Analysis of Drp 1 Receptors Important for Mitochondrial Fission

描述（由申请人提供）：在过去的十年中，线粒体融合和分裂已经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体中的重要性。这一建议的重点是线粒体分裂的机制，这是牵连在线粒体形态的控制，线粒体的自噬降解，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，Drp 1（动力蛋白相关蛋白1）从细胞质中被募集到线粒体表面。在这里，它组装成一个低聚复合物，包裹在线粒体小管周围，并使其收缩以介导膜断裂。为了将Drp 1募集到线粒体，线粒体外膜含有四种Drp 1受体：Fis 1，Mff，MiD 49和MiD 51。细胞研究表明，后三种分子对线粒体分裂功能最重要。该提案的重点是获得这些分子如何介导Drp 1招募的结构，生物化学和细胞理解。在目的1中，使用X射线晶体学来获得Mff、MiD 49和MiD 51的原子结构。目前的工作已经产生了MiD 51的高分辨率结构。令人惊讶的是，该结构表明MiD 51具有核苷酸转移酶结构域，其中保守涉及核苷酸结合和催化的关键残基。将获得与核苷酸复合的MiD 51的其他结构。在目标2中，生物化学研究用于推进对MiD 51和MiD 49功能的理解。鉴于MiD 51具有核苷酸基转移酶折叠，将使用定量结合试验测量MiD 51的核苷酸结合特异性。此外，将使用酶测定来确定MiD 51是否在核苷酸转移中具有催化功能。将用与MiD 51同源的MiD 49进行类似的研究。在目的3中，使用细胞培养物中的结构-功能分析来确定核苷酸结合、催化或二聚化对于MiD 51募集Drp和介导分裂的能力是否重要。MiD 51和MiD 49敲除细胞将通过使用基因组编辑技术产生，用于进一步的结构-功能分析。最后，将利用遗传学方法来定位Mff、MiD 49和MiD 51上的Drp 1结合位点。总之，这些研究将大大推进对Drp 1受体结构生物学的理解，并可能最终导致调节线粒体分裂的新方法。1

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