STRUCTURE AND FUNCTION OF THE SMALL HEAT SHOCK PROTEINS

本研究的长期目标是了解sHsp在体内的功能，并进一步确定sHsp分子伴侣活性的机制。 在真核生物和原核生物中保守的小热休克蛋白（sHsp）/α-晶状体蛋白家族蛋白在经历热应激的细胞中以显著水平产生，表明它们在高温生存中具有古老和保守的作用。 研究表明，sHsps作为分子伴侣，通过结合变性蛋白质并将这些底物呈递给其他伴侣进行ATP依赖性重折叠来防止不可逆的蛋白质聚集。 sHsp家族的成员也可能在没有热应激和疾病应激的细胞中发挥重要作用。 它们存在于不同的细胞和组织类型中，以响应发育、分化、生长条件和致癌状态的阶段。此外，sHsps/α-晶体蛋白在与神经变性疾病相关的细胞中表达，并且α-晶体蛋白突变与遗传性结蛋白肌病和白内障病症相关，所有这些都涉及蛋白质错误折叠。 该实验室已经开发了sHsps伴侣活性的检测方法，并正在使用核磁共振、X射线和其他物理技术获取sHsps的详细结构数据。 此外，已经开发了一个遗传系统来分析sHsp功能;蓝细菌集胞藻，其中完整的基因组序列是已知的，表现出一个条件性耐热性缺陷的sHsp缺失突变体。 因此，在sHsp研究中，第一次可以使用联合收割机结合遗传、生化和结构分析的工具来详细研究sHsp的功能。 目前的研究有四个具体目标。1)通过筛选集胞藻Hsp16.6的无功能突变体来确定sHsps体内活性所需的特征，并使用这些突变体来测试sHsp功能的伴侣蛋白模型的有效性。2)鉴定与集胞藻Hsp16.6相互作用的蛋白质，作为sHsp功能的底物或伴侣。3)进一步研究sHsps的结构，为了解其分子作用机制奠定基础。4)定义sHsps与底物结合的相互作用，并扩展sHsp分子伴侣活性模型。 对这些普遍存在的应激蛋白的拟议研究将是理解它们在正常和应激细胞中作用的关键。

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.