The molecular mechanisms of mitochondrial behavior.

线粒体行为的分子机制。

• 批准号: 8722591
• 负责人: Suzanne C Hoppins
• 金额: $ 24.17万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-16 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722591
• 关键词: Address; Apoptosis; Apoptotic; Bax protein; Behavior; Biochemical; Biological Assay; Cardiac; Cell Death; Cell physiology; Cells; Characteristics; Chimeric Proteins; Chronic; Complex; Coupled; Data; Deletion Mutation; Disease Progression; Dynamin; Embryo; Event; Foundations; GTP Binding; Grant; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Heart; Heart Diseases; Hydrolysis; In Vitro; Knowledge; Light; Link; Mammals; Maps; Mediating; Membrane; Membrane Fusion; Microtubules; Mitochondria; Molecular; Movement; Myocardial Infarction; Nature; Neurodegenerative Disorders; Organelles; Organism; Pathway interactions; Physiological; Play; Process; Property; Protein Family; Proteins; Regulation; Relative (related person); Role; Shapes; Signal Pathway; Signal Transduction; Testing; Therapeutic; Time; Tissues; Translating; abstracting; base; cell motility; chemical genetics; drug development; fusion gene; in vitro Assay; insight; member; novel; protein function; research study; therapeutic development

Project Summary/Abstract: Mitochondrial fusion regulates the shape, distribution and function of the organelle and plays critical roles in protection from cell death and therefore offers a valid target for theurapeutic approaches to protect the heart from progression of disease or myocardial infarction events. Mitochondrial fusion is mediated by members of the dynamin related protein (DRP) family, large GTPases that, through their ability to self-assemble and hydrolyze GTP, control membrane remodeling events. In mammals, MFN1 and MFN2 function in place of a single outer membrane DRP in simple organisms, both are expressed ubiquitously but the relative expression level of each varies in a tissue dependent manner. Although MFN1 and MFN2 both function in mitochondrial fusion, they are not completely redundant, although the nature of the functional differences are not known. Our data indicate that the MFN1-MFN2 heterotypic trans complex is significantly more efficacious for fusion than either homotypic complex. From this, we predict that MFN1 and MFN2 have distinct molecular properties and that the functional significance of the two outer membrane DRPs in mammals is to provide a relatively simple regulatory fusion mechanism via their respective and relative expression levels. Further, data suggest that specific regulatory mechanisms exist, as we have shown that soluble Bax stimulates only MFN2 homotypic complexes. In order to understand the functional significance of two outer membrane fusion proteins and the regulatory mechanisms that govern their activity, we propose to characterize the biochemical properties of each DRP including GTP binding, GTP hydrolysis and complex assembly. These biochemical approaches together will determine the mechanistic basis for why MFN1 only, MFN2 only and MFN1/MFN2 mediated fusion efficiencies are distinct and will likely point to why and how they are distinctly regulated in cells. To explore what properties of MFN2 are modulated by the pro-apoptotic Bcl2 protein Bax, we will test the effect of Bax on the biochemical properties of MFN2, of particular significance in the heart where MFN2 is the predominantly expressed fusion DRP. We will further characterize the regulation of fusion by Bax through identification of interaction domains and exploring their functional significance in vitro and in cells. Bax also negatively effects mitochondrial fusion following activation by apoptotic signals and we will investigate whether the inhibition of fusion is direct or mediated through outer membrane permeabilization. As observed with the apoptotic pathway and mitochondrial dynamics, an interdependence also exists between mitochondrial fusion and mitochondrial motility, but the significance of this is relatively unexplored. We propose that mitochondrial tethering to microtubules and short range motility are both important regulators of mitochondrial fusion. We will determine the role that mitochondrial movement and tethering play in regulating mitochondrial fusion and investigate the molecular basis for the requirement of MFNs in these processes using an in vitro mitochondrial fusion-motility assay and complimentary biochemical approaches.

项目概要/摘要： 线粒体融合调节细胞器的形状、分布和功能，并在以下方面发挥关键作用： 保护细胞免于死亡，因此为保护心脏的紧急方法提供了有效的靶点 疾病进展或心肌梗死事件。线粒体融合是由 动力蛋白相关蛋白（DRP）家族，即大GTP酶，通过其自组装能力， 水解GTP，控制膜重塑事件。在哺乳动物中，MFN 1和MFN 2在哺乳动物中的功能是替代MFN 1和MFN 2。 在简单的生物体中，单个外膜DRP，两者都普遍表达，但相对表达 每一种的水平以组织依赖性方式变化。虽然MFN 1和MFN 2都在线粒体中起作用， 尽管不知道功能差异的性质，但它们并非完全多余。我们 数据表明，MFN 1-MFN 2异型反式复合物对于融合显著比 或是同型复合体由此，我们预测MFN 1和MFN 2具有不同的分子性质， 在哺乳动物中，两个外膜DRPs的功能意义是提供一个相对简单的 通过它们各自的和相对的表达水平调节融合机制。此外，数据表明， 存在特定的调节机制，因为我们已经表明可溶性Bax仅刺激MFN 2同型 配合物为了了解两种外膜融合蛋白的功能意义， 调节机制，支配他们的活动，我们建议表征的生化特性， 每个DRP包括GTP结合、GTP水解和复合物组装。这些生物化学方法 一起将确定为什么只有MFN 1，只有MFN 2和MFN 1/MFN 2介导的机制基础。 融合效率是不同的，并且可能指出它们在细胞中为什么以及如何被不同地调节。到 为了探索MFN 2的哪些特性受促凋亡Bcl 2蛋白Bax的调节，我们将测试 Bax对MFN 2的生物化学性质的影响，在心脏中特别重要，其中MFN 2是MFN 2的受体。 主要表达融合DRP。我们将通过以下方式进一步表征Bax对融合的调节： 鉴定相互作用结构域，并探索它们在体外和细胞中的功能意义。Bax也 在细胞凋亡信号激活后对线粒体融合产生负面影响，我们将研究是否 融合的抑制是直接的或通过外膜透化介导的。正如观察到的那样， 凋亡途径和线粒体动力学之间也存在相互依赖性， 和线粒体运动性，但其意义相对未被探索。我们认为线粒体 与微管的束缚和短程运动都是线粒体融合的重要调节因子。我们将 确定线粒体运动和束缚在调节线粒体融合中的作用， 研究在这些过程中使用体外线粒体MFN的要求的分子基础， 融合运动性测定和补充的生物化学方法。