Protein quality control, cardiomyopathy, cardiotoxicity and human isogenic iPSCs

蛋白质质量控​​制、心肌病、心脏毒性和人类同基因 iPSC

• 批准号: 9930312
• 负责人: Bruce R Conklin
• 金额: $ 4.41万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9930312
• 关键词: Affinity Chromatography; Autophagocytosis; BAG3 gene; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiotoxicity; Cell Line; Cell physiology; Cells; Chemotherapy-Oncologic Procedure; Client; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Defect; Development; Dilated Cardiomyopathy; Disease; Disease Pathway; Drug Targeting; Drug toxicity; Drug usage; Engineering; Foundations; Future; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Diseases; Genetic Epistasis; Genetic screening method; Genome engineering; Genomics; Goals; Heart; Heart Diseases; Heart failure; Heat shock proteins; Heat-Shock Proteins 70; Human; Lead; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mediating; Modeling; Molecular; Molecular Chaperones; Mutate; Mutation; Myocardium; Pathway interactions; Patients; Peripheral Nerves; Pharmaceutical Preparations; Phenotype; Play; Point Mutation; Process; Proteasome Inhibitor; Proteins; Quality Control; Reporting; Role; Sarcomeres; Skeletal Muscle; Stress; System; Testing; Therapeutic; Tissues; adverse event risk; base; cardiac muscle disease; clinical phenotype; clinically relevant; disease phenotype; gene interaction; genetic predictors; heart cell; human model; human stem cells; induced pluripotent stem cell; insight; link protein; misfolded protein; multicatalytic endopeptidase complex; mutant; patient subsets; prevent; protein aggregation; protein folding; scaffold; tool

PROJECT SUMMARY/ABSTRACT Dilated cardiomyopathy (DCM) is often associated with accumulation of misfolded proteins thought to result from aberrant protein quality control. Chemotherapeutic drugs that target the proteasome (a key component of protein quality control) are associated with cardiotoxicity and heart failure. We hypothesize that the DCM- associated BAG3 protein regulates protein quality control in the heart and plays a critical role in cardiomyopathy and cardiotoxicity. BAG3 serves as a scaffold that binds and coordinates two classes of molecular chaperones: heat shock proteins, HSPBs and HSP70s. The BAG3 complex is stress-inducible and orchestrates protein folding, proteasomal degradation, and autophagy—all critical steps in protein quality control. In cardiac and skeletal muscle, this chaperone complex is localized in the Z-disk, where it is poised to regulate specific sarcomere client proteins. Mutations in BAG3 cause DCM and mutation-specific clinical phenotypes, suggesting a link with distinct cellular processes and disease pathways. We will test specific models of BAG3 function by engineering point mutations in BAG3 in isogenic human induced pluripotent stem cells (iPSCs) to produce cardiomyocytes (iPS-CMs). We have made significant progress in iPSC genome engineering to produce iPS-CMs and model human cardiac disease. We are also developing gene regulation tools based on CRISPR inhibition (CRISPRi) to rapidly silence genes to validate putative BAG3 interactions. Our aims provide a clear path to these goals. Aim 1: Identify the cellular processes involved in BAG3 cardiomyopathy in isogenic iPSC lines bearing disease-associated BAG3 mutations. We are making heterozygous and homozygous isogenic iPS- CMs that harbor clinically relevant mutations in the endogenous BAG3 locus. Aim 2: Directly define the role of BAG3-binding partners in the development of a cardiomyopathy phenotype in iPS-CMs by silencing candidate BAG3 interactors with CRISPRi. We hypothesize that specific BAG3 protein-binding partners contribute to the DCM phenotype. Aim 3: Determine if proteasome inhibitors and other chemotherapeutics cause cardiotoxicity in a manner dependent on specific components of the protein QC network. We hypothesize that altered function of the BAG3 chaperone complex leads to enhanced cardiotoxicity of proteasome inhibitors. We propose to comprehensively determine the mechanistic role of the BAG3 network in human cardiomyocytes and in DCM. A fundamental understanding of BAG3-mediated cardiac protein quality control will provide insights into disease mechanism, drug toxicity, and potential therapeutic options. Our studies lay the foundation for predictive genetic testing to understand genetic disease and avoid cardiotoxicity. We are hopeful that mechanistic insights will lead to treatments for cardiomyopathy and diseases of aberrant protein quality control.

项目总结/文摘