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: 9384644
• 负责人: Bruce R Conklin
• 金额: $ 47.2万
• 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/9384644
• 关键词: Adverse event; 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; Genes; Genetic; Genetic Epistasis; Genetic screening method; Genome engineering; Genomics; Goals; Heart; Heart Diseases; Heart failure; Heat shock proteins; Heat-Shock Proteins 70; Hereditary Disease; Human; Lead; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mediating; Modeling; Molecular; Molecular Chaperones; Mutate; Mutation; Myocardium; Myopathy; Pathway interactions; Patients; Peripheral Nerves; Pharmaceutical Preparations; Phenotype; Play; Point Mutation; Process; Proteasome Inhibitor; Proteins; Quality Control; Reporting; Risk; Role; Sarcomeres; Skeletal Muscle; Stress; System; Testing; Therapeutic; Tissues; base; clinical phenotype; clinically relevant; disease phenotype; gene interaction; genetic predictors; heart cell; 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

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.

扩张型心肌病（DCM）通常与错误折叠蛋白质的积累有关， 异常的蛋白质质量控制。针对蛋白酶体（蛋白酶体的关键组成部分）的化疗药物 蛋白质质量控制）与心脏毒性和心力衰竭有关。我们假设DCM- 相关的BAG3蛋白调节心脏中的蛋白质质量控制， 心肌病和心脏毒性。BAG3充当结合和协调两类BAG3的支架。 分子伴侣：热休克蛋白，HSPB和HSP70。BAG3复合物是应激诱导的， 协调蛋白质折叠，蛋白酶体降解和自噬-蛋白质质量的所有关键步骤 控制在心肌和骨骼肌中，这种伴侣蛋白复合物定位于Z盘中，在那里它准备 调节特定的肌节客户蛋白。BAG3突变导致DCM和突变特异性临床 表型，表明与不同的细胞过程和疾病途径的联系。我们将测试具体的 通过在同基因人诱导多能干细胞中的BAG3中工程化点突变来建立BAG3功能模型 细胞（iPSC）以产生心肌细胞（iPS-CM）。我们在iPSC基因组方面取得了重大进展 工程化以生产iPS-CM并模拟人类心脏病。我们也在研究基因调控 基于CRISPR抑制（CRISPRi）的工具，用于快速沉默基因，以验证推定的BAG3相互作用。 我们的目标为实现这些目标提供了明确的途径。 目的1：在同基因iPSC系中鉴定参与BAG3心肌病的细胞过程 携带疾病相关的BAG3突变。我们正在制造杂合和纯合的同基因iPS- 在内源性BAG3基因座中具有临床相关突变的CM。 目的2：直接定义BAG3结合伴侣在心肌病发展中的作用 通过用CRISPRi沉默候选BAG3相互作用物来在iPS-CM中表达表型。我们假设 特异性BAG3蛋白结合配偶体促成DCM表型。 目的3：确定蛋白酶体抑制剂和其他化疗药物是否会导致心脏毒性。 方式取决于蛋白质QC网络的特定组件。我们假设 BAG3分子伴侣复合物的功能导致蛋白酶体抑制剂的心脏毒性增强。 我们建议全面确定BAG3网络在人类中的机制作用。 心肌细胞和DCM中。对BAG3介导的心脏蛋白质质量控制的基本认识 将提供疾病机制，药物毒性和潜在的治疗选择的见解。我们的研究 预测性基因检测的基础，以了解遗传疾病和避免心脏毒性。我们 希望机械的见解将导致心肌病和异常蛋白质疾病的治疗 质量控制