Fis1 regulation of mitochondrial fission

描述(申请人提供)：这项提案的长期目标是确定线粒体分裂的分子机制及其在细胞凋亡中的作用。线粒体分裂的缺陷会造成严重的后果，甚至死亡。然而，人们对裂变是如何工作的以及与其他细胞过程是如何协调的知之甚少。由其他研究人员进行的遗传和细胞研究，主要是对酿酒酵母的研究，已经导致了线粒体分裂模型的发展。在这个模型中，Fis1蛋白通过介导动力蛋白相关的GTPase DNM1和适配蛋白Mdv1在线粒体外膜收缩部位的组装来调节分裂。尽管来自定性模型的数据是提示性的，但该模型并没有解释这一重要过程是如何被调控的。通过将细胞生物学、生化和生物物理数据与裂变机械的低分辨率和高分辨率结构相结合，目前的提议旨在开发一个全面的线粒体分裂模型。第一个具体目标是通过实验定义支持这些相互作用的结构域，这些相互作用的化学计量和亲和力及其对DNM1活性的影响，从而定义溶液中对裂变重要的蛋白质-蛋白质相互作用。拟议中的裂变机械的低分辨率图像将通过电子显微镜获得。FIS1、FIS1/Mdv1和FIS1/Mdv1/DNM1三元配合物的高分辨结构将通过核磁共振光谱和X-射线结晶学进行研究。第二个特定的目的是鉴定酵母Fis1残基，这些残基对同源二聚、Mdv1结合和DNM1结合很重要。我们将鉴定影响寡聚的FIS1突变体，并测试这些突变体的变化活性，以确定它们在裂变机械组装中的重要性。第三个具体目的是通过对来自合成脂质和分离的线粒体的膜进行实验，确定FIS1是否影响膜上DNM1和Mdv1的组装。这些实验还应该允许确定组装的顺序。所有三种方法得到的数据都将被整合到线粒体分裂是如何完成的完整图景中。这些分析还有望对基于动力蛋白的膜动力学以及蛋白质-蛋白质和蛋白质-脂相互作用的基础有相当全面的了解。人类线粒体分裂机制的同系物存在，并被报道在调节细胞凋亡方面发挥重要作用，而细胞凋亡与许多重要疾病有关。因此，有关线粒体分裂的详细信息可能有助于设计抑制或诱导细胞凋亡的策略。 与公共健康相关：线粒体执行许多基本功能，这些功能被认为需要频繁的线粒体分裂和融合事件，这些事件是由不同的蛋白质机制完成的。线粒体分裂蛋白DNM1中的一个点突变导致了婴儿死亡。此外，线粒体裂变在细胞凋亡过程中增加，这一过程的错误调控导致了许多人类疾病。拟议的工作将阐明这些过程的机械细节，并代表着朝着发现人类疾病的新治疗策略迈出的重要一步。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify the molecular mechanism of mitochondrial fission and its role in apoptosis. Defects in mitochondrial fission have severe consequences and even death. Yet little is known about how fission works and is coordinated with other cellular processes. Genetic and cellular studies, primarily in Saccharomyces cerevisiae, by other investigators have led to the development of a model for mitochondrial fission. In this model, the protein Fis1 regulates fission by mediating the assembly of a dynamin- related GTPase, Dnm1, and an adaptor protein, Mdv1, at the sites of constriction on the mitochondrial outer membrane. Although the data from the qualitative model are suggestive, the model does not explain how this important process is regulated. By integrating cell biological, biochemical, and biophysical data with low- and high-resolution structures of the fission machinery, the current proposal aims to develop a comprehensive model for mitochondrial fission. The first specific aim is to define the protein-protein interactions in solution that are important to fission through experiments that will define domains that support these interactions, the stoichiometries and affinities of these interactions and their consequences on Dnm1 activity. Low-resolution images of the proposed fission machinery will be obtained by electron microscopy. High-resolution structures of Fis1, the binary Fis1/Mdv1 complex, and the ternary Fis1/Mdv1/Dnm1 complex will be pursued by NMR spectroscopy and x-ray crystallography. The second specific aim is to identify yeast Fis1 residues important for homodimerization, Mdv1 binding, and Dnm1 binding. We will identify mutants of Fis1 that affect oligomerization and test these mutants for altered activities to define their importance in assembly of the fission machinery. The third specific aim is to determine whether Fis1 affects the assembly of Dnm1 and Mdv1 on the membrane through experiments with membranes derived from synthetic lipids and isolated mitochondria. These experiments should also allow determination of the order of assembly. The resulting data from all three approaches will be integrated into a complete picture of how mitochondrial fission is accomplished. The analyses also promise considerable general insight into the basis of dynamin-based membrane dynamics, as well as protein-protein and protein-lipid interactions. Human homologues of the mitochondrial fission machinery exist and are reported to be important in regulating apoptosis, which is linked to many important diseases. Therefore, detailed information on mitochondrial fission might be helpful in designing strategies to inhibit or induce apoptosis. PUBLIC HEALTH RELEVANCE: Mitochondria perform many essential functions that are thought to require frequent mitochondrial fission and fusion events, which are accomplished by distinct protein machineries. A point mutant in the mitochondrial fission protein, Dnm1, caused infant death. Additionally, mitochondrial fission increases during apoptosis, a process whose misregulation contributes to many human diseases. The work proposed will illuminate mechanistic details of these processes and represents an important step towards the discovery of new therapeutic strategies for human diseases.