The molecular mechanisms of mitochondrial behavior.

线粒体行为的分子机制。

• 批准号: 8111493
• 负责人: Suzanne C Hoppins
• 金额: $ 9.69万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8111493
• 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; base; cell motility; chemical genetics; drug development; fusion gene; in vitro Assay; insight; member; novel; protein function; research study; therapeutic development; therapeutic target

DESCRIPTION (provided by applicant): 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 therapeutic 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. PUBLIC HEALTH RELEVANCE: The experiments proposed in this grant will shed light on the fundamental mechanism of mitochondrial fusion, providing a foundation to further understand the physiological roles this process plays in cardiac function, under all circumstances including the healthy heart, in chronic heart disease and during myocardial infarction events. By probing the mechanistic link between mitochondrial fusion and apoptosis and the interdependence of mitochondrial fusion and mitochondrial tethering and motility, we will gain insight into the regulatory mechanisms that exist in cells. These approaches will contribute to the identification of novel targets for drug development and therapeutics for heart disease.

描述（由申请人提供）：线粒体融合调节细胞器的形状、分布和功能，并在保护细胞免于死亡方面发挥关键作用，因此为保护心脏免于疾病进展或心肌梗死事件的治疗方法提供了有效靶点。线粒体融合由发动蛋白相关蛋白（DRP）家族成员介导，该家族是大GTP酶，通过其自组装和水解GTP的能力，控制膜重塑事件。在哺乳动物中，MFN 1和MFN 2在简单生物体中代替单个外膜DRP起作用，两者都广泛表达，但各自的相对表达水平以组织依赖性方式变化。虽然MFN 1和MFN 2都在线粒体融合中起作用，但它们并不是完全多余的，尽管功能差异的性质尚不清楚。我们的数据表明，MFN 1-MFN 2异型反式复合物对于融合比任一同型复合物显著更有效。由此，我们预测MFN 1和MFN 2具有不同的分子特性，并且两个外膜DRPs在哺乳动物中的功能意义是通过它们各自的和相对的表达水平提供相对简单的调节融合机制。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同型复合物。为了了解两个外膜融合蛋白的功能意义和管理其活动的调节机制，我们建议表征每个DRP的生化特性，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仅MFN 1、仅MFN 2和MFN 1/MFN 2介导的融合效率是不同的机制基础，并且可能指出它们在细胞中为什么和如何被不同地调节。为了探索促凋亡Bcl 2蛋白Bax调节MFN 2的什么性质，我们将测试Bax对MFN 2的生化性质的影响，这在心脏中特别重要，其中MFN 2是主要表达的融合DRP。我们将通过鉴定相互作用结构域并探索其在体外和细胞中的功能意义来进一步表征Bax对融合的调控。Bax也对凋亡信号激活后的线粒体融合产生负面影响，我们将研究融合的抑制是直接的还是通过外膜透化介导的。正如在凋亡途径和线粒体动力学中所观察到的，线粒体融合和线粒体运动之间也存在相互依赖性，但其意义相对未被探索。我们认为，线粒体系留微管和短程运动都是重要的调节线粒体融合。我们将确定线粒体运动和拴系在调节线粒体融合中发挥的作用，并使用体外线粒体融合运动试验和补充生物化学方法研究在这些过程中需要MFN的分子基础。 公共卫生相关性：这项资助中提出的实验将揭示线粒体融合的基本机制，为进一步了解这一过程在心脏功能中的生理作用提供基础，包括在健康心脏，慢性心脏病和心肌梗死事件的所有情况下。通过探索线粒体融合和凋亡之间的机制联系以及线粒体融合和线粒体束缚和运动之间的相互依赖性，我们将深入了解细胞中存在的调节机制。这些方法将有助于确定心脏病药物开发和治疗的新靶点。