Identifying the mechanisms behind non-apoptotic functions of mitochondrial matrix-localized MCL-1

项目总结 细胞凋亡的异常调节是人类病理的一个标志，包括神经退行性变、自身免疫、 和癌症。内源性细胞凋亡是由抗凋亡蛋白组成的bcl2家族调控的 (例如，BCL-2和MCL-1)隔离促凋亡的BH3蛋白(例如，BIM和BAD)或直接抑制 促凋亡效应分子BAX和BAK，防止其寡聚化。许多癌细胞过度表达抗- 促进异常存活的凋亡蛋白。MCL-1在抗凋亡蛋白中是独一无二的，因为它是 对早期胚胎发育和许多细胞系(例如，造血干细胞)的生存至关重要 细胞、淋巴细胞、中性粒细胞、神经元和心肌细胞)。我们的实验室之前报告了MCL-1有两个 异构体--一种定位于线粒体膜外膜(OMM)，另一种定位于 线粒体基质。虽然OMM MCL-1‘S的典型抗细胞凋亡功能已经被很好地表征，但 基质MCL-1的作用在很大程度上仍不清楚。有证据表明，基质MCL-1用于维持正常 线粒体分裂/融合、氧化磷酸化和冠状超微结构。这些生理学的基础 MCL-1的作用尚不清楚。MCL1缺陷(MCL1-/-)小鼠胚胎的代谢研究 成纤维细胞和缺乏基质结合MCL1的成纤维细胞对谷氨酰胺高度敏感 与野生型MEF相比，剥夺。此外，MCL-1蛋白水平在24小时后下降 野生型MEF中谷氨酰胺的戒断。这些数据表明MCL-1和谷氨酰胺代谢之间存在联系。 这可能与MCL1缺失时观察到的线粒体缺陷有关。这样做的目的是 建议确定MCL-1矩阵的功能，并对这些功能有一个机械性的理解。 我假设基质定位的mcl-1在维持 线粒体形态和生物能量学。为了解决这一假设，我将使用一种新的突变小鼠 内源性表达截短的MCL-1蛋白(MCL-1OM)，可阻止细胞凋亡，但不能 输入到线粒体基质中。首先，我将异位表达MCL1突变体回到MCL1-/-MEF中，以 确定哪个版本的mcl1(S)可以挽救谷氨酰胺戒断引发的死亡。我也会表演 MCL1-/-、MCL1+/+、MCL1-/+和MCL1-/OMMEF上的mRNAs-SEQ研究缺失引起的代谢重联 定位于基质的MCL-1。其次，我将评估线粒体功能(例如，海马XF Mito压力测试和 TMRE染色)，确定与基质定位的MCL-1相互作用的蛋白质，异位表达OPA1和 在MCL1-/-MEF中突变Drp1，并对这些细胞进行共聚焦成像，以确定基质- MCL-1定位于线粒体形态。最后，我将在MCL1-/OM小鼠身上进行活体实验，以 分析T细胞激活、效应器功能和记忆T细胞生成。这些发现将提供新的、 对基质定位的MCL-1的非凋亡作用的机械性见解，并可能揭示其潜在的作用 临床使用MCL-1抑制剂的后果。

PROJECT SUMMARY Aberrant regulation of apoptosis is a hallmark of human pathologies, including neurodegeneration, autoimmunity, and cancer. Intrinsic apoptosis is regulated by the BCL-2 family which is composed of anti-apoptotic proteins (e.g., BCL-2 and MCL-1) which sequester pro-apoptotic BH3-only proteins (e.g., BIM and BAD) or directly inhibit pro-apoptotic effectors, BAX and BAK, preventing their oligomerization. Many cancer cells overexpress anti- apoptotic proteins to promote aberrant survival. MCL-1 is unique among anti-apoptotic proteins because it is essential in early embryonic development and for the survival of many cell lineages (e.g., hematopoietic stem cells, lymphocytes, neutrophils, neurons, and cardiomyocytes). Our lab previously reported that MCL-1 has two isoforms –– one that localizes to the outer mitochondrial membrane (OMM) and one that localizes to the mitochondrial matrix. Although OMM MCL-1’s canonical anti-apoptotic function has been well characterized, the roles of matrix MCL-1 are still largely unknown. Evidence suggests that matrix MCL-1 serves to maintain normal mitochondrial fission/fusion, oxidative phosphorylation, and cristae ultrastructure. The basis for these physiologic roles of MCL-1 remains unknown. Metabolomic investigation of Mcl1–deficient (Mcl1–/–) murine embryonic fibroblasts (MEFs) and MEFs lacking matrix-localized Mcl1 revealed that they are highly sensitive to glutamine deprivation as compared to wild-type MEFs. Additionally, MCL-1 protein levels decrease after 24 hours of glutamine withdrawal in wild-type MEFs. These data suggest a link between MCL-1 and glutamine metabolism that could be connected to the mitochondrial defects that are observed upon Mcl1 deletion. The goal of this proposal is to determine the functions of matrix MCL-1 and gain a mechanistic understanding of these functions. I hypothesize that matrix-localized MCL-1 plays an essential, non-apoptotic role in maintaining mitochondrial morphology and bioenergetics. To address this hypothesis, I will use a novel mutant mouse that endogenously expresses a truncated MCL-1 protein (MCL-1OM) which blocks apoptosis but cannot be imported into the mitochondrial matrix. First, I will ectopically express Mcl1 mutants back into Mcl1–/– MEFs to determine which version(s) of Mcl1 can rescue the death triggered by glutamine withdrawal. I will also perform mRNA-Seq on Mcl1–/–, Mcl1+/+, Mcl1–/+, and Mcl1–/OM MEFs to interrogate the metabolic rewiring induced by loss of matrix-localized MCL-1. Second, I will assess mitochondrial function (e.g., Seahorse XF Mito Stress Test and TMRE staining), determine the proteins interacting with matrix-localized MCL-1, ectopically express Opa1 and Drp1 mutants in Mcl1–/– MEFs, and perform confocal imaging on these cells to determine the functions of matrix- localized MCL-1 in mitochondrial morphology. Finally, I will perform in vivo experiments on Mcl1-/OM mice to analyze T cell activation, effector function, and memory T cell generation. These findings will provide new, mechanistic insights into the non-apoptotic roles of matrix-localized MCL-1 and could shed light on the potential consequences of using MCL-1 inhibitors for clinical applications.