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

确定线粒体基质定位的 MCL-1 非凋亡功能背后的机制

基本信息

批准号：
10537709
负责人：
Tristen Wright
金额：
$ 4.28万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL GRADUATE SCHOOL OF BIOMEDICAL SCIENCES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10537709
关键词：
Address Adoptive Transfer Affect Affinity Antioxidants Apoptosis Apoptotic Autoimmunity BAX gene BCL2 gene BCL2L1 gene BCL2L11 gene Back Binding Bioenergetics Biological Assay CD8-Positive T-Lymphocytes Cardiac Myocytes Caspase Inhibitor Cell Death Cell Lineage Cells Cessation of life Citrates Citric Acid Cycle Clinical Trials Confocal Microscopy Crista ampullaris Data Defect Embryo Embryonic Development Environment Epitopes Family Fibroblasts Flow Cytometry Gene Expression Profile Generations Genus Hippocampus Glutamine Goals Hematology Hematopoietic stem cells Homeostasis Hour Human Pathology Immunoprecipitation In Vitro Investigation Knock-out Light Link Listeria monocytogenes Liver Lymphocyte MCL1 gene Malates Malignant Neoplasms Mass Spectrum Analysis Measures Memory Metabolic Metabolism Mitochondria Mitochondrial Matrix Morphology Mouse Strains Mus Mutant Strains Mice Nerve Degeneration Neurons OPA1 gene Outer Mitochondrial Membrane Ovalbumin Oxidative Phosphorylation Oxygen Consumption Pathology Pathway interactions Physiological Physiology Play Production Protein Dynamics Protein Family Protein Isoforms Proteins Reduced Glutathione Regulation Reporting Resources Respiration Role Saint Jude Children&apos s Research Hospital Scientist Solid Neoplasm Splenocyte Spottings Stains Stress Tests Structure Supplementation T memory cell T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Training Transgenic Mice Withdrawal alpha ketoglutarate cancer cell clinical application confocal imaging congenic deprivation experimental study in vivo inhibitor insight mRNA sequencing metabolomics mutant neutrophil novel overexpression prevent small molecule inhibitor tool transcriptome sequencing

Address Adoptive Transfer Affect Affinity Antioxidants Apoptosis Apoptotic Autoimmunity BAX gene BCL2 gene BCL2L1 gene BCL2L11 gene Back Binding Bioenergetics Biological Assay CD8-Positive T-Lymphocytes Cardiac Myocytes Caspase Inhibitor Cell Death Cell Lineage Cells Cessation of life Citrates Citric Acid Cycle Clinical Trials Confocal Microscopy Crista ampullaris Data Defect Embryo Embryonic Development Environment Epitopes Family Fibroblasts Flow Cytometry Gene Expression Profile Generations Genus Hippocampus Glutamine Goals Hematology Hematopoietic stem cells Homeostasis Hour Human Pathology Immunoprecipitation In Vitro Investigation Knock-out Light Link Listeria monocytogenes Liver Lymphocyte MCL1 gene Malates Malignant Neoplasms Mass Spectrum Analysis Measures Memory Metabolic Metabolism Mitochondria Mitochondrial Matrix Morphology Mouse Strains Mus Mutant Strains Mice Nerve Degeneration Neurons OPA1 gene Outer Mitochondrial Membrane Ovalbumin Oxidative Phosphorylation Oxygen Consumption Pathology Pathway interactions Physiological Physiology Play Production Protein Dynamics Protein Family Protein Isoforms Proteins Reduced Glutathione Regulation Reporting Resources Respiration Role Saint Jude Children&apos s Research Hospital Scientist Solid Neoplasm Splenocyte Spottings Stains Stress Tests Structure Supplementation T memory cell T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Training Transgenic Mice Withdrawal alpha ketoglutarate cancer cell clinical application confocal imaging congenic deprivation experimental study in vivo inhibitor insight mRNA sequencing metabolomics mutant neutrophil novel overexpression prevent small molecule inhibitor tool transcriptome sequencing

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项目摘要

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.

项目摘要凋亡的异常调节是人类病理的标志，包括神经变性，自身免疫性，和癌症。固有凋亡受Bcl-2家族的调节，Bcl-2家族由抗凋亡蛋白组成（例如，Bcl-2和MCl-1）哪个隔离pro-凋亡BH3仅蛋白质（例如BIM和坏蛋白）或直接抑制促凋亡作用，Bax和Bak，以防止其低聚。许多癌细胞过表达抗凋亡蛋白促进异常生存。 Mcl-1在抗凋亡蛋白中是独一无二的早期胚胎发育和许多细胞谱系的生存至关重要（例如造血茎细胞，淋巴细胞，神经粒，神经元和心肌细胞）。我们的实验室以前报道MCL-1有两个同工型 - 一种定位于外部线粒体膜（OMM）的同工型，一种定位于线粒体矩阵。尽管OMM MCL-1的规范抗凋亡功能已经很好地表征了矩阵MCL-1的作用仍然很大未知。有证据表明矩阵MCL-1可以保持正常线粒体裂变/融合，氧化磷酸化和Cristae超微结构。这些生理学的基础 MCL-1的角色仍然未知。 Mcl1-缺陷型（Mcl1 - / - ）鼠类胚胎的代谢组研究成纤维细胞（MEF）和缺乏基质量化MCL1的MEF显示它们对谷氨酰胺高度敏感剥夺与野生型MEF相比。另外，24小时后MCL-1蛋白水平降低野生型MEF的谷氨酰胺提取。这些数据表明MCL-1和谷氨酰胺代谢之间有联系可以将其连接到MCL1缺失时观察到的线粒体缺陷。目标的目标建议是确定矩阵MCL-1的功能并获得对这些功能的机械理解。我假设基质定位的mcl-1在维持维持线粒体形态和生物能学。为了解决这一假设，我将使用一种新型的突变鼠标该内源表达截短的Mcl-1蛋白（MCL-1OM），该蛋白会阻塞凋亡，但不能是进口到线粒体矩阵中。首先，我将向Mcl1突变体向MCL1 - / - MEFS表达确定哪种版本的MCL1可以挽救谷氨酰胺提取触发的死亡。我也会表演 mcl1 –/ - ，mcl1+/+，mcl1 - /+和mcl1 - /om mefs上的mRNA-seq，以询问由损失引起的代谢重新布线矩阵定位的mcl-1。第二，我将评估线粒体功能（例如，Seahorse XF Mito应力测试和 TMRE染色），确定与基质 - 定位Mcl-1相互作用的蛋白质，异位表达OPA1和 Mcl1 - / - MEF中的DRP1突变体，并在这些细胞上执行共共聚焦成像，以确定基质的功能线粒体形态中的局部MCL-1。最后，我将在MCL1-/OM小鼠上进行体内实验分析T细胞激活，效应子功能和记忆T细胞的产生。这些发现将提供新的机械洞察基质化的MCL-1的非凋亡作用，并可能阐明电势将MCL-1抑制剂用于临床应用的后果。