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Richard Bayliss - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like
2016Co-Authors: Death Domains, Anja Winter, Ralf Schmid, Richard BaylissAbstract:Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal cat-alytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of Separases and the details of their regulation and substrate recognition are poorly under-stood. Here we undertook an in-depth bioinformatical analysis of Separases from different species with respect to their similarity in amino acid sequence and protein fold in compari-son to caspases, MALT-1 proteins (mucosa-associated lymphoidtissue lymphoma translo-cation protein 1) and gingipain-R. A comparative model of the single C-terminal caspase-like domain in Separase from C. elegans suggests similar binding modes of substrate pep-tides between these protein subfamilies, and enables differences in substrate specificity of Separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel con
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A closed conformation of the Caenorhabditis elegans Separase–securin complex
Open Biology, 2016Co-Authors: Gudrun Bachmann, Mark W Richards, Anja Winter, Fabienne Beuron, Edward P Morris, Richard BaylissAbstract:The protease Separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, Separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the Separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans Separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 A that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of Separase, whereas the second lobe readily accommodates homology models of the Separase C-terminal death and caspase-like domains. The globular structure of the C. elegans Separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of Separase activity through conformational change.
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a closed conformation of the caenorhabditis elegans Separase securin complex
Open Biology, 2016Co-Authors: Gudrun Bachmann, Mark W Richards, Anja Winter, Fabienne Beuron, Edward P Morris, Richard BaylissAbstract:The protease Separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, Separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the Separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans Separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 A that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of Separase, whereas the second lobe readily accommodates homology models of the Separase C-terminal death and caspase-like domains. The globular structure of the C. elegans Separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of Separase activity through conformational change.
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Separases from all species share a similar topology with highly conserved regions, which includes a caspase-like domain in their C-terminal region.
2015Co-Authors: Anja Winter, Ralf Schmid, Richard BaylissAbstract:(A) Consolidated secondary structure prediction of Separase from C. elegans using both PsiPred and JPred predicts a largely helical N-terminus (dark grey) with an unstructured region around residue 400 and a region of three β-strands from residues 720 to 750 (light grey). The conserved C-terminal half harbours the caspase-like domain (black), residues 900 to 1140. The catalytic dyad is indicated as white lines. (B) Multiple sequence alignment of the caspase-like domain of Separase from C. elegans in comparison with human caspase 3, gingipain-R and MALT-1. Alignment was manually adjusted using Jalview to match secondary structure elements from predictions (PsiPred) of Separases to structural elements as observed in caspase 3 (3EDQ), MALT-1 (3UO8) and gingipain R (1CVR). α-helices are shown in dark grey and β-strands in light grey. The conserved catalytic dyad (C, H) is shown in bold letters.
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Interactions made between the predicted substrate peptide MEVER and the homology model of the C. elegans Separase caspase domain.
2015Co-Authors: Anja Winter, Ralf Schmid, Richard BaylissAbstract:(A) Residues in Separases that interact with the substrate peptide are highlighted in an alignment. P1-Arg is anchored via hydrogen bonds to Glu917 (~) and Asp1082 towards the N-terminus of helix α4 (+). The main chain amide-NH of residue P1 interacts with main chain oxygen atom of Thr1075 (#). The side chain of P2-Glu forms hydrogen bonds to Arg1120 (which is located in a well-conserved region preceding β6) and Arg1044 (which is located in a loop region between helix α3’ and β4) which are both highlighted with * in the alignment. Aside from hydrophobic interactions, P3-Val interacts with both the main chain and side chain oxygen atoms of Thr1077 (^). P4-Glu forms hydrogen bonds to the main chain amide of Lys1118 and guanidino group of Arg1116 (–). α-helices are shown in dark grey and β-strands in light grey. The catalytic dyad (C, H) is shown in bold letters. (B) Schematic representation of interactions between C. elegans Separase and the proposed substrate peptide EVER. Corresponding interacting residues in Separases from other species are taken from Fig 4A. Core recognition sites of Scc1 proteins are shown in the boxed inset.
Olaf Stemmann - One of the best experts on this subject based on the ideXlab platform.
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securin independent regulation of Separase by checkpoint induced shugoshin mad2
Nature, 2020Co-Authors: Susanne Hellmuth, Laura Gomezh, Alberto M Pendas, Olaf StemmannAbstract: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. Shugoshin and MAD2 regulate Separase-mediated chromosome separation during mitosis, in parallel to a previously identified mechanism involving the anaphase inhibitor securin.
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securin independent regulation of Separase by checkpoint induced shugoshin mad2
Nature, 2020Co-Authors: Susanne Hellmuth, Laura Gomezh, Alberto M Pendas, Olaf StemmannAbstract: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.
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identification of bioactive small molecule inhibitors of Separase
ACS Chemical Biology, 2019Co-Authors: Lars Henschke, Susanne Hellmuth, Olaf Stemmann, Matthias Frese, Andreas Marx, Thomas U MayerAbstract:: 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 51 009-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.
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pp2a delays apc c dependent degradation of Separase associated but not free securin
The EMBO Journal, 2014Co-Authors: Susanne Hellmuth, Franziska Bottger, Matthias Mann, Olaf StemmannAbstract: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.
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anaphase topsy turvy cdk1 a securin Separase a cki
Cell Cycle, 2006Co-Authors: Olaf Stemmann, Ingo H. Gorr, Dominik BoosAbstract:Chromosome segregation in mitosis and meiosis is triggered by activation of a large protease, Separase. While it has been known for some time that the anaphase inhibitor securin regulates Separase activity recent work shows that this is only half the story. In vertebrates Cdk1-dependent inhibition of Separase represents a second, securin-independent branch of anaphase regulation. Furthermore, an unanticipated ability of Separase to inhibit Cdk1 suggests additional, non-proteolytic functions of Separase.
Frank Uhlmann - One of the best experts on this subject based on the ideXlab platform.
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Separase securin complex a cunning way to control chromosome segregation
Nature Structural & Molecular Biology, 2017Co-Authors: Martin R Singleton, Frank UhlmannAbstract:Separases are crucial cell cycle proteases that control the metaphase-to-anaphase transition by cleaving chromosomal cohesin rings. Two new high-resolution structures of Separase bound by its inhibitory chaperone securin illustrate intriguing regulatory mechanisms.
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the dual mechanism of Separase regulation by securin
Current Biology, 2002Co-Authors: Nadine C D Hornig, Philip P Knowles, Neil Q Mcdonald, Frank UhlmannAbstract:Abstract Background: Sister chromatid separation and segregation at anaphase onset are triggered by cleavage of the chromosomal cohesin complex by the protease Separase. Separase is regulated by its binding partner securin in two ways: securin is required to support Separase activity in anaphase; and, at the same time, securin must be destroyed via ubiquitylation before Separase becomes active. The molecular mechanisms underlying this dual regulation of Separase by securin are unknown. Results: We show that, in budding yeast, securin supports Separase localization. Separase enters the nucleus independently of securin, but securin is required and sufficient to cause accumulation of Separase in the nucleus, where its known cleavage targets reside. Securin also ensures that Separase gains full proteolytic activity in anaphase. We also show that securin, while present, directly inhibits the proteolytic activity of Separase. Securin prevents the binding of Separase to its substrates. It also hinders the Separase N terminus from interacting with and possibly inducing an activating conformational change at the protease active site 150 kDa downstream at the protein's C terminus. Conclusions: Securin inhibits the proteolytic activity of Separase in a 2-fold manner. While inhibiting Separase, securin is able to promote nuclear accumulation of Separase and help Separase to become fully activated after securin's own destruction at anaphase onset.
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Orchestrating anaphase and mitotic exit: Separase cleavage and localization of Slk19
Nature Cell Biology, 2001Co-Authors: Matt Sullivan, Christine Lehane, Frank UhlmannAbstract:Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease Separase. Here we show that Separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by Separase in the orchestration of high-fidelity anaphase.
Mark W Richards - One of the best experts on this subject based on the ideXlab platform.
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A closed conformation of the Caenorhabditis elegans Separase–securin complex
Open Biology, 2016Co-Authors: Gudrun Bachmann, Mark W Richards, Anja Winter, Fabienne Beuron, Edward P Morris, Richard BaylissAbstract:The protease Separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, Separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the Separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans Separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 A that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of Separase, whereas the second lobe readily accommodates homology models of the Separase C-terminal death and caspase-like domains. The globular structure of the C. elegans Separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of Separase activity through conformational change.
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a closed conformation of the caenorhabditis elegans Separase securin complex
Open Biology, 2016Co-Authors: Gudrun Bachmann, Mark W Richards, Anja Winter, Fabienne Beuron, Edward P Morris, Richard BaylissAbstract:The protease Separase plays a key role in sister chromatid disjunction and centriole disengagement. To maintain genomic stability, Separase activity is strictly regulated by binding of an inhibitory protein, securin. Despite its central role in cell division, the Separase and securin complex is poorly understood at the structural level. This is partly owing to the difficulty of generating a sufficient quantity of homogeneous, stable protein. Here, we report the production of Caenorhabditis elegans Separase–securin complex, and its characterization using biochemical methods and by negative staining electron microscopy. Single particle analysis generated a density map at a resolution of 21–24 A that reveals a close, globular structure of complex connectivity harbouring two lobes. One lobe matches closely a homology model of the N-terminal HEAT repeat domain of Separase, whereas the second lobe readily accommodates homology models of the Separase C-terminal death and caspase-like domains. The globular structure of the C. elegans Separase–securin complex contrasts with the more elongated structure previously described for the Homo sapiens complex, which could represent a different functional state of the complex, suggesting a mechanism for the regulation of Separase activity through conformational change.
Susanne Hellmuth - One of the best experts on this subject based on the ideXlab platform.
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securin independent regulation of Separase by checkpoint induced shugoshin mad2
Nature, 2020Co-Authors: Susanne Hellmuth, Laura Gomezh, Alberto M Pendas, Olaf StemmannAbstract: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. Shugoshin and MAD2 regulate Separase-mediated chromosome separation during mitosis, in parallel to a previously identified mechanism involving the anaphase inhibitor securin.
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securin independent regulation of Separase by checkpoint induced shugoshin mad2
Nature, 2020Co-Authors: Susanne Hellmuth, Laura Gomezh, Alberto M Pendas, Olaf StemmannAbstract: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.
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identification of bioactive small molecule inhibitors of Separase
ACS Chemical Biology, 2019Co-Authors: Lars Henschke, Susanne Hellmuth, Olaf Stemmann, Matthias Frese, Andreas Marx, Thomas U MayerAbstract:: 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 51 009-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.
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pp2a delays apc c dependent degradation of Separase associated but not free securin
The EMBO Journal, 2014Co-Authors: Susanne Hellmuth, Franziska Bottger, Matthias Mann, Olaf StemmannAbstract: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.
