The role of mitochondria-associated RING finger proteins in mitochondrial quality

描述（由申请人提供）：线粒体对多种细胞功能至关重要，包括ATP生产，脂质生物合成和钙稳态。此外，许多主要的细胞信号通路，包括细胞凋亡，都需要线粒体。与线粒体在控制细胞功能中的主要作用一致，线粒体缺陷导致各种恶性病理。因此，控制线粒体稳态的分子成分可能是细胞命运和整个生物体健康的主要决定因素。然而，尽管它们很重要，线粒体质量控制的机制，以及线粒体和其他细胞间的信号传导机制，在很大程度上是未知的。我们发现了两种新的线粒体E3泛素连接酶MARCH5和IBRDC2，并确定这两种蛋白分别在细胞凋亡和线粒体分裂中调控线粒体功能。这些结果暗示一个新的线粒体膜相关环指E3泛素连接酶家族通过泛素依赖机制调节线粒体稳态。本研究旨在阐明IBRDC2和MARCH5的功能，以及它们在健康细胞和凋亡过程中线粒体蛋白调节和膜动力学中的作用。生物化学和细胞研究，使用延时成像方法的成像调查，PI开发的新荧光工具（包括光活化荧光蛋白），以及各种分子遗传学方法将用于解决以下三个问题：1)IBRDC2和MARCH5的生化特性是什么？将确定IBRDC2和MARCH5的亚线粒体定位、膜拓扑结构和底物特异性。2) IBRDC2和MARCH5如何在活细胞线粒体中起作用？利用功能获得和功能丧失方法的研究将测试IBRDC2和MARCH5在蛋白酶体依赖性线粒体蛋白降解中的作用，以及在膜动力学和凋亡相关线粒体蛋白复合物的调节中的作用。这些研究还将鉴定受IBRDC2和MARCH5调控的线粒体蛋白。3) IBRDC2和MARCH5对凋亡级联中特定分子事件的影响是什么？这些研究将确定IBRDC2和MARCH5活性在凋亡不同步骤的进展中所需要的程度。解决这些问题将提高我们对线粒体功能的总体理解，从长远来看，可能有助于开发新的药理学方法来治疗线粒体功能障碍引起的疾病。提出的研究是我们了解线粒体正常功能和线粒体缺陷如何导致疾病的长期努力的一部分。

DESCRIPTION (provided by applicant): Mitochondria are essential for a variety of cellular functions, including ATP production, lipid biosynthesis and calcium homeostasis. Moreover, a number of major cell signaling pathways, including apoptosis, require mitochondria. Consistent with a major role of mitochondria in the control of cell function, mitochondrial defects result in a variety of malignant pathologies. Thus, the molecular components that control mitochondrial homeostasis are likely to be major determinants of cell fate and the well being of the whole organism. However, despite their importance, the mechanisms of mitochondrial quality control, as well as the signaling mechanisms between mitochondria and other cell compartments, are largely unknown. We have identified MARCH5 and IBRDC2, two novel mitochondrial E3 ubiquitin ligases, and have determined that these two proteins are essential for the regulation of mitochondrial function in apoptosis and mitochondrial division, respectively. These results implicate a family of novel mitochondrial membrane-associated RING finger E3 ubiquitin ligases in the regulation of mitochondrial homeostasis through ubiquitin-dependent mechanisms. The present proposal seeks to elucidate the functions of IBRDC2 and MARCH5, and their roles in mitochondrial protein regulation and in membrane dynamics, both in healthy cells and during apoptosis. Biochemical and cellular studies, imaging investigations using time-lapse methods, new fluorescent tools developed by the PI (including photoactivable fluorescent proteins), and a variety of molecular genetic methodologies will be utilized to address the following three questions: 1) What are the biochemical properties of IBRDC2 and MARCH5? The sub-mitochondrial localization, membrane topology and substrate specificity of IBRDC2 and MARCH5 will be determined. 2) How do IBRDC2 and MARCH5 work in the mitochondria of living cells? Studies exploiting gain- and loss-of-function approaches will test the roles of IBRDC2 and MARCH5 in proteasome-dependent mitochondrial protein degradation, as well as in the regulation of membrane dynamics and apoptosis-related mitochondrial protein complexes. These studies will also identify mitochondrial proteins that are under regulatory control of IBRDC2 and MARCH5. 3) What is the influence of IBRDC2 and MARCH5 on specific molecular events in the apoptotic cascade? These studies will determine to what degree IBRDC2 and MARCH5 activities are required for progression of distinct steps of apoptosis. Addressing these questions will improve our general understanding of mitochondrial function and, in the long term, are likely to contribute to the development of novel pharmacological approaches to treat diseases stemming from mitochondrial dysfunction. The proposed studies are part of our long- term effort to understand the normal functions of mitochondria and how mitochondrial defects contribute to disease. Public Health Relevance: We have identified MARCH5 and IBRDC2, two novel mitochondrial E3 ubiquitin ligases, and have determined that these two proteins are essential for the regulation of mitochondrial function in apoptosis and mitochondrial division, respectively. These results implicate a family of novel mitochondrial membrane-associated RING finger E3 ubiquitin ligases in the regulation of mitochondrial homeostasis through ubiquitin-dependent mechanisms. The present proposal seeks to elucidate the functions of IBRDC2 and MARCH5, and their roles in mitochondrial protein regulation and in membrane dynamics, both in healthy cells and during apoptosis. The results of these studies, should improve our general understanding of mitochondrial function and, in the long term, are likely to contribute to the development of novel pharmacological approaches to treat diseases stemming from mitochondrial dysfunction.