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.

项目摘要： 当哺乳动物细胞准备分裂时，脱落检查点检查了残留有丝分裂错误和延迟 脱落直到满足检查站。然后细胞完成细胞因子，两个子细胞是 不可逆转地分开。最近的工作表明，脱落检查点的丢失导致积累 DNA损伤并与几种不同类型的人类癌症的倾向相关，表明 检查点起着重要的保护作用。尽管我们对检查站缺乏完全的理解 信号传导，很明显，运输所需的内体分类复合物（ESCRTS）是关键 检查点的监管目标。在细胞因子过程中，将ESCRT复合物募集到并在 分裂细胞之间的中体，它们在其中收缩膜并介导子细胞分离。 但是，这些ESCRT活动必须受到负调节，以防止脱落，直到检查站为 使满意。我们最近将蛋白酶calpain-7确定为一种新颖的基本检查点成分。在 初步研究，我们表征了Calpain-7 MIT结构域如何结合ESCRT-III蛋白IST1， 确定相关的Calpain-7 MIT-IST1复合物的结构，确定的点突变抑制了这一点 相互作用，表明IST1将Calpain-7募集到中体，并证明CALPAIN-7催化活性 需要进行ESCRT-III结合才能维持脱落检查点。我们现在建议获得 通过识别calpain-7的底物，对Calpain-7如何维护检查点的机械理解 蛋白水解和表征其功能（AIM 1）以及通过进行生化和结构分析的 Calpain-7法规（AIM 2）。 AIM 1中的实验将利用公正的基于近端的涡轮/质量 识别候选Calpain-7底物的光谱法方法。然后，这些候选人将通过 在体外测试Calpain-7处理，并表征其在脱落检查点维护中的作用 培养的细胞。 AIM 2中的实验将为Calpain-7调节提供结构和生化见解， 包括自身抑制的机制以及蛋白质寡聚在IST1结合和中体中的作用 招聘。这些研究将共同​​为监管网络提供重要的新机械见解 和中体组件，以响应有丝分裂错误而阻止细胞分裂。