Analysis of the mitotic activator separase

有丝分裂激活剂分离酶的分析

• 批准号: 7593725
• 负责人: Orna Cohen-Fix
• 金额: $ 19.43万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7593725
• 关键词: Active Sites; Affect; Alleles; Binding; Cell Cycle Progression; Cell Line; Cell Nucleus; Chromosome Segregation; Chromosomes; Cleaved cell; Collaborations; Complex; Condition; Cytoplasm; Defect; Endopeptidases; Eukaryota; Eukaryotic Cell; Genetic; Goals; Knowledge; Lead; Life; Link; Masks; Meiosis; Microtubules; Mitosis; Mitotic; Molecular Chaperones; Mus; Mutation; Nuclear; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphorylation; Process; Protease Domain; Protein Region; Proteins; Regulation; Role; Saccharomycetales; Schedule; Sister Chromatid; Tertiary Protein Structure; Work; Yeasts; base; cell fixing; cell type; cohesin; cohesion; falls; human PTTG1 protein; inhibitor/antagonist; mutant; protein degradation; segregation; separase; tool

Separase (Esp1 in budding yeast) is a conserved protease that is necessary for chromosome segregation. Mice lacking separase function fail to survive and cell lines in which separase is inactivated show gross chromosome mis-segregation. During mitosis and meiosis, separase cleaves one of the subunits of the cohesin complex that links the two sister chromatids together. This cleavage leads to the dissolution of cohesion, allowing the sister chromatids to be pulled apart by the spindle microtubules. Separase is regulated at multiple levels, including phosphorylation, auto-cleavage, and sub-cellular localization. In addition, the activity of separase is regulated by an inhibitor, called securin (Pds1 in budding yeast), which binds to separase and blocks its active site. Evidence from both yeast and higher eukaryotes suggests that securin/Pds1 is not only an inhibitor but that its binding to separase is needed for separase activation. The mechanism of separase activation by Pds1, or any other protein, is unknown. In this project, we aim to uncover the mechanisms for separase activation, and in particular we are focusing on proteins and pathways that control separase's nuclear localization. Previous work from our labs and others showed that Pds1 is needed for Esp1s nuclear localization, but it is not known if Esp1 enters the nucleus unaccompanied by Pds1 and then gets sequestered in the nucleus through Pds1 binding, or whether Pds1 shuttles in and out of the nucleus, interacting with Esp1 in the cytoplasm and promoting its nuclear localization. There is also evidence to suggest that other proteins are involved in Esp1's nuclear localization: in mutants lacking Pds1, Esp1 enters the nucleus on schedule, albeit at reduced levels, and in wild type cells Esp1 lingers in the nucleus after Pds1 is removed by protein degradation. To gain a better understanding of how separase is activated and to uncover proteins involved in the nuclear localization of the budding yeast Esp1, we have taken a two pronged approach. The first, which is being done in collaboration with Dr. Mark Winey, is a genetic based approach making use of several different esp1 mutant alleles. The mutations in these alleles all fall in different ESP1 regions and result in different mutant phenotypes in terms of cell cycle progression and conditions for inactivation. It should be noted that apart from the protease domain, the functions of most of the Esp1 protein domains are unknown. We are currently undertaking a high copy suppressor screen, the idea being that by over expressing proteins that normally interact with Esp1 or lead to its activation, we will be able to overcome the defects of the various mutant alleles. Indeed, so far we have isolated multiple suppressors; some of which are common to all esp1 alleles and some are allele specific. Some suppressors act as protein chaperons, some increase the levels of Esp1 and some suppress by a yet unknown mechanism. Using this approach we will identify proteins that contribute to Esp1 activation and further our knowledge of separase function in higher eukaryotes. To study the regulation of Esp1s nuclear localization we are currently developing tools to detect Esp1 in live and fixed cells. We will then use these tools to determine the protein regions of Esp1 that are necessary for its nuclear localization, either in the presence or absence of Pds1. We will then identify proteins that facilitate the nuclear localization of Esp1 and determine the precise role of Pds1 in this process.

分离酶（Esp 1）是一种保守的蛋白酶，是染色体分离所必需的。缺乏分离酶功能的小鼠无法存活，分离酶失活的细胞系显示出严重的染色体错误分离。在有丝分裂和减数分裂期间，分离酶切割将两个姐妹染色单体连接在一起的粘着蛋白复合体的一个亚基。这种分裂导致凝聚力的溶解，使姐妹染色单体被纺锤体微管拉开。分离酶在多个水平上受到调节，包括磷酸化、自切割和亚细胞定位。此外，分离酶的活性受到一种抑制剂的调节，这种抑制剂被称为securin（芽殖酵母中的Pds 1），它与分离酶结合并阻断其活性位点。 来自酵母和高等真核生物的证据表明，securin/Pds 1不仅是一种抑制剂，而且其与分离酶的结合是分离酶激活所必需的。Pds 1或任何其他蛋白质激活分离酶的机制尚不清楚。在这个项目中，我们的目标是揭示分离酶激活的机制，特别是我们专注于控制分离酶的核定位的蛋白质和途径。我们实验室和其他人以前的工作表明，Pds 1是Esp 1核定位所必需的，但目前尚不清楚Esp 1是否在没有Pds 1的陪同下进入细胞核，然后通过Pds 1结合被隔离在细胞核中，或者Pds 1是否穿梭进出细胞核，与细胞质中的Esp 1相互作用并促进其核定位。也有证据表明，其他蛋白质参与Esp 1的核定位：在缺乏Pds 1的突变体中，Esp 1按计划进入细胞核，尽管水平降低，而在野生型细胞中，Esp 1在Pds 1被蛋白质降解去除后徘徊在细胞核中。 为了更好地了解分离酶如何被激活并发现参与芽殖酵母Esp 1核定位的蛋白质，我们采取了双管齐下的方法。第一个是与Mark Winey博士合作完成的，是一种基于遗传的方法，利用了几种不同的esp 1突变等位基因。这些等位基因中的突变都落在不同的ESP 1区域，并在细胞周期进展和失活条件方面导致不同的突变表型。应该注意的是，除了蛋白酶结构域，大多数Esp 1蛋白结构域的功能是未知的。 我们目前正在进行高拷贝抑制筛选，其想法是通过过度表达通常与Esp 1相互作用或导致其激活的蛋白质，我们将能够克服各种突变等位基因的缺陷。事实上，到目前为止，我们已经分离出了多种抑制因子;其中一些是所有esp 1等位基因共有的，一些是等位基因特异性的。一些抑制因子作为蛋白伴侣，一些增加Esp 1的水平，一些通过未知的机制抑制。使用这种方法，我们将确定有助于Esp 1激活的蛋白质，并进一步了解分离酶在高等真核生物中的功能。 为了研究Esp 1 s核定位的调节，我们目前正在开发检测活细胞和固定细胞中Esp 1的工具。然后，我们将使用这些工具来确定Esp 1的蛋白质区域，无论是在存在或不存在Pds 1的情况下，这些区域对于Esp 1的核定位是必要的。 然后，我们将确定促进Esp 1的核定位的蛋白质，并确定Pds 1在这一过程中的确切作用。