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. PUBLIC HEALTH RELEVANCE: Defects in the balance of cell survival and death can lead to a variety of human diseases including autoimmunity, immunodeficiency, and cancer. MCL-1 is a critical regulator of blood cell survival whose expression is tightly controlled in normal cells, but in cancerous cells such control is lost leading to inappropriate survival. This proposal is relevant to the goals of the National Institutes of Health because it investigates a novel role for MCL-1 in normal blood cells and will assess how MCL-1 can render cancer cells resistant to death.