STRUCTURE AND FUNCTION OF THE SMALL HEAT SHOCK PROTEINS

这项研究的长期目标是了解SHSP在体内的功能，并进一步确定SHSP伴侣的活性机制。小热休克蛋白(ShSP)/α-晶体蛋白家族在真核生物和原核生物中都是保守的，在经历热应激的细胞中大量产生，这表明它们在高温生存中具有古老而保守的作用。研究表明，sHsps作为分子伴侣，通过与变性蛋白质结合并将这些底物呈递给其他依赖于ATP的伴侣进行重折叠，来防止蛋白质的不可逆聚集。SHSP家族的成员也可能在没有热应激和细胞受到疾病应激的情况下发挥重要作用。它们存在于不同的细胞和组织类型中，对发育阶段、分化、生长条件和致癌状态做出反应。此外，sHsps/α-晶体蛋白在与神经退行性疾病相关的细胞中表达，α-晶体蛋白突变与遗传性结蛋白肌病和白内障有关，所有这些都与蛋白质错误折叠有关。这个实验室已经开发了sHsps伴侣活性的分析方法，并正在利用核磁共振、X射线和其他物理技术获得sHsps的详细结构数据。此外，还建立了分析SHSP功能的遗传系统；已知其完整基因组序列的蓝藻聚胞藻在SHSP缺失突变体中表现出条件性耐热性缺陷。因此，在SHSP研究中，第一次可以利用工具结合遗传、生化和结构分析来详细研究SHSP的功能。目前的研究有四个具体目标。1)通过筛选聚球藻Hsp16.6的非功能突变体，确定体内活性所需的sHsps的特征，并利用这些突变体检验SHSP功能的伴侣模型的有效性。2)鉴定与聚球藻Hsp16.6相互作用的蛋白质，作为SHSP功能的底物或合作伙伴。3)进一步研究sHSPs的结构，为了解其作用的分子机制奠定基础。4)确定底物与sHsps的结合作用，推广sHsps的伴侣活性模型。对这些普遍存在的应激蛋白的拟议研究将是了解它们在正常和应激细胞中的作用的关键。

The long term goals of the proposed research are to understand sHsp function in vivo and to define further the mechanism of sHsp chaperone activity. The small Hsp (sHsp)/alpha-crystallin family of proteins, which are conserved in both eukaryotes and prokaryotes, are produced at significant levels in cells experiencing heat stress, indicating they have an ancient and conserved role in survival of high temperature. Research indicates that sHsps act as molecular chaperones to prevent irreversible protein aggregation by binding to denaturing proteins and presenting these substrates to other chaperones for ATP-dependent refolding. Members of the sHsp family may also play important roles in the absence of heat stress and in cells stressed by disease. They are found in different cell and tissue types in response to stage of development, differentiation, growth conditions and oncogenic status. Furthermore, sHsps/alpha- crystallins are expressed in cells associated with neurodegenerative diseases, and alpha-crystallin mutations are linked to an inherited desmin myopathy and to a cataract condition, all of which involve protein misfolding. Assays for sHsp chaperone activity have been developed in this laboratory, and detailed structural data on sHsps, using NMR, X-ray and other physical techniques, are being acquired. In addition, a genetic system to analyze sHsp function has been developed; the cyanobacterium Synechocystis, for which the complete genome sequence is known, exhibits a conditional thermotolerance defect in an sHsp deletion mutant. Thus, for the first time in sHsp research, the tools are available to combine genetic, biochemical and structural analysis to study the function of an sHsp in detail. The current research has four specific aims. 1) To define features of the sHsps that are required for activity in vivo by screening for non-functional mutants of Synechocystis Hsp16.6, and to use these mutants to test the validity of the chaperone model for sHsp function. 2) To identify proteins that interact with Synechocystis Hsp16.6 either as substrates or partners in sHsp function. 3) To investigate further the structure of the sHsps as a basis for understanding their molecular mechanism of action. 4) To define substrate binding interactions with the sHsps and to extend the model of sHsp chaperone activity. The proposed studies of these ubiquitous stress proteins will be key to understanding their role in normal and stressed cells.