Identification and functional analysis of UNC-45 chaperone partners in muscle

肌肉中UNC-45分子伴侣的鉴定和功能分析

基本信息

批准号：
9319213
负责人：
Daniel Smith
金额：
$ 6.1万
依托单位：
SAN DIEGO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9319213
关键词：
Address Adolescent Affect Alleles Animal Model Biochemical Biochemistry Biological Biological Assay Biological Models Biology C-terminal Caenorhabditis elegans Candidate Disease Gene Cataract Cells Cellular biology Chimeric Proteins Chromosomes Citrate (si)-Synthase Client Co-Immunoprecipitations Complex Confocal Microscopy Defect Development Disease Drosophila genus Educational process of instructing Electron Microscopy Exhibits Family Fluorescence Microscopy Genes Genetic Genetic Screening Genetic Techniques Goals Haploidy Heart failure Human Immunoprecipitation In Situ Hybridization Inclusion Bodies Individual Investigation Knowledge Life Cycle Stages Link Maps Mass Spectrum Analysis Mediating Mentors Methodology Molecular Chaperones Motor Muscle Muscle Cells Muscle Development Muscle function Myocardium Myopathy Myosin ATPase N-terminal Organism Phenotype Protein-Folding Disease Proteins RNA Interference Regulation Reporter Reporting Research Role Sarcomeres Scheme Skeletal Muscle Specificity Stress Striated Muscles Supervision Techniques Tertiary Protein Structure Testing Therapeutic Therapeutic Uses Training Transgenic Organisms Writing base biological adaptation to stress experimental study fly gene product genetic approach human disease in vitro activity in vivo insight interest knock-down member muscle stress mutant novel prospective public health relevance response scaffold screening skeletal abnormality trait

项目摘要

DESCRIPTION (provided by applicant): The UNC-45 protein chaperone is a member of the UNC-45/CRO1/She4p (UCS) family. Its loss results in abnormal skeletal and cardiac muscle development in several animal models, suggesting a conserved evolutionary function. UNC-45 interacts with chaperones Hsp90 and Hsp70 through its N-terminal TPR domain and with the myosin motor region through its C-terminal UCS domain. While its mechanism is unclear, UNC-45 appears to act as both scaffold and chaperone during myosin motor folding. Alterations in UNC-45 expression levels are associated with inclusion body myopathy and cardiac failure, linking UNC-45 to human disease. Recently, the first human UNC-45 mutant was shown to cause juvenile cataracts. Finally, our group recently found that UNC-45 is a viable therapeutic in protein folding diseases. The goal of this proposal is to expand our understanding of UNC-45 function and its mechanism of action by identifying novel UNC-45 interacting partners using complementary genetic and biochemical approaches. To this end, we crossed chromosomal-segment haploid deficiency lines onto an UNC-45 haploid (sensitized) background and screened flight ability as a readout of muscle function. We identified several candidate deficiency lines that exhibit poor flight ability in conjunction with haploid loss of UNC-45. Aim 1 of this project will utilize the powerful mutant allele and RNAi knockdown techniques in Drosophila to map specific genes that exhibit defective muscle development in the UNC-45 sensitized background. This will test the hypothesis that candidate chromosomal-segment deficiency lines harbor specific genes whose products functionally interact with UNC-45 during muscle development. Aim 2 will employ a complementary biochemical approach to isolate and identify UNC-45 interacting partners using co-immunoprecipitation followed by mass spectrometry. A transgenic fly expressing an UNC-45:GFP fusion protein will allow us to capture and analyze UNC-45 complexes formed during fly development and organism stress. This will test the hypothesis that UNC-45 interacting partners change during muscle development and stress. Aim 3a will localize identified UNC-45 interacting partners in vivo using confocal microscopy and will use RNAi knockdown to assess their roles in muscle development. Aim 3b will functionally interrogate the ability of identified partners to modulate UNC-45 chaperone activity using myosin aggregation protection and citrate synthase folding assays. We will also investigate whether identified proteins act as UNC-45 client proteins using heat-induced aggregation protection assays. Aim 3 will address the hypothesis that UNC-45 interacting partners affect UNC-45 chaperone activity or act as UNC-45 clients within muscle cells. Overall, our project will provide an integrative analysis of UNC-45 interacting partners and their functional roles in UNC-45 biology during muscle development. It will offer outstanding training in Drosophila genetics, cell biology and biochemistry as well as mentoring in scientific writing, research lab supervision and teaching.

描述（由申请人提供）：UNC-45 蛋白伴侣是 UNC-45/CRO1/She4p (UCS) 家族的成员，其缺失导致多种动物模型骨骼和心肌发育异常，表明其具有保守的进化功能。 UNC-45 通过其 N 端 TPR 结构域与伴侣 Hsp90 和 Hsp70 相互作用，并通过其 C 端 UCS 与肌球蛋白运动区相互作用虽然其机制尚不清楚，但 UNC-45 似乎在肌球蛋白运动折叠过程中充当支架和伴侣。UNC-45 表达水平的改变与包涵体肌病和心力衰竭有关，最近将 UNC-45 与人类疾病联系起来。，第一个人类 UNC-45 突变体被证明会导致青少年白内障。最后，我们的小组最近发现 UNC-45 是蛋白质折叠疾病的一种可行的治疗方法。该提案的目标是扩大我们对蛋白质折叠疾病的了解。通过使用遗传互补和生化方法鉴定新的 UNC-45 相互作用伙伴，UNC-45 的功能及其作用机制为此，我们将染色体片段单倍体缺陷系杂交到 UNC-45 单倍体（致敏）背景上并筛选飞行能力。作为肌肉功能的读数，我们确定了几个候选缺陷系，这些缺陷系表现出与 UNC-45 单倍体丢失相关的较差飞行能力。该项目的研究人员将利用果蝇中强大的突变等位基因和 RNAi 敲低技术来绘制在 UNC-45 致敏背景下表现出肌肉发育缺陷的特定基因，这将检验候选染色体片段缺陷系含有其产物具有功能性的特定基因的假设。目标 2 将采用互补的生化方法，通过免疫共沉淀和质量分析来分离和鉴定 UNC-45 相互作用的伙伴。表达 UNC-45:GFP 融合蛋白的转基因果蝇将使我们能够捕获和分析果蝇发育和生物体应激过程中形成的 UNC-45 复合物，这将检验 UNC-45 相互作用伙伴在肌肉发育和应激过程中发生变化的假设。 Aim 3a 将使用共聚焦显微镜定位已识别的 UNC-45 伴侣在体内相互作用，并将使用 RNAi 敲低来评估它们在肌肉发育中的作用。 Aim 3b 将在功能上询问已识别的能力。我们还将使用热诱导聚集保护测定来研究已识别的蛋白质是否充当 UNC-45 客户蛋白，目标 3 将解决 UNC-45 的假设。相互作用的伙伴影响 UNC-45 伴侣活性或充当肌肉细胞内的 UNC-45 客户总体而言，我们的项目将提供 UNC-45 的综合分析。它将提供果蝇遗传学、细胞生物学和生物化学方面的出色培训，以及科学写作、研究实验室监督和教学方面的指导。