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

 描述(申请人提供)：UNC-45蛋白伴侣是UNC-45/CRO1/She4p(UCS)家族的成员。在几个动物模型中，它的缺失导致骨骼和心肌发育异常，表明进化功能保守。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-末端UCS域与肌球蛋白运动区相互作用。虽然其机制尚不清楚，但在肌球蛋白马达折叠过程中，UNC-45似乎既是支架又是伴侣。UNC-45表达水平的改变与包涵体肌病和心力衰竭相关，将UNC-45与人类疾病联系起来。最近，第一个人类UNC-45突变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是一种治疗蛋白质折叠疾病的可行药物。这项建议的目的是通过利用互补的遗传和生化方法确定新的UNC-45相互作用伙伴来扩大我们对UNC-45功能及其作用机制的理解。为此，我们将染色体片段单倍体缺陷系与UNC-45单倍体(敏化)背景杂交，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，这些缺陷系表现出较差的飞行能力，并伴随着UNC-45的单倍体丢失。目标1 该项目将利用果蝇中强大的突变等位基因和RNAi敲除技术来定位在UNC-45敏化背景下显示肌肉发育缺陷的特定基因。这将检验一种假设，即候选染色体片段缺陷系含有特定基因，其产物在肌肉发育过程中与UNC-45功能相互作用。AIM 2将使用一种互补的生化方法来分离和鉴定UNC-45相互作用的伙伴，方法是免疫共沉淀和质谱分析。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机体应激过程中形成的UNC-45复合体。这将检验UNC-45互动伙伴在肌肉发育和压力过程中发生变化的假设。AIM 3a将使用共聚焦显微镜在体内定位已识别的UNC-45相互作用伙伴，并将使用RNAi敲除来评估它们在肌肉发育中的作用。Aim 3b将通过肌球蛋白聚集保护和柠檬酸合成酶折叠分析，从功能上询问已确定的合作伙伴调节UNC-45伴侣活性的能力。我们还将使用热诱导聚集保护分析来研究识别的蛋白质是否作为UNC-45客户蛋白。目标3将解决这样的假设，即UNC-45相互作用的伙伴影响UNC-45的伴侣活动或在肌肉细胞内充当UNC-45客户。总体而言，我们的项目将对UNC-45相互作用的伙伴及其在肌肉发育过程中UNC-45生物学中的功能作用进行综合分析。它将提供果蝇遗传学、细胞生物学和生物化学方面的杰出培训，以及在科学写作、研究实验室监督和教学方面的指导。

 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.