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

描述(申请人提供)：线粒体对多种细胞功能是必不可少的，包括三磷酸腺苷的产生、脂质的生物合成和钙的稳态。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。与线粒体在细胞功能控制中的主要作用一致，线粒体缺陷会导致多种恶性病理。因此，控制线粒体动态平衡的分子成分很可能是细胞命运和整个生物体福祉的主要决定因素。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室之间的信号机制，在很大程度上是未知的。我们已经鉴定了两个新的线粒体E3泛素连接酶March5和IBRDC2，并确定这两个蛋白分别对线粒体在细胞凋亡和线粒体分裂中的功能起着至关重要的调节作用。这些结果暗示了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的机制来调节线粒体的动态平衡。本研究试图阐明IBRDC2和March5的功能，以及它们在线粒体蛋白调节和膜动力学中的作用，无论是在健康细胞中还是在凋亡过程中。将利用生物化学和细胞研究、使用延时方法的成像研究、PI开发的新的荧光工具(包括可光激活的荧光蛋白)以及各种分子遗传学方法来解决以下三个问题：1)IBRDC2和March5的生化特性是什么？IBRDC2和March5的亚线粒体定位、膜拓扑结构和底物特异性将被确定。2)IBRDC2和March5如何在活细胞的线粒体中发挥作用？利用获得和功能丧失方法的研究将测试IBRDC2和March5在蛋白酶体依赖的线粒体蛋白质降解中的作用，以及在调节膜动力学和与凋亡相关的线粒体蛋白质复合体中的作用。这些研究还将确定受IBRDC2和March5调控的线粒体蛋白质。3)IBRDC2和March5对细胞凋亡级联中的特定分子事件有何影响？这些研究将确定IBRDC2和March5活性在多大程度上是细胞凋亡不同步骤进展所必需的。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由线粒体功能障碍引起的疾病。拟议的研究是我们长期努力的一部分，目的是了解线粒体的正常功能以及线粒体缺陷是如何导致疾病的。 公共卫生相关性：我们已经鉴定了两个新的线粒体E3泛素连接酶March5和IBRDC2，并确定这两个蛋白分别对线粒体在细胞凋亡和线粒体分裂中的功能调节至关重要。这些结果暗示了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的机制来调节线粒体的动态平衡。本研究试图阐明IBRDC2和March5的功能，以及它们在线粒体蛋白调节和膜动力学中的作用，无论是在健康细胞中还是在凋亡过程中。这些研究的结果，应该会提高我们对线粒体功能的总体理解，并从长远来看，可能有助于开发新的药理学方法来治疗由线粒体功能障碍引起的疾病。

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.