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

项目摘要 细胞凋亡的异常调节是人类病理学的标志，包括神经变性，自身免疫， 和癌症内源性凋亡受BCL-2家族的调控，BCL-2家族由抗凋亡蛋白组成 (e.g., BCL-2和MCL-1），其螯合促凋亡的仅BH 3蛋白（例如，BIM和BAD）或直接抑制 促凋亡效应物BAX和巴克，防止其寡聚化。许多癌细胞过度表达抗- 凋亡蛋白促进异常存活。MCL-1在抗凋亡蛋白中是独特的，因为它是 在早期胚胎发育和许多细胞谱系的存活中是必需的（例如，造血干 细胞、淋巴细胞、嗜中性粒细胞、神经元和心肌细胞）。我们的实验室先前报道MCL-1有两个 同种型-一种定位于线粒体外膜（OMM），另一种定位于 线粒体基质尽管OMM MCL-1的典型抗凋亡功能已经得到很好的表征， 基质MCL-1的作用仍然很大程度上未知。有证据表明，基质MCL-1有助于维持正常的 线粒体分裂/融合、氧化磷酸化和嵴超微结构。这些生理基础 MCL-1的作用仍然未知。Mcl 1-/-小鼠胚胎的代谢组学研究 成纤维细胞（MEFs）和缺乏基质定位Mcl 1的MEFs显示，它们对谷氨酰胺高度敏感 与野生型MEF相比，此外，MCL-1蛋白水平在24小时后降低， 野生型MEFs中谷氨酰胺的退出。这些数据表明MCL-1和谷氨酰胺代谢之间的联系 这可能与Mcl 1缺失后观察到的线粒体缺陷有关。这个目标 建议是确定矩阵MCL-1的功能，并获得这些功能的机械理解。 我推测，基质定位的MCL-1在维持细胞凋亡中起着重要的非凋亡作用。 线粒体形态学和生物能量学。为了解决这个假设，我将使用一种新的突变小鼠， 其内源性表达截短的MCL-1蛋白（MCL-1 OM），其阻断细胞凋亡但不能被 输入到线粒体基质中。首先，我将异位表达Mcl 1突变体回到Mcl 1-/- MEFs中， 确定哪种版本的Mcl 1可以挽救谷氨酰胺停药引发的死亡。我也会表演 Mcl 1-/-、Mcl 1 +/+、Mcl 1-/+和Mcl 1-/OM MEFs上的mRNA-Seq，以询问由损失诱导的代谢重新连接 基质定位的MCL-1其次，我将评估线粒体功能（例如，海马XF水户压力测试和 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Opa 1， Mcl 1-/- MEFs中Drp 1突变体，并对这些细胞进行共聚焦成像，以确定基质- 定位MCL-1的线粒体形态。最后，我将对Mcl 1-/OM小鼠进行体内实验， 分析T细胞活化、效应子功能和记忆T细胞生成。这些发现将提供新的， 对基质定位的MCL-1的非凋亡作用的机制见解，并可以揭示其潜力 将MCL-1抑制剂用于临床应用的后果。

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.