The molecular mechanisms of mitochondrial behavior.

线粒体行为的分子机制。

• 批准号: 8875045
• 负责人: Suzanne C Hoppins
• 金额: $ 23.99万
• 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/8875045
• 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; genetic approach; 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.

项目摘要/摘要： 线粒体融合调节细胞器的形状、分布和功能，并在 防止细胞死亡，因此为保护心脏的治疗方法提供了有效的目标 疾病进展或心肌梗塞事件。线粒体融合是由以下成员介导的 动力相关蛋白 (DRP) 家族，大型 GTP 酶，通过其自组装和 水解 GTP，控制膜重塑事件。在哺乳动物中，MFN1 和 MFN2 的功能是代替 简单生物体中的单外膜 DRP 均普遍表达，但相对表达量 每个的水平以组织依赖性方式变化。虽然 MFN1 和 MFN2 都在线粒体中发挥作用 尽管功能差异的本质尚不清楚，但它们并不是完全冗余。我们的 数据表明，MFN1-MFN2 异型反式复合物的融合效果明显优于 要么是同型复合体。由此，我们预测 MFN1 和 MFN2 具有不同的分子特性，并且 哺乳动物中两个外膜 DRP 的功能意义是提供一个相对简单的 通过它们各自和相对表达水平的调节融合机制。此外，数据表明 存在特定的调节机制，正如我们已经证明可溶性 Bax 仅刺激 MFN2 同型 复合物。为了了解两种外膜融合蛋白的功能意义以及 控制其活动的调节机制，我们建议表征 每个 DRP 包括 GTP 结合、GTP 水解和复合物组装。这些生化方法 一起将确定为什么仅 MFN1、仅 MFN2 和 MFN1/MFN2 介导的机制基础 融合效率是不同的，并且很可能表明它们在细胞中受到明显调节的原因和方式。到 探索促凋亡 Bcl2 蛋白 Bax 调节 MFN2 的哪些特性，我们将测试其效果 Bax 对 MFN2 的生化特性的研究，对于 MFN2 是心脏的心脏特别重要 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： 识别相互作用域并探索它们在体外和细胞中的功能意义。巴克斯也 细胞凋亡信号激活后会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 凋亡途径和线粒体动力学，线粒体融合之间也存在相互依赖性 和线粒体运动，但其意义相对尚未被探索。我们建议线粒体 微管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 使用体外线粒体研究这些过程中 MFN 需求的分子基础 融合运动测定和互补生化方法。