Proteins Interacting in Myosin Assembly

描述（由申请人提供）：肌凝蛋白作为肌动蛋白细胞骨架的蛋白质马达，在基本的生物活动中起作用，包括细胞分裂（细胞质分裂），质膜动力学和囊泡运输，以及更专门的生理作用，包括心脏和骨骼肌收缩。一个新发现的含有ucs结构域的蛋白家族（U: unc-45; C: Cro-1; S: She4p）在从酵母到人类的真核生物中表达，并与肌球蛋白的组装和功能有关。在从秀丽隐杆线虫到人类的动物中，两个额外的蛋白质区域是保守的：nh2末端tpr结构域（四联肽重复），它负责结合Hsp90，这是一种参与蛋白质折叠的分子伴侣。中心区域与cooh - ucs结构域一起是肌凝蛋白结合和伴侣活性的充分区域。这些动物蛋白通常被称为UNC-45，以典型的秀丽隐杆线虫分子命名。为了进一步了解UNC-45机器的工作机制，以及在小鼠和人类中发现的一般细胞和条纹肌同种型，我们提出了以下具体目标：1)在肌球蛋白S1折叠、全长肌球蛋白组装或肌球蛋白运动相关活性中，UNC-45作为伴侣和Hsp90共同伴侣的作用机制是什么？2)确定的UNC-45区域及其特定位点之间的结构-功能关系以及它们与Hsp90在肌凝蛋白S1折叠、全长肌凝蛋白组装或肌凝蛋白运动相关活性方面的相互作用？3)哺乳动物中UNC-45的一般细胞和横纹肌同种型之间的氨基酸序列和表达模式的差异对功能的影响是什么？为了回答这些问题，我们将使用秀丽隐杆线虫和小鼠以及特定小鼠敲除系的野生型和突变型重组UNC-45蛋白。对这些问题的理解可能不仅对细胞生物学的多个问题，而且对疾病也有广泛的意义。UNC-45真菌同源物是细胞分裂所必需的研究结果表明，UNC-45一般细胞异构体可能在癌症细胞分裂失调中起重要作用。秀丽隐杆线虫中unc-45突变体将肌球蛋白组装成细丝的缺陷表明，unc-45横纹肌异构体可能在收缩机制功能失调或缺陷的心力衰竭和骨骼肌萎缩中起重要作用。

DESCRIPTION (provided by applicant): The myosins function as protein motors of the actin cytoskeleton in fundamental biological activities including cell division (cytokinesis), plasma membrane dynamics and vesicle trafficking as well as more specialized physiological roles including cardiac and skeletal muscle contraction. A newly identified family of proteins containing UCS-domains (U: unc-45; C: Cro-1; S: She4p) is expressed in eukaryotic organisms from yeast to humans and has been implicated in myosin assembly and function. In animals from Caenorhabditis elegans to humans, two additional protein regions are conserved: an NH2-terminal TPR-domain (Tetratrico Peptide Repeat), which is responsible for binding Hsp90, a molecular chaperone implicated in protein folding. The central region together with the COOH-UCS-domain is sufficient for myosin binding and chaperone activity. The animal proteins are generically termed UNC-45 after the canonical C. elegans molecule. To further understand both the mechanisms by which UNC-45 machines work and the finding in mice and humans of general cell and striated muscle isoforms, we propose the following Specific Aims: 1) What mechanisms operate in UNC-45 activity as a chaperone and as a Hsp90 co-chaperone in myosin S1 folding, full-length myosin assembly, or myosin motor-related activity? 2) What are the structure-function relationships between identified regions of UNC-45 and specific sites within them and their interactions with Hsp90 for myosin S1 folding, full-length myosin assembly, or myosin motor-related activity? 3) What are the functional consequences of the differences in amino acid sequences and patterns of expression between the general cellular and striated muscle isoforms of UNC-45 in mammals? To answer these questions, we will use wild type and mutant recombinant UNC-45 proteins of C. elegans and mice and specific mouse knockout lines. The understanding of these questions is likely to have broad significance not only to multiple problems in cell biology but also with respect to disease. The findings that UNC-45 fungal homologues are necessary for cytokinesis suggests that the UNC-45 general cell isoform may be important in relation to the dysregulation of cell division of cancer. The defective assembly of myosin into filaments by unc-45 mutants in C. elegans suggests that the UNC-45 striated muscle isoform may be important in heart failure and skeletal muscle atrophy in which the contractile machinery is dysfunctional or deficient.