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Role of Calpain-7 in the Abscission Checkpoint

Calpain-7 在脱落检查点中的作用

基本信息

批准号：
10434700
负责人：
Elliott Paine
金额：
$ 3.83万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
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/10434700
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 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

项目摘要

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.

项目总结: