Role of Calpain-7 in the Abscission Checkpoint

Calpain-7 在脱落检查点中的作用

• 批准号: 10214578
• 负责人: Elliott Paine
• 金额: $ 3.76万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10214578
• 关键词: Affinity; Affinity Chromatography; Binding; Biochemical; Biological Process; Biotin; C-terminal; Calpain; Caspase; Cell Cycle; Cell Line; Cell Proliferation; Cell Separation; Cell division; Cells; Chimeric Proteins; Cleaved cell; Code; Collaborations; Complex; Cultured Cells; Cytokinesis; DNA Damage; Data; Ensure; Excision; Filament; Genome; Genomics; Goals; Human; In Vitro; Label; Learning; Length; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Manuscripts; Mass Spectrum Analysis; Mediating; Membrane; Microtubules; Mitosis; Mitotic; Molecular; Neck; Peptide Hydrolases; Play; Point Mutation; Polymers; Predisposition; Preparation; Process; Proteins; Proteolysis; Reaction; Recruitment Activity; Regulation; Residual state; Role; Series; Signal Transduction; Sorting - Cell Movement; Specificity; Structure; Tail; Testing; Thinness; Time; Work; base; combinatorial; constriction; daughter cell; experimental study; insight; mutant; novel; prevent; protein transport; recruit; response; trafficking

Project Summary: As mammalian cells prepare to divide, the abscission checkpoint checks for residual mitotic errors and delays abscission until the checkpoint is satisfied. Cells then complete cytokinesis and the two daughter cells are irreversibly separated. Recent work has shown that loss of the abscission checkpoint leads to accumulation of DNA damage and correlates with a predisposition to several different types of human cancer, indicating the checkpoint plays an important protective role. Although we lack a complete understanding of checkpoint signaling, it is already clear that the Endosomal Sorting Complexes Required for Transport (ESCRTs) are a key regulatory target of the checkpoint. During cytokinesis, ESCRT complexes are recruited to and function in the midbody between the dividing cells, where they constrict the membrane and mediate daughter cell separation. However, these ESCRT activities must be negatively regulated to prevent abscission until the checkpoint is satisfied. We have recently identified the protease Calpain-7 as a novel and essential checkpoint component. In preliminary studies, we have characterized how the Calpain-7 MIT domain binds the ESCRT-III protein IST1, determined the structure of the relevant Calpain-7 MIT-IST1 complex, identified point mutations that inhibit this interaction, shown that IST1 recruits Calpain-7 to the midbody, and demonstrated that Calpain-7 catalytic activity and ESCRT-III binding are required to maintain the abscission checkpoint. We now propose to gain a mechanistic understanding of how Calpain-7 maintains the checkpoint by identifying substrates of Calpain-7 proteolysis and characterizing their functions (Aim 1) and by performing biochemical and structural analyses of Calpain-7 regulation (Aim 2). Experiments in Aim 1 will utilize an unbiased, proximity-based TurboID/mass spectrometry approach to identify candidate Calpain-7 substrates. These candidates will then be validated by testing for Calpain-7 processing in vitro and characterizing their roles in abscission checkpoint maintenance in cultured cells. Experiments in Aim 2 will provide structural and biochemical insights into Calpain-7 regulation, including the mechanism of autoinhibition and the role of protein oligomerization in IST1 binding and midbody recruitment. Together, these studies will provide important new mechanistic insights into the regulatory networks and midbody assemblies that arrest cell division in response to mitotic errors.

项目概要： 当哺乳动物细胞准备分裂时，分裂检查点检查残留的有丝分裂错误和延迟 直到检查点满足为止。然后细胞完成胞质分裂， 不可逆转的分离最近的研究表明，失去了发射检查点， DNA损伤，并与几种不同类型的人类癌症的易感性相关，表明 检查点起着重要的保护作用。尽管我们对检查点缺乏全面的了解， 在信号传导中，已经清楚的是，转运所需的内体分选复合物（ESCRT）是一个关键的信号传导途径。 检查站的监管目标。在胞质分裂期间，ESCRT复合物被募集到细胞中并在细胞中发挥作用。 在分裂细胞之间的中间体，在那里它们收缩膜并介导子细胞分离。 然而，这些ESCRT活动必须受到负调控，以防止在检查点被激活之前发生解离。 满意我们最近已经确定蛋白酶Calpain-7作为一种新的和必不可少的检查点组件。在 在初步研究中，我们已经表征了钙蛋白酶-7 MIT结构域如何结合ESCRT-III蛋白IST 1， 确定了相关的Calpain-7 MIT-IST 1复合物的结构，确定了抑制这种复合物的点突变， 相互作用，表明IST 1招募钙蛋白酶-7到中间体，并证明钙蛋白酶-7的催化活性， 和ESCRT-III结合是维持解离检查点所必需的。我们现在建议获得一个 通过识别钙蛋白酶-7的底物，了解钙蛋白酶-7如何维持检查点 蛋白水解和表征其功能（目的1），并通过进行生化和结构分析， 钙蛋白酶-7调节（目的2）。目标1中的实验将利用无偏的、基于邻近度的TurboID/mass 光谱方法鉴定候选钙蛋白酶-7底物。然后，这些候选人将通过 在体外测试Calpain-7的加工，并表征它们在维持细胞凋亡检查点中的作用。 培养细胞目标2中的实验将提供对钙蛋白酶-7调节的结构和生化见解， 包括自抑制机制和蛋白寡聚化在IST 1结合和中间体中的作用 招聘总之，这些研究将为调节网络提供重要的新机制见解 以及响应有丝分裂错误而阻止细胞分裂的中间体组装。