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

描述（由申请人提供）：抗凋亡BCL-2家族成员调节造血功能，当失调时会导致癌症、免疫缺陷和自身免疫。MCL-1在造血发育的多个阶段都是细胞存活所必需的。相比之下，基因消融Bcl-2或Bcl-X在促进造血存活方面具有特殊作用。为什么MCL-1在维持生存中起着如此关键的作用，以及为什么伴随的其他抗凋亡调节因子的内源性表达不能补偿MCL-1的损失仍未得到解决。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控。为了了解MCL-1在发育过程中的需求，我们最近发现它定位于线粒体外膜，在那里MCL-1结合和隔离促凋亡分子，并以N端截断的形式定位于线粒体内膜。这两种定位对MCL-1功能的贡献尚不清楚。本提案的目的是剖析MCL-1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中的个体作用。我们的中心假设是MCL-1的外膜形式像“经典”BCL-2家族成员一样拮抗促凋亡，而MCL-1的内膜形式通过调节线粒体功能来调节细胞存活。我们提出的研究将揭示MCL-1以前未被认识的作用，并揭示抗凋亡BCL-2家族成员促进线粒体功能的新机制。目的1：明确不同线粒体形式MCL-1在调节造血中的作用。MCL-1在造血过程的许多阶段表现出深刻的需求，但原因尚不清楚。目的2：明确线粒体内MCL-1在调节细胞凋亡中的作用。我们的初步研究表明，只有外线粒体形式的MCL-1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如何促进其“经典”抗凋亡性质。目的3：确定MCL-1如何调节线粒体生理。我们的初步研究表明，即使细胞未发生凋亡，mcl -1缺乏也会导致线粒体生理异常。我的实验室已经做出了许多开创性的发现，定义了MCL-1在促进造血过程中生存的作用。此外，我们在成功开展这些研究方面具有独特的优势，因为我们已经确定了新的定位，产生了可以解剖不同形式MCL-1的MCL-1突变体，并且在MCL-1生物学方面拥有丰富的经验。在本研究结束时，我们将阐明MCL-1在促进线粒体功能方面以前未被认识到的作用。

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.