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Analysis of Drp 1 Receptors Important for Mitochondrial Fission

对线粒体裂变重要的 Drp 1 受体分析

基本信息

批准号：
9197655
负责人：
David C Chan
金额：
$ 36.13万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-04-01 至 2017-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9197655
关键词：
Affect Apoptosis Autophagocytosis Autosomal Dominant Optic Atrophy Binding Binding Sites Biochemical Biological Biological Assay Caliber Catalysis Cell Culture Techniques Cell Line Cells Cellular Assay Charcot-Marie-Tooth Disease Complex Crystallization Cytosol Defect Dimerization Dominant-Negative Mutation Dynamin Failure Functional disorder Genes Genetic Goals Health Human Kinetics Knock-out Lead Maps Measures Mediating Membrane Membrane Proteins Methods Microcephaly Mitochondria Molecular Monitor Morphology Mus Neonatal Neurodegenerative Disorders Neurons Nucleotides Organelles Outer Mitochondrial Membrane Population Pregnancy Process Proteins Recruitment Activity Regulation Resolution Specificity Structure Surface System Technology Testing Thermodynamics Transferase Work X-Ray Crystallography base cell type chemical property design dimer genetic analysis genetic approach genome editing human disease insight mitochondrial dysfunction monomer mutant programs public health relevance receptor segregation structural biology

项目摘要

DESCRIPTION (provided by applicant): In the past decade, mitochondrial fusion and fission have emerged as major regulators of mitochondrial function. Defects in these processes are associated with human disease, and mouse studies indicate their importance in maintaining a healthy mitochondrial population within cells. This proposal focuses on the mechanism of mitochondrial fission, which is implicated in control of mitochondrial morphology, degradation of mitochondria by autophagy, control of mitochondrial distribution, and regulation of apoptosis. During the process of mitochondrial fission, Drp1 (dynamin-related protein 1) is recruited from the cytosol onto the mitochondrial surface. Here it assembles into an oligomeric complex that wraps around the mitochondrial tubule and constricts it to mediate membrane scission. In order for Drp1 to be recruited to mitochondria, the mitochondrial outer membrane contains four Drp1 receptors: Fis1, Mff, MiD49, and MiD51. Cellular studies indicate that the latter three molecules are the most functionally important for mitochondrial fission. This proposal focuses on obtaining a structural, biochemical, and cellular understanding of how these molecules mediate Drp1 recruitment. In Aim 1, X-ray crystallography is used to obtain atomic structures of Mff, MiD49, and MiD51. Current work has already yielded a high-resolution structure of MiD51. Surprisingly, this structure indicates that MiD51 has a nucleotidyl transferase domain, with conservation of key residues that are implicated in nucleotide binding and catalysis. Additional structures of MiD51 in complex with nucleotide will be obtained. In Aim 2, biochemical studies are used to advance the understanding of MiD51 and MiD49 function. Given that MiD51 has a nucleotidyl transferase fold, the nucleotide binding specificity of MiD51 will be measured with quantitative binding assays. In addition, enzymatic assays will be used to determine if MiD51 has a catalytic function in nucleotide transfer. Similar studies will be done with MiD49, which is homologous to MiD51. In Aim 3, structure-function analysis in cell culture is used to determine whether nucleotide binding, catalysis, or dimerization is important for the ability of MiD51 to recruit Drp and mediate fission. MiD51 and MiD49 knockout cells for further structure-function analysis will be generated through the use of genome editing technology. Finally, a genetic approach will be utilized to map the Drp1 binding site on Mff, MiD49, and MiD51. Taken together, these studies will greatly advance the understanding of the structural biology of Drp1 receptors and may ultimately lead to new methods to modulate mitochondrial fission. 1

描述（由申请人提供）：在过去的十年中，线粒体融合和裂变已成为线粒体功能的主要调节因子。这些过程中的缺陷与人类疾病有关，小鼠研究表明它们在维持细胞内健康的线粒体群方面的重要性。该提案重点关注线粒体裂变的机制，该机制涉及线粒体形态的控制、自噬降解线粒体、线粒体分布的控制和细胞凋亡的调节。在线粒体分裂过程中，Drp1（动力相关蛋白 1）从胞质溶胶募集到线粒体表面。在这里，它组装成寡聚复合物，包裹线粒体小管并使其收缩以介导膜分裂。为了将 Drp1 募集到线粒体，线粒体外膜包含四种 Drp1 受体：Fis1、Mff、MiD49 和 MiD51。细胞研究表明，后三种分子对于线粒体裂变功能最重要。该提案的重点是获得对这些分子如何介导 Drp1 募集的结构、生化和细胞理解。在目标 1 中，使用 X 射线晶体学获得 Mff、MiD49 和 MiD51 的原子结构。目前的工作已经产生了 MiD51 的高分辨率结构。令人惊讶的是，该结构表明 MiD51 具有核苷酸基转移酶结构域，并且保留了与核苷酸结合和催化有关的关键残基。将获得与核苷酸复合的MiD51的额外结构。在目标 2 中，生物化学研究用于增进对 MiD51 和 MiD49 功能的理解。鉴于 MiD51 具有核苷酸转移酶折叠，将通过定量结合测定来测量 MiD51 的核苷酸结合特异性。此外，酶测定将用于确定 MiD51 是否在核苷酸转移中具有催化功能。类似的研究也将针对与 MiD51 同源的 MiD49 进行。在目标 3 中，细胞培养物中的结构功能分析用于确定核苷酸结合、催化或二聚化对于 MiD51 募集 Drp 和介导裂变的能力是否重要。将通过使用基因组编辑技术生成用于进一步结构功能分析的 MiD51 和 MiD49 敲除细胞。最后，将利用遗传方法绘制 Mff、MiD49 和 MiD51 上的 Drp1 结合位点图谱。总而言之，这些研究将极大地促进对 Drp1 受体结构生物学的理解，并可能最终带来调节线粒体裂变的新方法。 1