In addition, irrespective of the grade, Ccne1, Ran, CDC20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or CDC20 appear to be more tightly associated with a poor prognosis (p<0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.
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tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer
International Journal of Cancer, 2006Co-Authors: Veronique Ouellet, Marieclaude Guyot, Cecile Le Page, Abdelali Filalimouhim, Christian Lussier, Patricia N Tonin, Diane Provencher, A MesmassonAbstract:Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0–3) and stages (I–IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p < 0.01). In addition, irrespective of the grade, Ccne1, Ran, CDC20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or CDC20 appear to be more tightly associated with a poor prognosis (p < 0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management. © 2006 Wiley-Liss, Inc.
Janmichael Peters - One of the best experts on this subject based on the ideXlab platform.
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cryo em of mitotic checkpoint complex bound apc c reveals reciprocal and conformational regulation of ubiquitin ligation
Molecular Cell, 2016Co-Authors: Masaya Yamaguchi, Ryan Vanderlinden, Florian Weissmann, Nicholas G. Brown, Renping Qiao, Prakash Dube, David Haselbach, Wei Zhang, Sachdev S Sidhu, Janmichael PetersAbstract:The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpoint termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly and conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCC's CDC20 molecule. Access of UBE2C is limiting for subsequent polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.
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structure of the anaphase promoting complex cyclosome interacting with a mitotic checkpoint complex
Science, 2009Co-Authors: Franz Herzog, Ivana Primorac, Holger Stark, B. Sander, Peter Lenart, Karl Mechtler, Prakash Dube, Janmichael PetersAbstract:Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator CDC20 (APC/CCDC20). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/CCDC20. We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to CDC20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the CDC20 binding site on APC/C, locks the otherwise flexible APC/C in a “closed” state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.
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the wd40 propeller domain of cdh1 functions as a destruction box receptor for apc c substrates
Molecular Cell, 2005Co-Authors: Claudine Kraft, Hartmut C Vodermaier, Sebastian Maurerstroh, Frank Eisenhaber, Janmichael PetersAbstract:Activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 and Cdh1 leads to ubiquitin-dependent degradation of securin and cyclin B and thereby promotes the initiation of anaphase and exit from mitosis. Cyclin B and securin ubiquitination depend on a destruction box (D box) sequence in these proteins, but how APC/C bound to CDC20 or Cdh1 recognizes the D box is poorly understood. By using site-specific photocrosslinking in combination with mutational analyses, we show that the D box directly interacts with an evolutionarily conserved surface on the predicted WD40 propeller structure of Cdh1 and that this interaction is essential for processive substrate ubiquitination. We further show that Cdh1 specifically crosslinks to the APC/C subunit Cdc27 and that Cdh1 binding to APC/C depends on the presence of Cdc27. Our data imply that APC/C is activated by the association of Cdh1 with Cdc27, which enables APC/C to recognize the D box of substrates via Cdh1's propeller domain.
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mitotic regulation of the human anaphase promoting complex by phosphorylation
The EMBO Journal, 2003Co-Authors: Claudine Kraft, Franz Herzog, Karl Mechtler, Christian Gieffers, Anja Hagting, Jonathon Pines, Janmichael PetersAbstract:The anaphase‐promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and CDC20 binding. We have identified 43 phospho‐sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro , at least 15 of the mitotic phospho‐sites can be generated by cyclin‐dependent kinase 1 (Cdk1), and 3 by Polo‐like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased CDC20 binding and APC activation. Immunofluorescence microscopy using phospho‐antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho‐APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo . These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.
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activation of the human anaphase promoting complex by proteins of the CDC20 fizzy family
Current Biology, 1998Co-Authors: Edgar R Kramer, Christian Gieffers, Gabriele Holzl, Markus Hengstschlager, Janmichael PetersAbstract:Abstract The initiation of anaphase and exit from mitosis depend on the activation of the cyclosome/anaphase-promoting complex (APC) that ubiquitinates regulatory proteins such as anaphase inhibitors and mitotic cyclins [1–4]. Genetic experiments have demonstrated that two related WD40-repeat proteins – called CDC20p and Hct1p/Cdh1p in budding yeast and Fizzy and Fizzy-related in Drosophila – are essential for APC-dependent proteolysis [5–11]. Human orthologs of these proteins – hCDC20/p55 CDC [12] and hCDH1 – have recently been found to associate with APC in a cell-cycle-dependent manner [13,14]. Here, we show that the amount of hCDC20 and hCDH1 bound to APC correlates with a high ubiquitination activity of APC and that binding of recombinant hCDC20 and hCDH1 can activate APC in vitro . Our results suggest that the association between hCDH1 and APC is regulated by post-translational mechanisms, whereas the amount of hCDC20 bound to APC may in addition be controlled by hCDC20 synthesis and destruction [15]. The temporally distinct association of hCDC20 and hCDH1 with APC suggests that these proteins are, respectively, mitosis-specific and G1-specific activating subunits of APC.
Patricia N Tonin - One of the best experts on this subject based on the ideXlab platform.
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tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer
International Journal of Cancer, 2006Co-Authors: Veronique Ouellet, Marieclaude Guyot, Cecile Le Page, Abdelali Filalimouhim, Christian Lussier, Patricia N Tonin, Diane Provencher, A MesmassonAbstract:Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0-3) and stages (I-IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p<0.01). In addition, irrespective of the grade, Ccne1, Ran, CDC20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or CDC20 appear to be more tightly associated with a poor prognosis (p<0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management.
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tissue array analysis of expression microarray candidates identifies markers associated with tumor grade and outcome in serous epithelial ovarian cancer
International Journal of Cancer, 2006Co-Authors: Veronique Ouellet, Marieclaude Guyot, Cecile Le Page, Abdelali Filalimouhim, Christian Lussier, Patricia N Tonin, Diane Provencher, A MesmassonAbstract:Molecular profiling is a powerful approach to identify potential clinical markers for diagnosis and prognosis as well as providing a better understanding of the biology of epithelial ovarian cancer. On the basis of the analysis of HuFL expression data, we have previously identified genes that distinguish low malignant potential and invasive serous epithelial ovarian tumors. In this study, we used immunohistochemistry to monitor a subset of differently expressed candidates (Ahr, Paep, Madh3, Ran, Met, Mek1, Ccne1, Ccd20, Cks1 and Cas). A tissue array composed of 244 serous tumors of different grades (0–3) and stages (I–IV) was used in this analysis. All markers assayed presented differential protein expression between serous tumors of low and high grade. Significant differences in Ccne1 and Ran expression were observed in a comparison of low malignant potential and grade 1 tumor samples (p < 0.01). In addition, irrespective of the grade, Ccne1, Ran, CDC20 and Cks1 showed significant differences of expression in association with the clinical stage of disease. While high level of Ccne1 have previously been associated with poor outcomes, here we found that high level of either Ran or CDC20 appear to be more tightly associated with a poor prognosis (p < 0.001, 0.03, respectively). The application of these biomarkers in both the initial diagnosis and prognostic attributes of patients with epithelial ovarian tumors should prove to be useful in patient management. © 2006 Wiley-Liss, Inc.
Jakob Nilsson - One of the best experts on this subject based on the ideXlab platform.
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coupling of CDC20 inhibition and activation by bubr1
Journal of Cell Biology, 2021Co-Authors: Jamin B Hein, Dimitriya H Garvanska, Isha Nasa, Arminja N Kettenbach, Jakob NilssonAbstract:Tight regulation of the APC/C-CDC20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits CDC20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of CDC20 by cyclin B1-Cdk1 independently inhibits APC/C-CDC20 activation. This creates a conundrum for how CDC20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both CDC20 inhibition and activation activities, allowing for cross-talk between the two CDC20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient CDC20 dephosphorylation in the MCC. A mutant CDC20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting CDC20 dephosphorylation to the MCC is important. Collectively, our work reveals how CDC20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.
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coupling of CDC20 inhibition and activation by bubr1
bioRxiv, 2020Co-Authors: Jamin B Hein, Dimitriya H Garvanska, Isha Nasa, Arminja N Kettenbach, Jakob NilssonAbstract:Tight regulation of the APC/C-CDC20 ubiquitin ligase that targets Cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits CDC20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of CDC20 by Cyclin B1-Cdk1 independently inhibits APC/C-CDC20 activation. This creates a conundrum for how CDC20 gets activated prior to Cyclin B1 degradation. Here we show that the MCC component BubR1 harbours both CDC20 inhibition and activation activities, allowing for cross-talk between the two CDC20 inhibition pathways. Specifically BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient CDC20 dephosphorylation in the MCC. A mutant CDC20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest arguing that restricting CDC20 dephosphorylation to the MCC is important. Collectively our work reveals how CDC20 can be dephosphorylated in the presence of Cyclin B1-Cdk1 activity without causing premature anaphase onset.
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The closed form of Mad2 is bound to Mad1 and CDC20 at unattached kinetochores
2018Co-Authors: Gang Zhang, Jakob NilssonAbstract:The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation by delaying anaphase onset in response to unattached kinetochores. Anaphase is delayed by the generation of the mitotic checkpoint complex (MCC) composed of the checkpoint proteins Mad2 and BubR1/Bub3 bound to the protein CDC20. Current models assume that MCC production is catalyzed at unattached kinetochores and that the Mad1/Mad2 complex is instrumental in the conversion of Mad2 from an open form (O-Mad2) to a closed form (C-Mad2) that can bind to CDC20. Importantly the levels of Mad2 at kinetochores correlate with SAC activity but whether C-Mad2 at kinetochores exclusively represents its complex with Mad1 is not fully established. Here we use a recently established C-Mad2 specific monoclonal antibody to show that CDC20 and C-Mad2 levels correlate at kinetochores and that depletion of CDC20 reduces Mad2 but not Mad1 kinetochore levels. Importantly reintroducing wild type CDC20 but not CDC20 R132A, a mutant form that cannot bind Mad2, restores Mad2 levels. In agreement with this live cell imaging of fluorescent tagged Mad2 reveals that CDC20 depletion strongly reduces Mad2 localization to kinetochores. These results support the presence of Mad2-CDC20 complexes at kinetochores in agreement with current models of the SAC but also argue that Mad2 levels at kinetochores cannot be used as a direct readout of Mad1 levels.
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interphase apc c CDC20 inhibition by cyclin a2 cdk2 ensures efficient mitotic entry
Nature Communications, 2016Co-Authors: Jamin B Hein, Jakob NilssonAbstract:Proper cell-cycle progression requires tight temporal control of the Anaphase Promoting Complex/Cyclosome (APC/C), a large ubiquitin ligase that is activated by one of two co-activators, Cdh1 or CDC20. APC/C and CDC20 are already present during interphase but APC/C-CDC20 regulation during this window of the cell cycle, if any, is unknown. Here we show that cyclin A2-Cdk2 binds and phosphorylates CDC20 in interphase and this inhibits APC/C-CDC20 activity. Preventing CDC20 phosphorylation results in pre-mature activation of the APC/C-CDC20 and several substrates, including cyclin B1 and A2, are destabilized which lengthens G2 and slows mitotic entry. Expressing non-degradable cyclin A2 but not cyclin B1 restores mitotic entry in these cells. We have thus uncovered a novel positive feedback loop centred on cyclin A2-Cdk2 inhibition of interphase APC/C-CDC20 to allow further cyclin A2 accumulation and mitotic entry.
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the internal CDC20 binding site in bubr1 facilitates both spindle assembly checkpoint signalling and silencing
Nature Communications, 2014Co-Authors: Tiziana Lischetti, Gang Zhang, Garry G Sedgwick, Victor M Bolanosgarcia, Jakob NilssonAbstract:Improperly attached kinetochores activate the spindle assembly checkpoint (SAC) and by an unknown mechanism catalyse the binding of two checkpoint proteins, Mad2 and BubR1, to CDC20 forming the mitotic checkpoint complex (MCC). Here, to address the functional role of CDC20 kinetochore localization in the SAC, we delineate the molecular details of its interaction with kinetochores. We find that BubR1 recruits the bulk of CDC20 to kinetochores through its internal CDC20 binding domain (IC20BD). We show that preventing CDC20 kinetochore localization by removal of the IC20BD has a limited effect on the SAC because the IC20BD is also required for efficient SAC silencing. Indeed, the IC20BD can disrupt the MCC providing a mechanism for its role in SAC silencing. We thus uncover an unexpected dual function of the second CDC20 binding site in BubR1 in promoting both efficient SAC signalling and SAC silencing.
Xuelian Luo - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of human CDC20 supports multisite degron recognition by apc c
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Wei Tian, Diana R. Tomchick, Ross Warrington, Xuelian LuoAbstract:The anaphase-promoting complex/cyclosome (APC/C) promotes anaphase onset and mitotic exit through ubiquitinating securin and cyclin B1. The mitotic APC/C activator, the cell division cycle 20 (CDC20) protein, directly interacts with APC/C degrons––the destruction (D) and KEN boxes. APC/CCDC20 is the target of the spindle checkpoint. Checkpoint inhibition of APC/CCDC20 requires the binding of a BubR1 KEN box to CDC20. How APC/C recognizes substrates is not understood. We report the crystal structures of human CDC20 alone or bound to a BubR1 KEN box. CDC20 has a disordered N-terminal region and a C-terminal WD40 β propeller with a preformed KEN-box-binding site at its top face. We identify a second conserved surface at the side of the CDC20 β propeller as a D-box-binding site. The D box of securin, but not its KEN box, is critical for securin ubiquitination by APC/CCDC20. Although both motifs contribute to securin ubiquitination by APC/CCdh1, securin mutants lacking either motif are efficiently ubiquitinated. Furthermore, D-box peptides diminish the ubiquitination of KEN-box substrates by APC/CCdh1, suggesting possible competition between the two motifs. Our results indicate the lack of strong positive cooperativity between the two degrons of securin. We propose that low-cooperativity, multisite target recognition enables APC/C to robustly ubiquitinate diverse substrates and helps to drive cell cycle oscillations.
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Insights Into MAD2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric MAD2 Dimer.
PLoS Biology, 2008Co-Authors: Maojun Yang, Josep Rizo, Diana R. Tomchick, Mischa Machius, Chyong Jy Liu, Xuelian LuoAbstract:In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator CDC20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to CDC20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/CCDC20 inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2–C-Mad2 (C–C) or asymmetric O-Mad2–C-Mad2 (O–C) dimers. We also report the crystal structure of the symmetric C–C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C–C dimer is functional in vitro and in living cells. Finally, the Mad1–Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.
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conformation specific binding of p31comet antagonizes the function of mad2 in the spindle checkpoint
The EMBO Journal, 2004Co-Authors: Guohong Xia, Xuelian Luo, Josep Rizo, Toshiyuki Habu, Tomohiro MatsumotoAbstract:The spindle checkpoint ensures accurate chromosome segregation by delaying anaphase in response to misaligned sister chromatids during mitosis. Upon checkpoint activation, Mad2 binds directly to CDC20 and inhibits the anaphase-promoting complex or cyclosome (APC/C). CDC20 binding triggers a dramatic conformational change of Mad2. Consistent with an earlier report, we show herein that depletion of p31comet (formerly known as Cmt2) by RNA interference in HeLa cells causes a delay in mitotic exit following the removal of nocodazole. Purified recombinant p31comet protein antagonizes the ability of Mad2 to inhibit APC/CCDC20 in vitro and in Xenopus egg extracts. Interestingly, p31comet binds selectively to the CDC20-bound conformation of Mad2. Binding of p31comet to Mad2 does not prevent the interaction between Mad2 and CDC20 in vitro. During checkpoint inactivation in HeLa cells, p31comet forms a transient complex with APC/CCDC20-bound Mad2. Purified p31comet enhances the activity of APC/C isolated from nocodazole-arrested HeLa cells without disrupting the Mad2–CDC20 interaction. Therefore, our results suggest that p31comet counteracts the function of Mad2 and is required for the silencing of the spindle checkpoint.
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The Mad2 spindle checkpoint protein has two distinct natively folded states.
Nature Structural & Molecular Biology, 2004Co-Authors: Xuelian Luo, Katja Wassmann, Zhanyun Tang, Tomohiro Matsumoto, Guohong Xia, Josep RizoAbstract:The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to CDC20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/CCDC20. We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.
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Structure of the Mad2 spindle assembly checkpoint protein and its interaction with CDC20.
Nature Structural Biology, 2000Co-Authors: Xuelian Luo, Guowei Fang, Marc W Kirschner, Melissa Coldiron, Yingxi Lin, Gerhard WagnerAbstract:The checkpoint protein Mad2 inhibits the activity of the anaphase promoting complex by sequestering CDC20 until all chromosomes are aligned at the metaphase plate. We report the solution structure of human Mad2 and its interaction with CDC20. Mad2 possesses a novel three-layered α/β fold with three α-helices packed between two β-sheets. Using deletion mutants we identified the minimal Mad2-binding region of human CDC20 as a 40-residue segment immediately N-terminal to the WD40 repeats. Mutagenesis and NMR titration experiments show that a C-terminal flexible region of Mad2 is required for binding to CDC20. Mad2 and CDC20 form a tight 1:1 heterodimeric complex in which the C-terminal segment of Mad2 becomes folded. These results provide the first structural insight into mechanisms of the spindle assembly checkpoint.