Regulation of Cell Death and Mitochondrial Physiology by Anti-Apoptotic MCL-1

描述(由申请人提供)：抗凋亡的bcl2家族成员调节造血，当失调时会导致癌症、免疫缺陷和自身免疫。MCL-1是细胞在多个造血发育阶段生存所必需的。相反，基因去除Bcl2或BclX在促进造血细胞存活方面显示了特定的作用。为什么MCL-1在维持生存中起着如此关键的作用，以及为什么伴随而来的其他抗凋亡调节因子的内源性表达不能弥补MCL-1的丢失，至今仍未解决。因此，我的实验室的长期目标是了解MCL-1在造血发育和生存过程中的功能和调节。为了了解MCL-1在发育过程中的需求，我们最近发现它既定位于线粒体外膜，在那里它结合和隔离促凋亡分子，又定位于线粒体内膜，以N端截断的形式存在。这两种定位对MCL-1‘S功能的贡献尚不清楚。这项建议的目的是剖析mcl1的两种线粒体形式的功能作用，并评估这些定位在促进细胞存活方面的单独作用。我们的中心假设是，MCL-1的外膜形式类似于经典的bcl-2家族成员，以对抗促凋亡分子，而内膜形式的MCL-1通过调节线粒体功能来调节细胞生存。我们提出的研究将揭示MCL-1以前未知的作用，并阐明抗凋亡的BCL-2家族成员促进线粒体功能的新机制。目的1：明确mcl1不同线粒体形态在造血调控中的作用(S)。MCL-1在造血的许多阶段都表现出深刻的需求，但原因尚不清楚。目的2：明确线粒体内MCL-1在细胞凋亡调控中的作用(S)。我们的初步研究表明，只有MCL-1的外部线粒体形式能够结合和隔离促凋亡分子。然而，目前尚不清楚MCL-1的内部线粒体形式是否会改变对死亡刺激的反应。因此，我们将研究不同形式的MCL-1如何对其经典的抗细胞凋亡特性做出贡献。目的3：研究MCL-1对线粒体生理的调节作用。我们的初步研究表明，Mcl-1缺乏会导致线粒体生理异常，即使细胞没有发生凋亡。我的实验室已经取得了许多开创性的发现，确定了MCL-1在促进造血过程中的存活方面的作用。此外，我们具有得天独厚的优势，可以成功地进行这些研究，因为我们已经确定了新的定位，产生了可以剖析不同形式的MCL-1的突变体，并且在MCL-1生物学方面拥有丰富的经验。在这项研究的最后，我们将阐明以前未被认识到的MCL-1在促进线粒体功能中的作用。

DESCRIPTION (provided by applicant): Anti-apoptotic BCL-2 family members regulate hematopoiesis and when dysregulated contribute to cancer, immunodeficiency, and autoimmunity. MCL-1 is absolutely required for cell survival at multiple stages of hematopoietic development. In contrast, genetic ablation of Bcl-2 or Bcl-X has revealed specific roles in promoting hematopoietic survival. Why MCL-1 plays such a critical role in maintaining survival and why the concomitant endogenous expression of other anti-apoptotic regulators cannot compensate for MCL-1 loss has remained unresolved. Therefore, the long term goal of my laboratory is to understand how MCL-1 functions and is regulated during hematopoietic development and survival. To understand the requirement for MCL-1 in development, we have recently identified that it localizes to both the outer mitochondrial membrane, where it MCL-1 binds and sequesters pro-apoptotic molecules, and to the mitochondrial inner membrane as an N- terminally truncated form. The contribution of both of these localizations to MCL-1's function is unclear. The objective of this proposal is to dissect the functional roles of the two mitochondrial forms of MCL-1 and to assess the individual roles of these localizations in promoting cellular survival. Our central hypothesis is that the outer membrane form of MCL-1 acts like "classical" BCL-2 family members to antagonize pro-apoptotics, while the inner membrane form of MCL-1 regulates cell survival by modulating mitochondrial function. The studies we propose will reveal a previously unrecognized role for MCL-1 and shed light onto a new mechanism by which an anti-apoptotic BCL-2 family member promotes mitochondrial function. Aim 1: Define the role(s) for the different mitochondrial forms of MCL-1 in regulating hematopoiesis. MCL-1 exhibits a profound requirement during many stages of hematopoiesis, but it is unclear why. Aim 2: Define the role(s) of inner mitochondrial MCL-1 in regulating apoptosis. Our preliminary studies indicate that only the outer mitochondrial form of MCL-1 is capable of binding and sequestering pro-apoptotic molecules. However, it is unclear whether the inner mitochondrial form of MCL-1 may alter response to death stimuli. Therefore, we will investigate how the different forms of MCL-1 contribute to its "classical" anti-apoptotic nature. Aim 3: Identify how MCL-1 regulates mitochondrial physiology. Our preliminary studies indicate that Mcl-1-deficiency results in abnormalities in mitochondrial physiology even when cells are not undergoing apoptosis. My laboratory has made many of the seminal findings defining the role of MCL-1 in promoting survival during hematopoiesis. Furthermore, we are uniquely positioned to successfully perform these studies as we have identified the novel localization, generated MCL-1 mutants that can dissect the different forms of MCL-1, and have extensive experience in MCL-1 biology. At the end of this study, we will have illuminated a previously unrecognized role for MCL-1 in promoting mitochondrial function.