The Experts below are selected from a list of 2487 Experts worldwide ranked by ideXlab platform
Maria Tortolero - One of the best experts on this subject based on the ideXlab platform.
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a single mutation in Securin induces chromosomal instability and enhances cell invasion
European Journal of Cancer, 2013Co-Authors: Mar Morasantos, Cristina M Limonmortes, Maria Tortolero, Carmen Saez, Miguel A Japon, Servando Giraldez, Joaquin Herreroruiz, Carolina Castilla, Francisco RomeroAbstract:Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1- bTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phos- phorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sort- ing analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chro- mosomal instability and cell invasion. These results show, for the first time, that a single muta- tion in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer. 2012 Elsevier Ltd. All rights reserved.
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glycogen synthase kinase 3β gsk3β negatively regulates pttg1 human Securin protein stability and gsk3β inactivation correlates with Securin accumulation in breast tumors
Journal of Biological Chemistry, 2011Co-Authors: Mar Morasantos, Cristina M Limonmortes, Maria Tortolero, Carmen Saez, Miguel A Japon, Servando Giraldez, Joaquin Herreroruiz, F J RomeroAbstract:Abstract PTTG1, also known as Securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, Securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, Securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for Securin in tumor progression. Stability of Securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting Securin for proteolysis have not been identified, and mechanistic insight into the cause of Securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates Securin to promote its proteolysis via SCFβTrCP E3 ubiquitin ligase. Importantly, a strong correlation between Securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for Securin accumulation in breast cancers.
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Glycogen Synthase Kinase-3β (GSK3β) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3β Inactivation Correlates with Securin Accumulation in Breast Tumors
Journal of Biological Chemistry, 2011Co-Authors: Mar Mora-santos, Maria Tortolero, Carmen Saez, Miguel A Japon, Servando Giraldez, M. Cristina Limón-mortés, Joaquín Herrero-ruiz, Francisco RomeroAbstract:Abstract PTTG1, also known as Securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, Securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, Securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for Securin in tumor progression. Stability of Securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting Securin for proteolysis have not been identified, and mechanistic insight into the cause of Securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates Securin to promote its proteolysis via SCFβTrCP E3 ubiquitin ligase. Importantly, a strong correlation between Securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for Securin accumulation in breast cancers.
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uv induced degradation of Securin is mediated by skp1 cul1 βtrcp e3 ubiquitin ligase
Journal of Cell Science, 2008Co-Authors: Cristina M Limonmortes, Maria Tortolero, Jose Antonio Pintortoro, Mar Morasantos, Agueda G Espina, Antonio Lopezroman, Francisco RomeroAbstract:Securin is a chaperone protein with bifunctional properties. It binds to separase to inhibit premature sister chromatid separation until the onset of anaphase, and it also takes part in cell-cycle arrest after UV irradiation. At metaphase-to-anaphase transition, Securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome (APC/C), allowing activation of separase. However, although Securin is reported to undergo proteasome-dependent degradation after UV irradiation, the ubiquitin ligase responsible for Securin ubiquitylation has not been well characterized. In this study, we show that UV radiation induced a marked reduction of Securin in both the nucleus and cytoplasm. Moreover, we show that GSK-3β inhibitors prevent Securin degradation, and that CUL1 and βTrCP are involved in this depletion. We also confirmed that SKP1-CUL1-βTrCP (SCFβTrCP) ubiquitylates Securin in vivo, and identified a conserved and unconventional βTrCP recognition motif (DDAYPE) in the Securin primary amino acid sequence of humans, nonhuman primates and rodents. Furthermore, downregulation of βTrCP caused an accumulation of Securin in non-irradiated cells. We conclude that SCFβTrCP is the E3 ubiquitin ligase responsible for Securin degradation after UV irradiation, and that it is involved in Securin turnover in nonstressed cells.
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proliferative potential after dna damage and non homologous end joining are affected by loss of Securin
Cell Death & Differentiation, 2008Co-Authors: Juan A Bernal, Maria Tortolero, Agueda G Espina, M Roche, Cristina Mendezvidal, Jose Antonio PintortoroAbstract:The faithful repair of DNA damage, especially chromosomal double-strand breaks (DSBs), is crucial for genomic integrity. We have previously shown that Securin interacts with the Ku70/80 heterodimer of the DSB non-homologous DNA end-joining (NHEJ) repair machinery. Here we demonstrate that Securin deficiency compromises cell survival and proliferation, but only after genotoxic stress. Securin−/− cells show a significant increase in gross chromosomal rearrangements and chromatid breaks after DNA damage, and also reveal an altered pattern of end resection in an NHEJ assay in comparison with Securin+/+ cells. These data suggest that Securin has a key role in the maintenance of genomic stability after DNA damage, thereby providing a previously unknown mechanism for regulating tumour progression.
Pauliina Kronqvist - One of the best experts on this subject based on the ideXlab platform.
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prognostic implications of Securin expression and sub cellular localization in human breast cancer
Cellular Oncology, 2016Co-Authors: Natalia Gurvits, Marjukka Nykanen, Kati Talvinen, Teijo Kuopio, Heli Repo, Eliisa Loyttyniemi, J Anttinen, Pauliina KronqvistAbstract:Purpose Securin belongs to a class of cell cycle regulators that prevent metaphase-to-anaphase transition until sister chromatid separation is complete. Evidence is accumulating that Securin has a prognostic impact on a variety of malignancies but, thus far, the role and regulation of Securin expression and its sub-cellular localization have not been systematically addressed in breast cancer.
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cdc20 and Securin overexpression predict short term breast cancer survival
British Journal of Cancer, 2014Co-Authors: Henna Karra, Reino Pitkanen, Teijo Kuopio, Mirva Soderstrom, Ilmari Ahonen, Minnamaija Lintunen, Heli Repo, Eliisa Loyttyniemi, Pauliina KronqvistAbstract:Cdc20 is an essential component of cell division and responsible for anaphase initiation regulated by Securin degradation. Cdc20 function is strongly regulated by the spindle assembly checkpoint to ensure the timely separation of sister chromatids and integrity of the genome. We present the first results on Cdc20 in a large clinical breast cancer material. The study was based on 445 breast cancer patients with up to 20 years of follow-up (mean 10.0 years). DNA content was determined by image cytometry on cell imprints, and Cdc20 and Securin immunohistochemistry on tissue microarrays of breast cancer tissue. In our results, high Cdc20 and Securin expression was associated with aneuploid DNA content. In prognostic analyses, high Cdc20 immunoexpression alone and in combination with high Securin immunoexpression indicated aggressive course of disease and up to 6.8-fold (P<0.001) risk of breast cancer death. Particularly, high Cdc20 and Securin immunoexpression identified a patient subgroup with extremely short, on average 2.4 years, breast cancer survival and triple-negative breast cancer (TNBC) subtype. We report for the first time the association of high Cdc20 and Securin immunoexpression with extremely poor outcome of breast cancer patients. Our experience indicates that Cdc20 and Securin are promising candidates for clinical applications in breast cancer prognostication, especially in the challenging prognostic decisions of TNBC.
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Cdc20 and Securin overexpression predict short-term breast cancer survival
British Journal of Cancer, 2014Co-Authors: H Karra, Reino Pitkanen, Teijo Kuopio, Mirva Soderstrom, Ilmari Ahonen, Minnamaija Lintunen, Heli Repo, Eliisa Loyttyniemi, Pauliina KronqvistAbstract:Cdc20 is an essential component of cell division and responsible for anaphase initiation regulated by Securin degradation. Cdc20 function is strongly regulated by the spindle assembly checkpoint to ensure the timely separation of sister chromatids and integrity of the genome. We present the first results on Cdc20 in a large clinical breast cancer material. The study was based on 445 breast cancer patients with up to 20 years of follow-up (mean 10.0 years). DNA content was determined by image cytometry on cell imprints, and Cdc20 and Securin immunohistochemistry on tissue microarrays of breast cancer tissue. In our results, high Cdc20 and Securin expression was associated with aneuploid DNA content. In prognostic analyses, high Cdc20 immunoexpression alone and in combination with high Securin immunoexpression indicated aggressive course of disease and up to 6.8-fold (P
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Securin and breast cancer
2014Co-Authors: Pauliina KronqvistAbstract:Some of the most essential steps of cell cycle progression occur during metaphase-anaphase transition. In this event, genetic stability is controlled through a complex signalling network aiming at maintaining chromosomal cohesion by blocking mitosis and Securing the mitotic spindle until sister chromatid segregation is complete. Disruption of this intrigue interaction results in incorrect DNA content and structure predicting particularly aggressive behaviour of malignant disease. Also in breast cancer, deregulated proliferation has been recognized among the most important factors predicting the clinical outcome of the disease.
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low cdc27 and high Securin expression predict short survival for breast cancer patients
Apmis, 2013Co-Authors: Kati Talvinen, Henna Karra, Reino Pitkanen, Marjukka Nykanen, Teijo Kuopio, Mirva Soderstrom, Ilmari Ahonen, Minnamaija Lintunen, Pauliina KronqvistAbstract:: Cell cycle regulators cdc27 and Securin participate in control of the mitotic checkpoint and survey the mitotic spindle to maintain chromosomal integrity. This is achieved by their functions in metaphase-anaphase transition, DNA damage repair, enhancement of mitotic arrest and apoptosis. We report on the roles of cdc27 and Securin in aneuploidy and prognosis of breast cancer. The study comprises 429 breast cancer patients with up to 22 years of follow-up. DNA content was determined by image cytometry, and immunopositivity for cdc27 and Securin was based on tissue microarrays. An inverse association between cdc27 and Securin expression was observed in both image cytometric and immunohistochemical analyses. Low cdc27 and high Securin expression identified patients with significant difference in disease outcome. Cdc27 and Securin immunoexpression identified patients at risk of early cancer death within five years from diagnosis. In multivariate analysis, the combination of cdc27 and Securin immunohistochemistry was the strongest predictor of cancer death after lymph node status. We demonstrate, for the first time in human breast cancer, the prognostic value of cdc27 and Securin immunohistochemistry. Cdc27 and Securin appear promising biomarkers for applications in predicting disease progression, prognostication of individual patients and potential in anti-mitotic drug development.
Francisco Romero - One of the best experts on this subject based on the ideXlab platform.
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a single mutation in Securin induces chromosomal instability and enhances cell invasion
European Journal of Cancer, 2013Co-Authors: Mar Morasantos, Cristina M Limonmortes, Maria Tortolero, Carmen Saez, Miguel A Japon, Servando Giraldez, Joaquin Herreroruiz, Carolina Castilla, Francisco RomeroAbstract:Pituitary tumour transforming gene (pttg1) encodes Securin, a protein involved in the inhibition of sister chromatid separation binding to Separase until the onset of anaphase. Separase is a cysteine-protease that degrades cohesin to segregate the sister chromatids to opposite poles of the cell. The amount of Securin is strongly regulated because it should allow Separase activation when it is degraded by the anaphase promoting complex/cyclosome, should arrest the cell cycle after DNA damage, when it is degraded through SKP1-CUL1- bTrCP ubiquitin ligase, and its overexpression induces tumour formation and correlates with metastasis in multiple tumours. Securin is a phosphoprotein that contains 32 potentially phos- phorylatable residues. We mutated and analysed most of them, and found a single mutant, hSecT60A, that showed enhanced oncogenic properties. Our fluorescence activated cell sort- ing analysis, fluorescence in situ hybridisation assays, tumour cell migration and invasion experiments and gene expression by microarrays analysis clearly involved hSecT60A in chro- mosomal instability and cell invasion. These results show, for the first time, that a single muta- tion in pttg1 is sufficient to trigger the oncogenic properties of Securin. The finding of this point mutation in patients might be used as an effective strategy for early detection of cancer. 2012 Elsevier Ltd. All rights reserved.
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Glycogen Synthase Kinase-3β (GSK3β) Negatively Regulates PTTG1/Human Securin Protein Stability, and GSK3β Inactivation Correlates with Securin Accumulation in Breast Tumors
Journal of Biological Chemistry, 2011Co-Authors: Mar Mora-santos, Maria Tortolero, Carmen Saez, Miguel A Japon, Servando Giraldez, M. Cristina Limón-mortés, Joaquín Herrero-ruiz, Francisco RomeroAbstract:Abstract PTTG1, also known as Securin, is an inactivating partner of separase, the major effector for chromosome segregation during mitosis. At the metaphase-to-anaphase transition, Securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome, allowing activation of separase. In addition, Securin is overexpressed in metastatic or genomically instable tumors, suggesting a relevant role for Securin in tumor progression. Stability of Securin is regulated by phosphorylation; some phosphorylated forms are degraded out of mitosis, by the action of the SKP1-CUL1-F-box protein (SCF) complex. The kinases targeting Securin for proteolysis have not been identified, and mechanistic insight into the cause of Securin accumulation in human cancers is lacking. Here, we demonstrate that glycogen synthase kinase-3β (GSK3β) phosphorylates Securin to promote its proteolysis via SCFβTrCP E3 ubiquitin ligase. Importantly, a strong correlation between Securin accumulation and GSK3β inactivation was observed in breast cancer tissues, indicating that GSK3β inactivation may account for Securin accumulation in breast cancers.
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uv induced degradation of Securin is mediated by skp1 cul1 βtrcp e3 ubiquitin ligase
Journal of Cell Science, 2008Co-Authors: Cristina M Limonmortes, Maria Tortolero, Jose Antonio Pintortoro, Mar Morasantos, Agueda G Espina, Antonio Lopezroman, Francisco RomeroAbstract:Securin is a chaperone protein with bifunctional properties. It binds to separase to inhibit premature sister chromatid separation until the onset of anaphase, and it also takes part in cell-cycle arrest after UV irradiation. At metaphase-to-anaphase transition, Securin is targeted for proteasomal destruction by the anaphase-promoting complex or cyclosome (APC/C), allowing activation of separase. However, although Securin is reported to undergo proteasome-dependent degradation after UV irradiation, the ubiquitin ligase responsible for Securin ubiquitylation has not been well characterized. In this study, we show that UV radiation induced a marked reduction of Securin in both the nucleus and cytoplasm. Moreover, we show that GSK-3β inhibitors prevent Securin degradation, and that CUL1 and βTrCP are involved in this depletion. We also confirmed that SKP1-CUL1-βTrCP (SCFβTrCP) ubiquitylates Securin in vivo, and identified a conserved and unconventional βTrCP recognition motif (DDAYPE) in the Securin primary amino acid sequence of humans, nonhuman primates and rodents. Furthermore, downregulation of βTrCP caused an accumulation of Securin in non-irradiated cells. We conclude that SCFβTrCP is the E3 ubiquitin ligase responsible for Securin degradation after UV irradiation, and that it is involved in Securin turnover in nonstressed cells.
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protein phosphatase 2a stabilizes human Securin whose phosphorylated forms are degraded via the scf ubiquitin ligase
Molecular and Cellular Biology, 2006Co-Authors: Ana M Gilbernabe, Francisco Romero, Cristina M Limonmortes, Maria TortoleroAbstract:Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone Securin. Activation of separase occurs at anaphase onset, when Securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human Securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.
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Securin is a target of the uv response pathway in mammalian cells
Molecular and Cellular Biology, 2004Co-Authors: Francisco Romero, Ana M Gilbernabe, Carmen Saez, Miguel A Japon, Jose Antonio Pintortoro, Maria TortoleroAbstract:The segregation of sister chromatids to opposite poles of the cell during anaphase ensures that a complete set of chromosomes is transmitted from one generation to another. Sister chromatid separation is an irreversible process and is therefore tightly regulated. The separation of all sister chromatid pairs is delayed when individual chromosomes are damaged or have failed to attach to the mitotic spindle. Mistakes in such regulatory mechanisms in somatic cells are thought to contribute to the aneuploidy characteristic of many tumor cells (6), whereas defects during meiosis generate trisomies (34). Sister chromatid cohesion is mediated by a conserved complex called cohesin, which is composed of the Scc1, Scc3, Smc1, and Smc3 proteins in the budding yeast Saccharomyces cerevisiae (17). The separation of sister chromatids at the metaphase-to-anaphase transition is triggered by proteolytic cleavage of the Scc1 cohesin subunit by a conserved cysteine protease called Separase (Esp1 in S. cerevisiae) (54). Scc1 cleavage destroys the bridge between sister chromatids and thereby enables spindle microtubules to move the sister chromatids toward opposite poles of the cell. For most of the cell cycle, Separase is bound by an inhibitor called Securin (Pds1 in S. cerevisiae), which is destroyed by ubiquitin-mediated proteolysis shortly before the metaphase-to-anaphase transition. Moreover, Scc1 cleavage is also regulated by the phosphorylation of Separase recognition sites by the Polo-like kinase Cdc5 (2). In vertebrates, there are at least two cohesin complexes with different Scc3-like subunits (50). Cohesin is removed from chromosomes in two steps (57). During prophase and prometaphase, the bulk of cohesin dissociates from the arms of condensing chromosomes (31) via a mechanism that depends neither on the Securin-Separase pathway nor on the cleavage of the human ortholog of Scc1 (50). Dissociation appears to be mediated by a Polo kinase-dependent mechanism (51). However, a small amount of cohesin remains in centromeric regions until metaphase and is removed from chromosomes only at the onset of anaphase (57). At least two mechanisms prevent Separase activation, one by causing Separase phosphorylation (cyclin B/Cdk1) and the other by binding to and inhibiting the protease domain (Securin) (48). Securin is ubiquitinated by a multisubunit ubiquitin protein ligase, the anaphase-promoting complex or cyclosome (APC/C) (8, 14, 62), whose activity is controlled by Mad2, a component of the mitotic checkpoint which ensures that all kinetochores become attached to microtubules (45). In addition, to display full ubiquitin ligase activity, APC/C must bind to the Cdc20 and Cdh1 accessory factors, which are responsible for Securin and cyclin B degradation (36). The role of Securin in Separase activity has been explained in two ways. Securin has been implicated in the subcellular localization of Separase (21, 25) and enables the full catalytic activity of Separase after the destruction of Securin in anaphase (18). That Securin regulates chromatid separation during cell division suggests that aneuploidy caused by defective sister chromatid separation is involved in tumor development (20, 27). In fact, Securin is highly expressed in many tumors that have been analyzed (10, 40, 41). In S. cerevisiae, Securin not only is required for efficient chromosome segregation but also is needed to prevent anaphase in response to spindle and DNA damage (58, 59). In fact, surveillance mechanisms that sense DNA damage arrest cell cycle progression by causing the stabilization of Pds1, thereby blocking sister chromatid separation (52). Moreover, spindle damage blocks sister chromatid separation solely by inhibiting Cdc20-APC/C-dependent Pds1 proteolysis (3). On the other hand, in mammalian cells, Securin interacts with the regulatory subunit of the DNA-dependent protein kinase, which is involved in nonhomologous end joining (13, 28); this fact suggests that Securin may connect DNA damage response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs (39). To identify the mechanisms that involve human Securin (hSecurin) in DNA damage, we studied the effect of UV light on hSecurin. Human cells responded to UV light by rapid proteasome-dependent hSecurin degradation caused by unexpected APC/C activation after UV-induced DNA damage and by specific hSecurin protein synthesis inhibition. Moreover, here we show that hSecurin plays a role in the cellular response to UV radiation, being necessary for cell proliferation arrest after UV treatment.
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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positive and negative regulation of vertebrate separase by cdk1 cyclin b1 may explain why Securin is dispensable
Journal of Biological Chemistry, 2015Co-Authors: Susanne Hellmuth, Franziska Bottger, Christopher Pohlmann, Andreas Brown, Mathias Sprinzl, Olaf StemmannAbstract: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.
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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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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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Securin and not cdk1 cyclin b1 regulates sister chromatid disjunction during meiosis ii in mouse eggs
Developmental Biology, 2008Co-Authors: Ibtissem Nabti, Olaf Stemmann, Alexandra Reis, Mark Levasseur, Keith T JonesAbstract: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.
Richard Bayliss - 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.
