Fis1 regulation of mitochondrial fission

描述（由申请人提供）：本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。线粒体分裂的缺陷有严重的后果，甚至死亡。然而，人们对裂变是如何工作的以及如何与其他细胞过程协调的知之甚少。其他研究人员的遗传和细胞研究，主要是在酿酒酵母中，导致了线粒体分裂模型的发展。在该模型中，蛋白质Fis 1通过介导动力蛋白相关的GTdR（Dnm 1）和衔接蛋白（Mdv 1）在线粒体外膜上的收缩位点处的组装来调节裂变。虽然定性模型的数据具有启发性，但该模型并没有解释这一重要过程是如何调节的。通过将细胞生物学、生物化学和生物物理学数据与裂变机制的低分辨率和高分辨率结构相结合，目前的提案旨在开发一个线粒体裂变的综合模型。第一个具体的目标是通过实验来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以及它们对Dnm 1活性的影响，来定义对裂变很重要的溶液中的蛋白质-蛋白质相互作用。将通过电子显微镜获得提议的裂变机制的低分辨率图像。Fis 1、二元Fis 1/Mdv 1复合物和三元Fis 1/Mdv 1/Dnm 1复合物的高分辨率结构将通过核磁共振光谱学和X射线晶体学来研究。第二个具体的目的是确定酵母Fis 1残基的同源二聚体，Mdv 1的结合，和Dnm 1的结合重要。我们将确定影响寡聚化的Fis 1突变体，并测试这些突变体的改变活动，以确定其在裂变机制组装中的重要性。第三个具体的目的是确定Fis 1是否影响Dnm 1和Mdv 1的组装膜上通过实验与膜衍生自合成脂质和分离的线粒体。这些实验还应允许确定组装顺序。从所有三种方法得到的数据将被整合到线粒体裂变是如何完成的完整图片中。分析还承诺相当的一般洞察力的基础上动力蛋白为基础的膜动力学，以及蛋白质-蛋白质和蛋白质-脂质相互作用。线粒体分裂机制的人类同源物存在并且据报道在调节细胞凋亡中是重要的，细胞凋亡与许多重要疾病有关。因此，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。 公共卫生相关性：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，这是由不同的蛋白质机制完成的。线粒体分裂蛋白Dnm 1的点突变导致婴儿死亡。此外，线粒体分裂在细胞凋亡过程中增加，这是一个失调导致许多人类疾病的过程。这项工作将阐明这些过程的机制细节，并代表着为人类疾病发现新的治疗策略的重要一步。

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.