Analysis of Drp 1 Receptors Important for Mitochondrial Fission

描述(申请人提供)：在过去的十年中，线粒体融合和分裂已成为线粒体功能的主要调节因素。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种群方面具有重要意义。这一建议侧重于线粒体分裂的机制，它涉及到控制线粒体的形态、自噬导致的线粒体退化、控制线粒体的分布和调节细胞凋亡。在线粒体分裂的过程中，动力蛋白相关蛋白1(Dynamin-Related Protein 1)被从胞浆招募到线粒体表面。在这里，它组装成一个低聚复合体，包裹在线粒体管上，并收缩它，以介导膜的断裂。为了使Drp1被招募到线粒体，线粒体外膜包含四个Drp1受体：Fis1、Mff、MiD49和MiD51。细胞研究表明，后三个分子对线粒体分裂的功能最重要。这项建议侧重于获得结构、生化和细胞方面的了解，了解这些分子是如何调节Drp1的招募的。在目标1中，使用X射线结晶学获得了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，这一结构表明MiD51具有核苷酸转移酶结构域，并保守与核苷酸结合和催化有关的关键残基。将获得与核苷酸形成的复合体中MiD51的额外结构。在目标2中，利用生化研究来促进对MiD51和MiD49功能的理解。鉴于MiD51具有核苷酸转移酶折叠，将通过定量结合分析来衡量MiD51的核苷酸结合特异性。此外，将使用酶分析来确定MiD51是否在核苷酸转移中具有催化功能。与MiD51同源的MiD49也将进行类似的研究。在目标3中，细胞培养中的结构-功能分析被用来确定核苷酸结合、催化或二聚对MiD51招募DRP和介导分裂的能力是否重要。通过使用基因组编辑技术，将产生用于进一步结构功能分析的MiD51和MiD49基因敲除细胞。最后，将利用遗传方法定位MFF、MiD49和MiD51上的Drp1结合位点。综上所述，这些研究将极大地促进对Drp1受体结构生物学的理解，并最终可能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