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Proper chromosome segregation during cell division relies on the timely dissolution of chromosome cohesion. Separase (EC3.4.22.49), a cysteine protease, plays a critical role in mitosis by cleaving the kleisin subunit of cohesin, thereby presenting a promising target for cancer therapy. However, challenges in isolating active human separase suitable for high-throughput screening have limited the identification of effective inhibitors. Here, we conducted a high-throughput screening of small-molecule inhibitors using the protease domain of Chaetomium thermophilum separase (ctSPD), which not only shares significant sequence similarity with human separase but is also readily available. After conducting a primary screening of a library containing 9,172 compounds and subsequent validation using human separase, we identified walrycin B and its analogs, toxoflavin, 3-methyltoxoflavin, and 3-phenyltoxoflavin, as potent inhibitors of human separase. Subsequent microscale thermophoresis assays and molecular dynamics simulations revealed that walrycin B binds to the active site of separase and competes with substrates for binding. Additionally, cell-based studies showed that walrycin B and its analogs effectively induce cell cycle arrest at the M phase, activate apoptosis, and ultimately lead to cell death in mitosis. Finally, in a mouse xenograft model, walrycin B exhibited significant antitumor efficacy with minimal side effects. Together, these findings highlight the therapeutic potential of walrycin B for cancer treatment and its utility as a chemical tool in future studies involving separase., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Separase regulates multiple aspects of the metaphase-to-anaphase transition. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis. The anaphase-promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase is unknown. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C-mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase., (© 2024 Sorensen Turpin et al.)

Background: Extra spindle pole bodies-like 1 ( ESPL1 ) is known to play a crucial role in the segregation of sister chromatids during mitosis. Overexpression of ESPL1 is considered to have oncogenic effects in various human cancers. However, the specific biological function of ESPL1 in endometrial cancer (EC) remains unclear., Methods: The TCGA and GEO databases were utilized to assess the expression of ESPL1 in EC. Immunohistochemistry was utilized to detect separase expression in EC samples. Kaplan-Meier survival analysis and Cox regression analysis were performed to evaluate the diagnostic and prognostic significance of ESPL1 in EC. Gene Set Enrichment Analysis (GSEA) was employed to explore the potential signaling pathway of ESPL1 in EC. Cell proliferation and colony formation ability were analyzed using CCK-8 and colony formation assay., Results: Our analysis revealed that ESPL1 is significantly upregulated in EC, and its overexpression is associated with advanced clinical characteristics and unfavourable prognostic outcomes. Suppression of ESPL1 attenuated proliferation of EC cell line., Conclusion: The upregulation of ESPL1 is associated with advanced disease and poor prognosis in EC patients. These findings suggest that ESPL1 has the potential to serve as a diagnostic and prognostic biomarker in EC, highlighting its significance in the management of EC patients.

Separase is a key cysteine protease in the separation of sister chromatids through the digestion of the cohesin ring that inhibits chromosome segregation as a trigger of the metaphase-anaphase transition in eukaryotes. Its activity is highly regulated by binding with securin and cyclinB-CDK1 complex. These bindings prevent the proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties in biochemical examinations due to the instability of separase in vitro and the fact that few spatiotemporal resolution approaches exist for monitoring live separase activity throughout mitotic processes. Here, we have developed FRET-based molecular sensors, including GFP variants, with separase-cleavable sequences as donors and covalently attached fluorescent dyes as acceptor molecules. These are applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated the performance of equivalent molecular sensors, either localized or not localized inside the nucleus under cell cycle control, using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems that can be used specifically or statistically, which could lead to the elucidation of separase properties in detail.

Lysine acetylation of non-histone proteins plays crucial roles in many cellular processes. In this study, we examine the role of lysine acetylation during sister chromatid separation in mitosis. We investigate the acetylation of securin at K21 by cell-cycle-dependent acetylome analysis and uncover its role in separase-triggered chromosome segregation during mitosis. Prior to the onset of anaphase, the acetylated securin via TIP60 prevents its degradation by the APC/C CDC20 -mediated ubiquitin-proteasome system. This, in turn, restrains precocious activation of separase and premature separation of sister chromatids. Additionally, the acetylation-dependent stability of securin is also enhanced by its dephosphorylation. As anaphase approaches, HDAC1-mediated deacetylation of securin promotes its degradation, allowing released separase to cleave centromeric cohesin. Blocking securin deacetylation leads to longer anaphase duration and errors in chromosome segregation. Thus, this study illustrates the emerging role of securin acetylation dynamics in mitotic progression and genetic stability., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A tight synchrony between the DNA and centrosome cycle is essential for genomic integrity. Centriole disengagement, which licenses centrosomes for duplication, occurs normally during mitotic exit. We recently demonstrated that mild DNA replication stress typically seen in cancer cells causes premature centriole disengagement in untransformed mitotic human cells, leading to transient multipolar spindles that favour chromosome missegregation. How mild replication stress accelerates the centrosome cycle at the molecular level remained, however, unclear. Using ultrastructure expansion microscopy, we show that mild replication stress induces premature centriole disengagement already in G2 via the ATR-Chk1 axis of the DNA damage repair pathway. This results in a sub-critical Plk1 kinase activity that primes the pericentriolar matrix for Separase-dependent disassembly but is insufficient for rapid mitotic entry, causing premature centriole disengagement in G2. We postulate that the differential requirement of Plk1 activity for the DNA and centrosome cycles explains how mild replication stress disrupts the synchrony between both processes and contributes to genomic instability., (© 2023. Springer Nature Limited.)

Sister chromatid segregation is the final irreversible step of mitosis. It is initiated by a complex regulatory system that ultimately triggers the timely activation of a conserved cysteine protease named separase. Separase cleaves the cohesin protein ring that links the sister chromatids and thus facilitates their separation and segregation to the opposite poles of the dividing cell. Due to the irreversible nature of this process, separase activity is tightly controlled in all eukaryotic cells. In this mini-review, we summarize the latest structural and functional findings on the regulation of separase, with an emphasis on the regulation of the human enzyme by two inhibitors, the universal inhibitor securin and the vertebrate-specific inhibitor CDK1-cyclin B. We discuss the two fundamentally different inhibitory mechanisms by which these inhibitors block separase activity by occluding substrate binding. We also describe conserved mechanisms that facilitate substrate recognition and point out open research questions that will guide studies of this fascinating enzyme for years to come., (© 2023 The Author(s).)

Introduction: Extra spindle pole bodies like 1 (ESPL1) are required to continue the cell cycle, and its primary role is to initiate the final segregation of sister chromatids. Although prior research has revealed a link between ESPL1 and the development of cancer, no systematic pan-cancer analysis has been conducted. Combining multi-omics data with bioinformatics, we have thoroughly described the function of ESPL1 in cancer. In addition, we examined the impact of ESPL1 on the proliferation of numerous cancer cell lines. In addition, the connection between ESPL1 and medication sensitivity was verified using organoids obtained from colorectal cancer patients. All these results confirm the oncogene nature of ESPL1., Methods: Herein, we downloaded raw data from numerous publicly available databases and then applied R software and online tools to explore the association of ESPL1 expression with prognosis, survival, tumor microenvironment, tumor heterogeneity, and mutational profiles. To validate the oncogene nature of ESPL1, we have performed a knockdown of the target gene in various cancer cell lines to verify the effect of ESPL1 on proliferation and migration. In addition, patients' derived organoids were used to verify drug sensitivity., Results: The study found that ESPL1 expression was markedly upregulated in tumorous tissues compared to normal tissues, and high expression of ESPL1 was significantly associated with poor prognosis in a range of cancers. Furthermore, the study revealed that tumors with high ESPL1 expression tended to be more heterogeneous based on various tumor heterogeneity indicators. Enrichment analysis showed that ESPL1 is involved in mediating multiple cancer-related pathways. Notably, the study found that interference with ESPL1 expression significantly inhibited the proliferation of tumor cells. Additionally, the higher the expression of ESPL1 in organoids, the greater the sensitivity to PHA-793887, PAC-1, and AZD7762., Discussion: Taken together, our study provides evidence that ESPL1 may implicate tumorigenesis and disease progression across multiple cancer types, highlighting its potential utility as both a prognostic indicator and therapeutic target., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhong, Zheng, Zhang, Wu, Wang, Zhang, Feng and Wang.)

Receiving complete and undamaged genetic information is vital for the survival of daughter cells after chromosome segregation. The most critical steps in this process are accurate DNA replication during S phase and a faithful chromosome segregation during anaphase. Any errors in DNA replication or chromosome segregation have dire consequences, since cells arising after division might have either changed or incomplete genetic information. Accurate chromosome segregation during anaphase requires a protein complex called cohesin, which holds together sister chromatids. This complex unifies sister chromatids from their synthesis during S phase, until separation in anaphase. Upon entry into mitosis, the spindle apparatus is assembled, which eventually engages kinetochores of all chromosomes. Additionally, when kinetochores of sister chromatids assume amphitelic attachment to the spindle microtubules, cells are finally ready for the separation of sister chromatids. This is achieved by the enzymatic cleavage of cohesin subunits Scc1 or Rec8 by an enzyme called Separase. After cohesin cleavage, sister chromatids remain attached to the spindle apparatus and their poleward movement on the spindle is initiated. The removal of cohesion between sister chromatids is an irreversible step and therefore it must be synchronized with assembly of the spindle apparatus, since precocious separation of sister chromatids might lead into aneuploidy and tumorigenesis. In this review, we focus on recent discoveries concerning the regulation of Separase activity during the cell cycle.

Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes., (© 2022. The Author(s).)

The key to gametogenesis is the proper execution of a specialized form of cell division named meiosis. Prior to the meiotic divisions, the recombination of maternal and paternal chromosomes creates new genetic combinations necessary for fitness and adaptation to an ever-changing environment. Two rounds of chromosome segregation -meiosis I and II- have to take place without intermediate S-phase and lead to the creation of haploid gametes harboring only half of the genetic material. Importantly, the segregation patterns of the two divisions are fundamentally different and require adaptation of the mitotic cell cycle machinery to the specificities of meiosis. Separase, the enzyme that cleaves Rec8, a subunit of the cohesin complex constituting the physical connection between sister chromatids, has to be activated twice: once in meiosis I and immediately afterwards, in meiosis II. Rec8 is cleaved on chromosome arms in meiosis I and in the centromere region in meiosis II. This step-wise cohesin removal is essential to generate gametes of the correct ploidy and thus, embryo viability. Hence, separase control and Rec8 cleavage must be perfectly controlled in time and space. Focusing on mammalian oocytes, this review lays out what we know and what we still ignore about this fascinating mechanism., Competing Interests: The author declares no conflict of interest. The funders had no role in the writing of the manuscript; or in the decision to publish.

Centromeric histones (CenH3s) are essential for chromosome inheritance during cell division in most eukaryotes. CenH3 genes have rapidly evolved and undergone repeated gene duplications and diversification in many plant and animal species. In Caenorhabditis species, two independent duplications of CenH3 (named hcp-3 for HoloCentric chromosome-binding Protein 3) were previously identified in C. elegans and C. remanei. Using phylogenomic analyses in 32 Caenorhabditis species, we find strict retention of the ancestral hcp-3 gene and 10 independent duplications. Most hcp-3L (hcp-3-like) paralogs are only found in 1-2 species, are expressed in both males and females/hermaphrodites, and encode histone fold domains with 69-100% identity to ancestral hcp-3. We identified novel N-terminal protein motifs, including putative kinetochore protein-interacting motifs and a potential separase cleavage site, which are well conserved across Caenorhabditis HCP-3 proteins. Other N-terminal motifs vary in their retention across paralogs or species, revealing potential subfunctionalization or functional loss following duplication. An N-terminal extension in the hcp-3L gene of C. afra revealed an unprecedented protein fusion, where hcp-3L fused to duplicated segments from hcp-4 (nematode CENP-C). By extending our analyses beyond CenH3, we found gene duplications of six inner and outer kinetochore genes in Caenorhabditis, which appear to have been retained independent of hcp-3 duplications. Our findings suggest that centromeric protein duplications occur frequently in Caenorhabditis nematodes, are selectively retained for short evolutionary periods, then degenerate or are lost entirely. We hypothesize that unique challenges associated with holocentricity in Caenorhabditis may lead to this rapid "revolving door" of kinetochore protein paralogs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Soybean leaf-associated gemygorvirus-1 (SlaGemV-1) is a CRESS-DNA virus classified in the family Genomoviridae, which causes hypovirulence and abolishes sclerotia formation in infected fungal pathogens under the family Sclerotiniaceae. To investigate the mechanisms involved in the induction of hypovirulence, RNA-Seq was compared between virus-free and SlaGemV-1-infected Sclerotinia sclerotiorum strain DK3. Overall, 4639 genes were differentially expressed, with 50.5% up regulated and 49.5% down regulated genes. GO enrichments suggest changes in integral membrane components and transmission electron microscopy images reveal virus-like particles localized near the inner cell membrane. Differential gene expression analysis focused on genes responsible for cell cycle and DNA replication and repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis. Carbohydrate metabolism showed the most changes, with two glycoside hydrolase genes being the most down regulated by -2396.1- and -648.6-fold. Genes relating to pathogenesis showed consistent down regulation with the greatest being SsNep1, SsSSVP1, and Endo2 showing, -4555-, -14.7-, and -12.3-fold changes. The cell cycle and DNA replication/repair pathways were almost entirely up regulated including a putative cyclin and separase being up regulated 8.3- and 5.2-fold. The oxalate decarboxylase genes necessary for oxalic acid catabolism and oxalic acid precursor biosynthesis genes and its metabolism show down regulations of -17.2- and -12.1-fold changes. Sclerotial formation genes also appear differentially regulated including a melanin biosynthesis gene Pks 1 and a sclerotia formation gene Sl 2 with fold changes of 3.8 and -2.9.

To generate haploid gametes, cohesin is removed in a stepwise manner from chromosome arms in meiosis I and the centromere region in meiosis II to segregate chromosomes and sister chromatids, respectively. Meiotic cohesin removal requires cleavage of the meiosis-specific kleisin subunit Rec8 by the protease separase. 1 , 2 In yeast and C. elegans, Rec8 on chromosome arms has to be phosphorylated to be cleaved in meiosis I, 3-7 whereas Rec8 at the centromere is protected from cleavage by the action of PP2A-B56. 8-10 However, in mammalian meiosis, it is unknown whether Rec8 has to be equally phosphorylated for cleavage, and if so, the identity of the relevant kinase(s). This is due to technical challenges, as Rec8 is poorly conserved, preventing a direct translation of the knowledge gained from model systems such as yeast and C. elegans to mammals. Additionally, there is no turnover of Rec8 after cohesion establishment, preventing phosphomutant analysis of functional Rec8. To address the very basic question of whether Rec8 cleavage requires its phosphorylation in mammals, we adapted a biosensor that detects separase activity to study Rec8 cleavage in single mouse oocytes by live imaging. Crucially, through phosphomutant analysis, we identified phosphorylation sites in Rec8 promoting cleavage. We found that Rec8 cleavage depends on Aurora B/C kinase activities and identified an aminoacid residue that is phosphorylated in vivo. Accordingly, inhibition of Aurora B/C kinases during meiotic maturation impairs endogenous Rec8 phosphorylation and chromosome segregation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Cdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and can cooperate with the proteasome for their degradation. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase, resulting in co-degradation of separase along with securin. If true, Cdc48 would be important in mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Correct regulation of cohesin at chromosome arms and centromeres and accurate kinetochore-microtubule connections are significant for proper chromosome segregation. At anaphase of meiosis I, cohesin at chromosome arms is cleaved by separase, leading to the separation of homologous chromosomes. However, at anaphase of meiosis II, cohesin at centromeres is cleaved by separase, leading to the separation of sister chromatids. Shugoshin-2 (SGO2) is a member of the shugoshin/MEI-S332 protein family in mammalian cells, a crucial protein that protects centromeric cohesin from cleavage by separase and corrects wrong kinetochore-microtubule connections before anaphase of meiosis I. Shugoshin-1 (SGO1) plays a similar role in mitosis. Moreover, shugoshin can inhibit the occurrence of chromosomal instability (CIN), and its abnormal expression in several tumors, such as triple-negative breast cancer, hepatocellular carcinoma, lung cancer, colon cancer, glioma, and acute myeloid leukemia, can be used as biomarker for disease progression and potential therapeutic targets for cancers. Thus, this review discusses the specific mechanisms of shugoshin which regulates cohesin, kinetochore-microtubule connections, and CIN., (© 2023 S. Karger AG, Basel.)

Protection of peri-centromeric (periCEN) REC8 cohesin from Separase and sister kinetochore (KT) attachment to microtubules emanating from the same spindle pole (co-orientation) ensures that sister chromatids remain associated after meiosis I. Both features are lost during meiosis II, resulting in sister chromatid disjunction and the production of haploid gametes. By transferring spindle-chromosome complexes (SCCs) between meiosis I and II in mouse oocytes, we discovered that both sister KT co-orientation and periCEN cohesin protection depend on the SCC, and not the cytoplasm. Moreover, the catalytic activity of Separase at meiosis I is necessary not only for converting KTs from a co- to a bi-oriented state but also for deprotection of periCEN cohesion, and cleavage of REC8 may be the key event. Crucially, selective cleavage of REC8 in the vicinity of KTs is sufficient to destroy co-orientation in univalent chromosomes, albeit not in bivalents where resolution of chiasmata may also be required., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

L-type amino acid transporter 3 (LAT3, SLC43A1) is abundantly expressed in prostate cancer (PC) and is thought to play an essential role in PC progression through the cellular uptake of essential amino acids. Here, we analyzed the expression, function, and downstream target of LAT3 in PC. LAT3 was highly expressed in PC cells expressing androgen receptor (AR), and its expression was increased by dihydrotestosterone treatment and decreased by bicalutamide treatment. In chromatin immunoprecipitation sequencing of AR, binding of AR to the SLC43A1 region was increased by dihydrotestosterone stimulation. Knockdown of LAT3 inhibited cell proliferation, migration, and invasion, and the phosphorylation of p70S6K and 4EBP-1. Separase (ESPL1) was identified as a downstream target of LAT3 by RNA sequencing analysis. In addition, immunostaining of prostatectomy specimens was performed. In the multivariate analysis, high expression of LAT3 was an independent prognostic factor for recurrence-free survival (hazard ratio: 3.24; P = .0018). High LAT3 expression was correlated with the pathological T stage and a high International Society of Urological Pathology grade. In summary, our results suggest that LAT3 plays an important role in the progression of PC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

To generate haploid gametes, germ cells undergo two consecutive meiotic divisions requiring key changes to the cell division machinery. Here, we demonstrate that the protease separase rewires key cell division processes at the meiosis I/II transition by cleaving the meiosis-specific protein Meikin. Separase proteolysis does not inactivate Meikin but instead alters its function to create a distinct activity state. Full-length Meikin and the C-terminal Meikin separase cleavage product both localize to kinetochores, bind to Plk1 kinase, and promote Rec8 cleavage, but our results reveal distinct roles for these proteins in controlling meiosis. Mutations that prevent Meikin cleavage or that conditionally inactivate Meikin at anaphase I result in defective meiosis II chromosome alignment in mouse oocytes. Finally, as oocytes exit meiosis, C-Meikin is eliminated by APC/C-mediated degradation prior to the first mitotic division. Thus, multiple regulatory events irreversibly modulate Meikin activity during successive meiotic divisions to rewire the cell division machinery at two distinct transitions., Competing Interests: Declaration of interests I.M.C. is a member of the Editorial Advisory Board for Developmental Cell., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex 1 . Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD21 2-4 ). Separase is activated by degradation of its inhibitors, securin 5 and cyclin B 6 , but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans 7 and yeast 8 , human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK1 9,10 . Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine 6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Successful cell division relies on the timely removal of key cell cycle proteins such as securin. Securin inhibits separase, which cleaves the cohesin rings holding chromosomes together. Securin must be depleted before anaphase to ensure chromosome segregation occurs with anaphase. Here we find that in meiosis I, mouse oocytes contain an excess of securin over separase. We reveal a mechanism that promotes excess securin destruction in prometaphase I. Importantly, this mechanism relies on two phenylalanine residues within the separase-interacting segment (SIS) of securin that are only exposed when securin is not bound to separase. We suggest that these residues facilitate the removal of non-separase-bound securin ahead of metaphase, as inhibiting this period of destruction by mutating both residues causes the majority of oocytes to arrest in meiosis I. We further propose that cellular securin levels exceed the amount an oocyte is capable of removing in metaphase alone, such that the prometaphase destruction mechanism identified here is essential for correct meiotic progression in mouse oocytes., (© 2021. The Author(s).)

The conserved meiosis-specific kinetochore regulator, meikin (Moa1 in fission yeast) plays a central role in establishing meiosis-specific kinetochore function. However, the underlying molecular mechanisms remain elusive. Here, we show how Moa1 regulates centromeric cohesion protection, a function that has been previously attributed to shugoshin (Sgo1). Moa1 is known to associate with Plo1 kinase. We explore Plo1-dependent Rec8 phosphorylation and identify a key phosphorylation site required for cohesion protection. The phosphorylation of Rec8 by Moa1-Plo1 potentiates the activity of PP2A associated with Sgo1. This leads to dephosphorylation of Rec8 at another site, which thereby prevents cleavage of Rec8 by separase., (© 2021 Ma et al.; Published by Cold Spring Harbor Laboratory Press.)

During mitotic chromosome segregation, the protease separase severs cohesin between sister chromatids. A probe for separase activity has shown that separase undergoes abrupt activation shortly before anaphase onset, after being suppressed throughout metaphase; however, the relevance of this control remains unclear. Here, we report that separase activates precociously, with respect to anaphase onset, during prolonged metaphase in multiple types of cancer cell lines. The artificial extension of metaphase in chromosomally stable diploid cells leads to precocious activation and, subsequently, to chromosomal bridges in anaphase, which seems to be attributable to incomplete cohesin removal. Conversely, shortening back of a prolonged metaphase restores the activation of separase and ameliorates anaphase bridge formation. These observations suggest that retarded metaphase progression affects the separase activation profile and its enzymatic proficiency. Our findings provide an unanticipated etiology for chromosomal instability in cancers and underscore the relevance of swift mitotic transitions for fail-safe chromosome segregation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Separase is a large cysteine protease in eukaryotes and has crucial roles in many cellular processes, especially chromosome segregation during mitosis and meiosis, apoptosis, DNA damage repair, centrosome disengagement and duplication, spindle stabilization and elongation. It dissolves the cohesion between sister chromatids by cleaving one of the subunits of the cohesin ring for chromosome segregation. The activity of separase is tightly controlled at many levels, through direct binding of inhibitory proteins as well as posttranslational modification. Dysregulation of separase activity is linked to cancer and genome instability, making it a target for drug discovery. One of the best-known inhibitors of separase is securin, which has been identified in yeast, plants, and animals. Securin forms a tight complex with separase and potently inhibits its catalytic activity. Recent structures of the separase-securin complex have revealed the molecular mechanism for the inhibitory activity of securin. A segment of securin is bound in the active site of separase, thereby blocking substrate binding. Securin itself is not cleaved by separase as its binding mode is not compatible with catalysis. Securin also has extensive interactions with separase outside the active site, consistent with its function as a chaperone to stabilize this enzyme.

Objective: Endometrial cancer (EC) is one of the most common malignant gynaecological tumours worldwide. This study was aimed at identifying EC prognostic genes and investigating the molecular mechanisms of these genes in EC., Methods: Two mRNA datasets of EC were downloaded from the Gene Expression Omnibus (GEO). The GEO2R tool and Draw Venn Diagram were used to identify differentially expressed genes (DEGs) between normal endometrial tissues and EC tissues. Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Next, the protein-protein interactions (PPIs) of these DEGs were determined by the Search Tool for the Retrieval of Interacting Genes (STRING) tool and Cytoscape with Molecular Complex Detection (MCODE). Furthermore, Kaplan-Meier survival analysis was performed by UALCAN to verify genes associated with significantly poor prognosis. Next, Gene Expression Profiling Interactive Analysis (GEPIA) was used to verify the expression levels of these selected genes. Additionally, a reanalysis of the KEGG pathways was performed to understand the potential biological functions of selected genes. Finally, the associations between these genes and clinical features were analysed based on TCGA cancer genomic datasets for EC., Results: In EC tissues, compared with normal endometrial tissues, 147 of 249 DEGs were upregulated and 102 were downregulated. A total of 64 upregulated genes were assembled into a PPI network. Next, 14 genes were found to be both associated with significantly poor prognosis and highly expressed in EC tissues. Reanalysis of the KEGG pathways found that three of these genes were enriched in the cell cycle pathway. TTK , CDC25A , and ESPL1 showed higher expression in cancers with late stage and higher tumour grade., Conclusion: In summary, through integrated bioinformatics approaches, we found three significant prognostic genes of EC, which might be potential therapeutic targets for EC patients., Competing Interests: The authors have no conflicts of interests., (Copyright © 2020 Qiannan Yang et al.)

Mammalian cyclin A1 is prominently expressed in testis and essential for meiosis in the male mouse, however, it shows weak expression in ovary, especially during oocyte maturation. To understand why cyclin A1 behaves in this way in the oocyte, we investigated the effect of cyclin A1 overexpression on mouse oocyte meiotic maturation. Our results revealed that cyclin A1 overexpression triggered meiotic resumption even in the presence of germinal vesicle breakdown inhibitor, milrinone. Nevertheless, the cyclin A1-overexpressed oocytes failed to extrude the first polar body but were completely arrested at metaphase I. Consequently, cyclin A1 overexpression destroyed the spindle morphology and chromosome alignment by inducing premature separation of chromosomes and sister chromatids. Therefore, cyclin A1 overexpression will prevent oocyte maturation although it can promote meiotic resumption. All these results show that decreased expression of cyclin A1 in oocytes may have an evolutional significance to keep long-lasting prophase arrest and orderly chromosome separation during oocyte meiotic maturation., (© 2020 Wiley Periodicals, Inc.)

The DNA damage checkpoint halts cell cycle progression in G2 in response to genotoxic insults. Central to the execution of cell cycle arrest is the checkpoint-induced stabilization of securin-separase complex (yeast Pds1-Esp1). The checkpoint kinases Chk1 and Chk2 (yeast Chk1 and Rad53) are thought to critically contribute to the stability of securin-separase complex by phosphorylation of securin, rendering it resistant to proteolytic destruction by the anaphase promoting complex (APC). Dun1, a Rad53 paralog related to Chk2, is also essential for checkpoint-imposed arrest. Dun1 is required for the DNA damage-induced transcription of DNA repair genes; however, its role in the execution of cell cycle arrest remains unknown. Here, we show that Dun1's role in checkpoint arrest is independent of its involvement in the transcription of repair genes. Instead, Dun1 is necessary to prevent Pds1 destruction during DNA damage in that the Dun1-deficient cells degrade Pds1, escape G2 arrest and undergo mitosis despite the presence of checkpoint-active Chk1 and Rad53. Interestingly, proteolytic degradation of Pds1 in the absence of Dun1 is mediated not by APC but by the HECT domain-containing E3 ligase Rsp5. Our results suggest a regulatory scheme in which Dun1 prevents chromosome segregation during DNA damage by inhibiting Rsp5-mediated proteolytic degradation of securin Pds1., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tetraploidy, a common feature in cancer, results in the presence of extra centrosomes, which has been associated with chromosome instability (CIN) and aneuploidy. Deregulation in the number of centrosomes triggers tumorigenesis. However, how supernumerary centrosomes evolve during the emergence of tetraploid cells remains yet to be elucidated. Here, generating tetraploid isogenic clones in colorectal cancer and in non-transformed cells, we show that near-tetraploid clones exhibit a significant increase in the number of centrosomes. Moreover, we find that centrosome area in near-tetraploids is twice as large as in near-diploids. To evaluate whether centrosome clustering was occurring, we next analysed the number of centrioles revealing centriole amplification. Notwithstanding, more than half of the near-tetraploids maintained in culture do not present centrosome aberrations. To test whether cells progressively lost centrioles after becoming near-tetraploid, we transiently transfected diploid cells with siRNA against ESPL1/Separase, a protease responsible for triggering anaphase, to generate newly near-tetraploid cells. Finally, using this model, we assessed the number of centrioles at different time-points after tetraploidization finding that near-tetraploids rapidly lose centrosomes over time. Taken together, these data demonstrate that although most cells reduce supernumerary centrosomes after tetraploidization, a small fraction retains extra centrioles, potentially resulting in CIN.

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34 + cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34 + cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34 + cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

Timely sister chromatid separation, promoted by separase, is essential for faithful chromosome segregation. Separase is a member of the CD clan of cysteine proteases, which also includes the pro-apoptotic enzymes known as caspases. We report a role for the C. elegans separase SEP-1, primarily known for its essential activity in cell division and cortical granule exocytosis, in developmentally programmed cell death when the predominant pro-apoptotic caspase CED-3 is compromised. Loss of SEP-1 results in extra surviving cells in a weak ced-3(-) mutant, and suppresses the embryonic lethality of a mutant defective for the apoptotic suppressor ced-9/Bcl-2 implicating SEP-1 in execution of apoptosis. We also report apparent non-apoptotic roles for CED-3 in promoting germ cell proliferation, meiotic chromosome disjunction, egg shell formation, and the normal rate of embryonic development. Moreover, loss of the soma-specific (CSP-3) and germline-specific (CSP-2) caspase inhibitors result in CED-3-dependent suppression of embryonic lethality and meiotic chromosome non-disjunction respectively, when separase function is compromised. Thus, while caspases and separases have evolved different substrate specificities associated with their specialized functions in apoptosis and cell division respectively, they appear to have retained the residual ability to participate in both processes, supporting the view that co-option of components in cell division may have led to the innovation of programmed cell suicide early in metazoan evolution.

Prolonged mitosis often results in apoptosis 1 . Shortened mitosis causes tumorigenic aneuploidy, but it is unclear whether it also activates the apoptotic machinery 2 . Separase, a cysteine protease and trigger of all eukaryotic anaphases, has a caspase-like catalytic domain but has not previously been associated with cell death 3,4 . Here we show that human cells that enter mitosis with already active separase rapidly undergo death in mitosis owing to direct cleavage of anti-apoptotic MCL1 and BCL-XL by separase. Cleavage not only prevents MCL1 and BCL-XL from sequestering pro-apoptotic BAK, but also converts them into active promoters of death in mitosis. Our data strongly suggest that the deadliest cleavage fragment, the C-terminal half of MCL1, forms BAK/BAX-like pores in the mitochondrial outer membrane. MCL1 and BCL-XL are turned into separase substrates only upon phosphorylation by NEK2A. Early mitotic degradation of this kinase is therefore crucial for preventing apoptosis upon scheduled activation of separase in metaphase. Speeding up mitosis by abrogation of the spindle assembly checkpoint results in a temporal overlap of the enzymatic activities of NEK2A and separase and consequently in cell death. We propose that NEK2A and separase jointly check on spindle assembly checkpoint integrity and eliminate cells that are prone to chromosome missegregation owing to accelerated progression through early mitosis.

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 cohesin 1-3 . 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 securin 1 . In the absence of securin, mammalian chromosomes still segregate on schedule, but it is unclear how separase is regulated under these conditions 4,5 . Here we show that human shugoshin 2 (SGO2), an essential protector of meiotic cohesin with unknown functions in the soma 6,7 , 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 securin 8,9 , 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 p31 comet 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.

Aim: It has been shown that the integration of hepatitis B virus (HBV) gene into the host genome is a high-risk factor for development of hepatocellular carcinoma (HCC). However, the relationship between HBV S-integrated human extra spindle pole bodies-like 1 (ESPL1) gene and HCC is unknown. This study was designed to detect HBV S-integrated human ESPL1 fusion gene in patients with HCC for potentially using this fusion gene as a biomarker for HCC diagnosis., Patients and Methods: Nineteen and 70 patients with chronic hepatitis B (CHB) were recruited to the experimental and control groups, respectively, and both groups underwent an effective nucleoside/nucleotide analog therapy and follow-up for HCC occurrence for up to 11 years. HCC tissues were obtained by surgical resection from the experimental group, while liver tissues were collected by liver biopsy in the control group prior to treatment with nucleoside/nucleotide analogs. Alu polymerase chain reaction was used to assess HBV S gene integration in the liver tissues from both groups. HBV S-integrated human ESPL1 fusion gene was then detected in patients with HBV S gene integration using a gene database., Results: All patients in the experimental group developed HCC, whereas no HCC was diagnosed in the control group. HBV S gene integration was identified in 12 out of 19 HCC tissues in the experimental group, giving a detection rate of 63.2%, which was significantly greater than that of 15.7% (11/70) in the control group (p<0.001). We further showed that HBV S-integrated human ESPL1 fusion gene was detected in eight patients (rate of 66.7%) among the 12 patients with HCC with HBV S gene integration in the experimental group, whereas the fusion gene was not detectable in any of the patients in the control group (p=0.001)., Conclusion: This research demonstrates a high detection rate of HBV S-integrated human ESPL1 fusion gene in patients with HBV-related HCC and shows that this fusion gene appears to be associated with HCC development in patients with CHB. These findings suggest that HBV S-integrated human ESPL1 fusion gene may potentially serve as a biomarker for early detection of HCC in HBV-infected populations., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Chromosome segregation is driven by separase, activity of which is inhibited by binding to securin and cyclin B1/CDK1. In meiosis, premature separase activity will induce aneuploidy or abolish chromosome segregation owing to the untimely destruction of cohesin. Recently, we have proved that cyclin B2 can compensate for cyclin B1 in CDK1 activation for the oocyte meiosis G2/M transition. In the present study, we identify an interaction between cyclin B2/CDK1 and separase in mouse oocytes. We find that cyclin B2 degradation is required for separase activation during the metaphase I-anaphase I transition because the presence of stable cyclin B2 leads to failure of homologous chromosome separation and to metaphase I arrest, especially in the simultaneous absence of securin and cyclin B1. Moreover, non-phosphorylatable separase rescues the separation of homologous chromosomes in stable cyclin B2-arrested cyclin B1-null oocytes. Our results indicate that cyclin B2/CDK1 is also responsible for separase inhibition via inhibitory phosphorylation to regulate chromosome separation in oocyte meiosis, which may not occur in other cell types., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Chromosome segregation begins when the cysteine protease, separase, cleaves the Scc1 subunit of cohesin at the metaphase-to-anaphase transition. Separase is inhibited prior to metaphase by the tightly bound securin protein, which contains a pseudosubstrate motif that blocks the separase active site. To investigate separase substrate specificity and regulation, here we develop a system for producing recombinant, securin-free human separase. Using this enzyme, we identify an LPE motif on the Scc1 substrate that is distinct from the cleavage site and is required for rapid and specific substrate cleavage. Securin also contains a conserved LPE motif, and we provide evidence that this sequence blocks separase engagement of the Scc1 LPE motif. Our results suggest that rapid cohesin cleavage by separase requires a substrate docking interaction outside the active site. This interaction is blocked by securin, providing a second mechanism by which securin inhibits cohesin cleavage.

Exit from mitosis and completion of cytokinesis require the inactivation of mitotic cyclin-dependent kinase (Cdk) activity. In budding yeast, Cdc14 phosphatase is a key mitotic regulator that is activated in anaphase to counteract Cdk activity. In metaphase, Cdc14 is kept inactive in the nucleolus, where it is sequestered by its inhibitor, Net1. At anaphase onset, downregulation of PP2A Cdc55 phosphatase by separase and Zds1 protein promotes Net1 phosphorylation and, consequently, Cdc14 release from the nucleolus. The mechanism by which PP2A Cdc55 activity is downregulated during anaphase remains to be elucidated. Here, we demonstrate that Cdc55 regulatory subunit is phosphorylated in anaphase in a Cdk1-Clb2-dependent manner. Interestingly, cdc55-ED phosphomimetic mutant inactivates PP2A Cdc55 phosphatase activity towards Net1 and promotes Cdc14 activation. Separase and Zds1 facilitate Cdk-dependent Net1 phosphorylation and Cdc14 release from the nucleolus by modulating PP2A Cdc55 activity via Cdc55 phosphorylation. In addition, human Cdk1-CyclinB1 phosphorylates human B55, indicating that the mechanism is conserved in higher eukaryotes.

Proper chromosome segregation is crucial for maintaining genomic stability and dependent on separase, a conserved and essential cohesin protease. Securins are key regulators of separases, but remain elusive in many organisms due to sequence divergence. Here, we demonstrate that the separase homologue Esp1p in the ascomycete Candida albicans , an important pathogen of humans, is essential for chromosome segregation . However, C. albicans lacks a sequence homologue of securins found in model ascomycetes. We sought a functional homologue through identifying Esp1p interacting factors. Affinity purification of Esp1p and mass spectrometry revealed E sp1p- I nteracting P rotein 1 (Eip1p)/Orf19.955p, an uncharacterized protein specific to Candida species. Functional analyses demonstrated that Eip1p is important for chromosome segregation but not essential, and modulated in an APC Cdc20 -dependent manner, similar to securins. Eip1p is strongly enriched in response to methyl methanesulfate (MMS) or hydroxyurea (HU) treatment, and its depletion partially suppresses an MMS or HU-induced metaphase block. Further, Eip1p depletion reduces Mcd1p/Scc1p, a cohesin subunit and separase target. Thus, Eip1p may function as a securin. However, other defects in Eip1p-depleted cells suggest additional roles. Overall, the results introduce a candidate new securin, provide an approach for identifying these divergent proteins, reveal a putative anti-fungal therapeutic target, and highlight variations in mitotic regulation in eukaryotes.

Chromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments., Competing Interests: The authors have declared that no competing interests exist.

Acetic acid tolerance of the yeast Saccharomyces cerevisiae is manifested in several quantifiable parameters, of which the duration of the latency phase is one of the most studied. It has been shown recently that the latter parameter is mostly determined by a fraction of cells within the population that resumes proliferation upon exposure to acetic acid. The aim of the current study was to identify genetic determinants of the difference in this parameter between the highly tolerant strain MUCL 11987-9 and the laboratory strain CEN.PK113-7D. To this end, a combination of genetic mapping and pooled-segregant RNA sequencing was applied as a new approach. The genetic mapping data revealed four loci with a strong linkage to strain MUCL 11987-9, each containing still a large number of genes making the identification of the causal ones by traditional methods a laborious task. The genes were therefore prioritized by pooled-segregant RNA sequencing, which resulted in the identification of six genes within the identified loci showing differential expression. The relevance of the prioritized genes for the phenotype was verified by reciprocal hemizygosity analysis. Our data revealed the genes ESP1 and MET22 as two, so far unknown, genetic determinants of the size of the fraction of cells resuming proliferation upon exposure to acetic acid.

Separase halves eukaryotic chromosomes in M-phase by cleaving cohesin complexes holding sister chromatids together. Whether this essential protease functions also in interphase and/or impacts carcinogenesis remains largely unknown. Here, we show that mammalian separase is recruited to DNA double-strand breaks (DSBs) where it is activated to locally cleave cohesin and facilitate homology-directed repair (HDR). Inactivating phosphorylation of its NES, arginine methylation of its RG-repeats, and sumoylation redirect separase from the cytosol to DSBs. In vitro assays suggest that DNA damage response-relevant ATM, PRMT1, and Mms21 represent the corresponding kinase, methyltransferase, and SUMO ligase, respectively. SEPARASE heterozygosity not only debilitates HDR but also predisposes primary embryonic fibroblasts to neoplasia and mice to chemically induced skin cancer. Thus, tethering of separase to DSBs and confined cohesin cleavage promote DSB repair in G2 cells. Importantly, this conserved interphase function of separase protects mammalian cells from oncogenic transformation., (© 2018 The Authors.)

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

The cysteine protease separase opens the cohesin ring by cleaving its kleisin subunit and is a pivotal cell cycle factor for the transition from metaphase to anaphase. It is inhibited by forming a complex with the chaperone securin, and in vertebrates, also by the Cdk1-cyclin B1 complex. Separase is activated upon the destruction of securin or cyclin B1 by the proteasome, after ubiquitination by the anaphase-promoting complex/cyclosome (APC/C). Here we review recent structures of the active protease segment of Chaetomium thermophilum separase in complex with a substrate-mimic inhibitor and full-length Saccharomyces cerevisiae and Caenorhabditis elegans separase in complex with securin. These structures define the mechanism for substrate recognition and catalysis by separase, and show that securin has extensive contacts with separase, consistent with its chaperone function. They confirm that securin inhibits separase by binding as a pseudo substrate., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anaphase onset is an irreversible cell cycle transition that is triggered by the activation of the protease Separase. Separase cleaves the Mcd1 (also known as Scc1) subunit of Cohesin, a complex of proteins that physically links sister chromatids, triggering sister chromatid separation. Separase is regulated by the degradation of the anaphase inhibitor Securin which liberates Separase from inhibitory Securin/Separase complexes. In many organisms, Securin is not essential suggesting that Separase is regulated by additional mechanisms. In this work, we show that in budding yeast Cdk1 activates Separase (Esp1 in yeast) through phosphorylation to trigger anaphase onset. Esp1 activation is opposed by protein phosphatase 2A associated with its regulatory subunit Cdc55 (PP2ACdc55) and the spindle protein Slk19. Premature anaphase spindle elongation occurs when Securin (Pds1 in yeast) is inducibly degraded in cells that also contain phospho-mimetic mutations in ESP1, or deletion of CDC55 or SLK19. This striking phenotype is accompanied by advanced degradation of Mcd1, disruption of pericentric Cohesin organization and chromosome mis-segregation. Our findings suggest that PP2ACdc55 and Slk19 function redundantly with Pds1 to inhibit Esp1 within pericentric chromatin, and both Pds1 degradation and Cdk1-dependent phosphorylation of Esp1 act together to trigger anaphase onset.

Separase is a highly conserved protease required for chromosome segregation. Although observations that separase also regulates membrane trafficking events have been made, it is still not clear how separase achieves this function. Here, we present an extensive ENU mutagenesis suppressor screen aimed at identifying suppressors of sep-1(e2406) , a temperature-sensitive maternal effect embryonic lethal separase mutant that primarily attenuates membrane trafficking rather than chromosome segregation. We screened nearly a million haploid genomes and isolated 68 suppressed lines. We identified 14 independent intragenic sep-1(e2406) suppressed lines. These intragenic alleles map to seven SEP-1 residues within the N-terminus, compensating for the original mutation within the poorly conserved N-terminal domain. Interestingly, 47 of the suppressed lines have novel mutations throughout the entire coding region of the pph-5 phosphatase, indicating that this is an important regulator of separase. We also found that a mutation near the MEEVD motif of HSP-90, which binds and activates PPH-5, also rescues sep-1(e2406) mutants. Finally, we identified six potentially novel suppressor lines that fall into five complementation groups. These new alleles provide the opportunity to more exhaustively investigate the regulation and function of separase., (Copyright © 2018 Melesse et al.)

ESPL1/separase, a cysteine endopeptidase, is a key player in centrosome duplication and mitotic sister chromatid separation. Aberrant expression and/or altered separase proteolytic activity are associated with centrosome amplification, aneuploidy, tumorigenesis and disease progression. Since centrosome alterations are a common and early detectable feature in patients with myelodysplastic syndrome (MDS) and cytogenetic aberrations play an important role in disease risk stratification, we examined separase activity on single cell level in 67 bone marrow samples obtained from patients with MDS, secondary acute myeloid leukemia (sAML), de novo acute myeloid leukemia (AML) and healthy controls by a flow cytometric separase activity assay. The separase activity distribution (SAD) value, a calculated measure for the occurrence of cells with prominent separase activity within the analyzed sample, was tested for correlation with the centrosome, karyotype and gene mutation status. We found higher SAD values in bone marrow cells of sAML patients than in corresponding cells of MDS patients. This concurred with an increased incidence of aberrant centrosome phenotypes in sAML vs. MDS samples. No correlation was found between SAD values and the karyotype/gene mutation status. During follow-up of four MDS patients we observed increasing SAD values after transformation to sAML, in two patients SAD values decreased during azacitidine therapy. Cell culture experiments employing MDS-L cells as an in vitro model of MDS revealed that treatment with rigosertib, a PLK1 inhibitor and therapeutic drug known to induce G2/M arrest, results in decreased SAD values. In conclusion, the appearance of cells with unusual high separase activity levels, as indicated by increased SAD values, concurs with the transformation of MDS to sAML and may reflect separase dysregulation potentially contributing to clonal evolution during MDS progression. Separase activity measurement may therefore be useful as a novel additional molecular marker for disease monitoring.

Proper chromosome segregation is crucial for preserving genomic integrity, and errors in this process cause chromosome mis-segregation, which may contribute to cancer development. Sister chromatid separation is triggered by Separase, an evolutionary conserved protease that cleaves the cohesin complex, allowing the dissolution of sister chromatid cohesion. Here we provide evidence that Separase participates in genomic stability maintenance by controlling replication fork speed. We found that Separase interacted with the replication licensing factors MCM2-7, and genome-wide data showed that Separase co-localized with MCM complex and cohesin. Unexpectedly, the depletion of Separase increased the fork velocity about 1.5-fold and caused a strong acetylation of cohesin's SMC3 subunit and altered checkpoint response. Notably, Separase silencing triggered genomic instability in both HeLa and human primary fibroblast cells. Our results show a novel mechanism for fork progression mediated by Separase and thus the basis for genomic instability associated with tumorigenesis., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Separation of sister chromatids is a drastic and irreversible step in the cell cycle. The key biochemistry behind this event is the proteolysis mediated by the ubiquitin ligase called the anaphase promoting complex, or APC/C. Securin and cyclin B1 are the two established substrates for APC/C whose degradation releases separase and inactivates cyclin B1-dependent kinase 1 (cdk1), respectively, at the metaphase-to-anaphase transition. In this study, we have combined biochemical quantifications with mathematical simulations to characterize the kinetic regulation of securin and cyclin B1, in the cytoplasmic and chromosomal compartments, and found that they are differentially distributed and degraded with different rates. Modeling their interaction with separase predicted that activation timing of separase well coincides with the decline of securin-separase concentration in the cytoplasm. Notably, it also coincides with the peak of cyclin B1-separase level on chromosomes, which appeared crucial to coordinate the timing for separase activation and cdk1 inhibition. We have also conducted phosphoproteomic analysis and identified Ki67 as a chromosomal cdk1 substrate whose dephosphorylation is facilitated by cyclin B1-separase interaction in anaphase.

Separase proteolytically removes cohesin complexes from sister chromatid arms in meiosis I, which is essential for chromosome segregation. Regulation of separase activity is essential for proper cell cycle progression and correct chromosome segregation. Onset of endogenous separase activity has not yet been observed in live oocytes.We describe here a method for detecting separase activity in mouse oocytes in vivo. This method utilizes a previously described cleavage sensor made up of H2B-mCherry fused with Scc1(107-268 aa)-YFP. The cleavage sensor is loaded on the chromosomes through its H2B-tag, and the signal from both mCherry and YFP is visible. Upon separase activation the Scc1 fragment is cleaved and YFP dissociates from the chromosomes. The change in the ratio between mCherry and YFP fluorescence intensity is a readout of separase activity.

Cohesins establish sister chromatid cohesion during S phase and are removed when cohesin Scc1 is cleaved by separase at anaphase onset. During this process, cohesin Smc3 undergoes a cycle of acetylation: Smc3 acetylation by Eco1 in S phase stabilizes cohesin association with chromosomes, and its deacetylation by Hos1 in anaphase allows re-use of Smc3 in the next cell cycle. Here we find that Smc3 deacetylation by Hos1 has a more immediate effect in the early anaphase of budding yeast. Hos1 depletion significantly delayed sister chromatid separation and segregation. Smc3 deacetylation facilitated removal of cohesins from chromosomes without changing Scc1 cleavage efficiency, promoting dissolution of cohesion. This action is probably due to disengagement of Smc1-Smc3 heads prompted by de-repression of their ATPase activity. We suggest Scc1 cleavage per se is insufficient for efficient dissolution of cohesion in early anaphase; subsequent Smc3 deacetylation, triggered by Scc1 cleavage, is also required., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)