SGP-2 (CLUSTERIN) AND REPRODUCTION

SGP-2（簇蛋白）和繁殖

• 批准号: 6476780
• 负责人: MICHAEL D GRISWOLD
• 金额: $ 28.69万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-07-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476780
• 关键词: Sertoli cells; binding proteins; circular dichroism; clusterin; cytotoxicity; gene targeting; genetic mapping; genetic promoter element; genetic transcription; genetically modified animals; laboratory mouse; lipid transport; male; protein binding; protein metabolism; protein structure function; reproduction; solubility; tissue /cell culture

Clusterin is the major protein secreted by cultured Sertoli cells. Clusterin is heavily glycosylated protein consisting of disulfide linked heteromonomers and is expressed by many cells but primarily by those of epithelial origin. It is secreted into fluid-tissue interfaces and the basal level of secretion is greatly increased under conditions of cellular stress such as heat shock or anoxia. In addition, it accumulates in apoptotic germ cells in the testis but it is synthesized primarily by the Sertoli cells in response to the impeding death of the nearby cells. The function of clusterin is unknown but we present a hypothesis supported by data from the literature and our own work that clusterin is necessary for solubilizing insoluble macromolecular complexes and presenting them for endocytosis by the surviving cells. This function would make clusterin important for keeping fluid-tissue interfaces free from cellular debris and would have implications for stressed or damaged or aged tissue. The evidence supports the notion that the biological function of clusterin is a result of its ability to bind tightly to hydrophobic macromolecules and molecular complexes. In this proposal we present experiments to directly test our proposed function for clusterin. In the first specific aim we will thoroughly analyze the impact of a knock-out of the clusterin gene on male reproductive function under normal and toxic conditions. We will examine the basis for the binding of clusterin to a variety of target proteins. In the second specific aim we will analyze a number of structural properties of clusterin and attempt to relate structure to function. We will make deletion and site-specific mutations and analyze the binding of modified clusterin to a defined substrate. We will analyze structure by limited proteolysis, by CD spectroscopy and by fluorescence and cross-linking. Since clusterin is clearly implicated as a marker for cellular damage and pathology, the elucidation of its function is very important.

Sertoli细胞分泌的主要蛋白质是促性腺激素。白藜芦醇苷是由二硫键连接的杂单体组成的高度糖基化的蛋白质，并由许多细胞表达，但主要由上皮来源的细胞表达。 它被分泌到液体-组织界面，并且在细胞应激条件下，如热休克或缺氧，基础分泌水平大大增加。 此外，它在睾丸中的凋亡生殖细胞中积累，但它主要由支持细胞合成，以响应附近细胞的阻碍性死亡。 clusterin的功能是未知的，但我们提出了一个假设，从文献和我们自己的工作，clusterin是必要的溶解不溶性大分子复合物，并提出它们的存活细胞的内吞作用的数据支持。这一功能将使clusterin在保持液体-组织界面不受细胞碎片影响方面发挥重要作用，并将对受压或受损或老化的组织产生影响。 证据支持这样的观点，即丛生蛋白的生物学功能是其与疏水大分子和分子复合物紧密结合的能力的结果。 在这个建议中，我们提出的实验直接测试我们提出的clusterin函数。 在第一个具体的目标，我们将彻底分析在正常和有毒条件下的男性生殖功能的clusterin基因敲除的影响。 我们将研究丛生蛋白与各种靶蛋白结合的基础。 在第二个特定的目标，我们将分析一些clusterin的结构特性，并试图将结构与功能联系起来。 我们将进行删除和位点特异性突变，并分析修饰后的丛生蛋白与特定底物的结合。 我们将通过有限的蛋白水解，CD光谱，荧光和交联来分析结构。 由于clusterin是细胞损伤和病理学的标志物，阐明其功能是非常重要的。