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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：
10676766
负责人：
Tristen Wright
金额：
$ 4.37万
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/10676766
关键词：
Address Adoptive Transfer Affect Affinity Antioxidants Apoptosis Apoptotic Autoimmunity BAX gene BCL1 Oncogene BCL2L1 gene BCL2L11 gene Back Binding Bioenergetics Biological Assay Breeding 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 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 Phosphorylation 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

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.

项目总结