Regulation of Mitochondrial Fission and Fusion

细胞神经科的研究集中在一些神经退行性疾病的分子机制上，包括线粒体疾病、肌张力障碍和遗传性痉挛截瘫(HSPs)。这些疾病共同困扰着数以百万计的美国人，多年来还在潜移默化地恶化，其中许多人的治疗选择有限。我们的实验室正在研究这些疾病的遗传形式，使用分子和细胞生物学方法来研究疾病基因的突变最终是如何导致细胞功能障碍的。 在这个项目中，我们重点研究细胞内线粒体形态的调节。事实上，调节线粒体形态的融合和分裂事件对于正常的线粒体功能是必不可少的，它们的调节在不同的细胞功能中得到越来越多的认识。哺乳动物中的线粒体分裂事件至少由两种蛋白质协调：动力蛋白相关蛋白Drp1和整合膜蛋白Fis1。线粒体融合的相互过程也需要动力蛋白超家族的大GTP酶：OPA1和丝裂原Mfn1和Mfn2。由于已经在遗传性神经疾病患者中发现了DRp1、Mfn2和OPA1的突变，并且在程序性细胞死亡过程中存在显著的线粒体碎裂，因此对这些过程的调控的洞察具有高度的临床意义。 2007年，我们发表了一项研究，证明了依赖cAMP的蛋白磷酸化的Drp1蛋白调节其GTPase活性以及线粒体的形态。我们现在有一些正在进行的合作调查，重点是这种修饰在调节一些细胞内过程方面的相关性。此外，我们已经确定了Drp1蛋白上的总甲基化位置，目前正在使用显性-负性方法来确定总甲基化在Drp1功能中的功能作用。最后，我们最近完成了对Drp1 A395D突变的研究，该突变导致了一种新生儿致命的线粒体疾病，原因是线粒体分裂显著减少。通过酵母双杂交试验，我们能够证明这种突变导致了Drp1蛋白分子间和分子内相互作用的丧失，导致了正确的高阶组装的缺陷。在正在进行的研究中，我们已经确定了一些与Drp1相互作用的蛋白质，我们目前正在评估这些蛋白质。 尽管我们的大部分努力都集中在线粒体分裂的调控上，但我们也有几个与线粒体融合调控相关的合作项目。这些都特别集中在OPA1蛋白的蛋白质分解处理上。 总之，这些研究继续为细胞内线粒体形态的调节提供关键的见解，这是一个越来越具有临床相关性和重要性的领域。

Research in the Cellular Neurology Unit focuses on the molecular mechanisms underlying a number of neurodegenerative disorders, including mitochondrial disorders, dystonia, and the hereditary spastic paraplegias (HSPs). These disorders, which together afflict millions of Americans, worsen insidiously over a number of years, and treatment options are limited for many of them. Our laboratory is investigating inherited forms of these disorders, using molecular and cell biology approaches to study how mutations in disease genes ultimately result in cellular dysfunction. In this project, we are emphasizing investigations into the regulation of mitochondrial morphology within cells. Indeed, fusion and fission events that regulate mitochondrial morphology are essential for proper mitochondrial function, and their regulation is increasingly recognized in diverse cellular functions. Mitochondrial fission events in mammals are orchestrated by at least two proteins; the dynamin-related protein Drp1 and the integral membrane protein Fis1. The reciprocal process of mitochondrial fusion also requires large GTPases of the dynamin superfamily: OPA1 and the mitofusins Mfn1 and Mfn2. Since mutations in Drp1, Mfn2, and OPA1 have been identified in patients with inherited neurological disorders, and there is prominent fragmentation of mitochondria during programmed cell death, insights into the regulation of these processes is highly relevant clinically. In 2007, we published a study demonstrating that cAMP-dependent protein phosphorylation of the Drp1 protein modulates its GTPase activity as well as mitochondrial morphology. We now have a number of collaborative investigations underway focusing on the relevance of this modification in the regulation of a number of intracellular processes. In addition, we have identified the sites of sumoylation on the Drp1 protein, and are currently using dominant-negative approaches to determine the functional role of sumoylation in Drp1 function. Lastly, we have recently completed a study of the Drp1 A395D mutation that caused a neonatally fatal mitochondrial disorder due to markedly diminished mitochondrial fission. Using yeast two-hybrid tests, we were able to show that this mutation resulted in loss of inter- and intramolecular interactions of the Drp1 protein, resulting in a defect in proper higher-order assembly. In ongoing studies, we have identified a number of Drp1-interacting proteins that we are currently evaluating. Though most of our efforts are focused on the regulation of mitochondrial fission, we have several collaborative projects related to the regulation of mitochondrial fusion. These are particularly focused on the proteolytic processing of the OPA1 protein. Together, these studies are continuing to provide critical insights into the regulation of mitochondrial morphology within a cell, an area of increasing clinical relevance and importance.