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

• 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
• 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

 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（由适用提供）：UNC-45蛋白链酮是UNC-45/CRO1/SHE4P（UCS）家族的成员。它的损失导致多种动物模型中的骨骼骨骼和心肌发育异常，这表明其配置的进化功能。 UNC-45通过其N末端TPR结构域与链条HSP90和HSP70相互作用，并通过其C末端UCS域与肌球蛋白运动区域相互作用。尽管其机制尚不清楚，但UNC-45在肌球蛋白运动折叠过程中似乎是脚手架和伴侣。 UNC-45表达水平的改变与纳入身体肌病和心脏衰竭有关，将UNC-45与人类疾病联系起来。最近，第一个人类UNC-45突变体被证明会引起少年白内障。最后，我们的小组最近发现，UNC-45是蛋白质折叠疾病的可行疗法。该提案的目的是通过使用完整的遗传和生化方法来识别新型的UNC-45相互作用伴侣，扩大我们对UNC-45功能及其作用机理的理解。为此，我们将染色体段单倍体缺陷线越过UNC-45单倍体（敏化）背景和筛选飞行能力作为肌肉功能的读数。我们确定了几条候选缺陷线，这些缺陷线与UNC-45的单倍体损失有关，表现出较差的飞行能力。目标1 该项目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敏感背景中存在有缺陷的肌肉发育的特定基因。这将检验以下假设：候选染色体段缺陷线具有特定的基因，其产物在肌肉发育过程中与UNC-45相互作用。 AIM 2将采用完整的生化方法来隔离和鉴定UNC-45相互作用的伴侣使用共免疫沉淀，然后进行质谱。表达UNC-45：GFP融合蛋白的转基因蝇将使我们能够捕获和分析在苍蝇发育和生物胁迫下形成的UNC-45复合物。这将检验以下假设：unc-45相互作用的伴侣在肌肉发育和压力过程中发生变化。 AIM 3A将使用共聚焦显微镜在体内定位识别的UNC-45相互作用伙伴，并将使用RNAi AIM 3B在功能上询问已鉴定伴侣使用肌球蛋白聚集保护和柠檬酸酸酯合成酶折叠测定法调节UNC-45链酮活性的能力。我们还将使用热诱导的聚合保护测定法调查是否鉴定出蛋白质是UNC-45客户蛋白。 AIM 3将解决以下假设：UNC-45相互作用伙伴会影响UNC-45链酮活动或充当肌肉细胞中的UNC-45客户。总体而言，我们的项目将对UNC-45相互作用伙伴及其在肌肉发育过程中的UNC-45生物学中的功能作用提供综合分析。它将在果蝇遗传学，细胞生物学和生物化学以及科学写作，研究实验室监督和教学方面提供出色的培训。