The molecular mechanisms of mitochondrial behavior.

描述(申请人提供)：线粒体融合调节细胞器的形状、分布和功能，并在防止细胞死亡方面发挥关键作用，因此为保护心脏免受疾病进展或心肌梗死事件的治疗提供了有效的靶点。线粒体融合是由动力蛋白相关蛋白(DRP)家族的成员介导的，该家族是一种大的GTP酶，通过自组装和水解GTP的能力来控制膜重构事件。在哺乳动物中，Mfn1和Mfn2的功能取代了简单生物体中单一的外膜DRP，两者都广泛表达，但它们的相对表达水平随着组织的变化而变化。虽然Mfn1和Mfn2都在线粒体融合中发挥作用，但它们并不是完全冗余的，尽管功能差异的本质尚不清楚。我们的数据表明，Mfn1-Mfn2异型反式复合体的融合效率明显高于任何一种同型复合体。由此，我们预测Mfn1和Mfn2具有不同的分子特性，在哺乳动物中两个外膜DRP的功能意义在于通过它们各自和相对的表达水平提供一种相对简单的调节融合机制。此外，数据表明存在特定的调控机制，因为我们已经表明，可溶性Bax只刺激Mfn2同型复合体。为了了解两个外膜融合蛋白的功能意义和调控它们活性的机制，我们建议对每个DRP的生化性质进行表征，包括GTP结合、GTP水解和复杂组装。这些生化方法共同决定了为什么Mfn1、Mfn2和Mfn1/Mfn2介导的融合效率是不同的机制基础，并可能指出它们在细胞中受到明显调控的原因和方式。为了探索促凋亡的Bcl2蛋白Bax对Mfn2的哪些特性进行调节，我们将测试Bax对Mfn2的生化特性的影响，这在心脏中具有特别重要的意义，因为Mfn2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它们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还会在被凋亡信号激活后对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通透性介导的。从细胞凋亡途径和线粒体动力学的角度观察，线粒体融合与线粒体运动之间也存在相互依赖关系，但其意义尚不清楚。我们认为线粒体与微管的连接和短程运动都是线粒体融合的重要调节因素。我们将确定线粒体运动和拴系在调节线粒体融合中所起的作用，并使用体外线粒体融合运动试验和补充的生化方法来研究这些过程中需要MFN的分子基础。 公共卫生相关性：这项拨款中提议的实验将阐明线粒体融合的基本机制，为进一步了解这一过程在包括健康心脏在内的所有情况下、在慢性心脏病和心肌梗死期间对心脏功能所起的生理作用提供基础。通过探讨线粒体融合与细胞凋亡之间的机制联系，以及线粒体融合与线粒体系留和运动之间的相互依赖，我们将深入了解存在于细胞中的调控机制。这些方法将有助于确定心脏病药物开发和治疗的新目标。

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.