SGP-2 (CLUSTERIN) AND REPRODUCTION

簇蛋白是培养的支持细胞分泌的主要蛋白质。凝聚蛋白是由二硫键连接的异单体组成的高度糖基化的蛋白质，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-组织界面中，并且在热休克或缺氧等细胞应激条件下，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，以响应附近细胞的死亡。 凝聚素的功能尚不清楚，但我们提出了一个由文献和我们自己的工作数据支持的假设，即凝聚素对于溶解不溶性大分子复合物并将其呈现给存活细胞的内吞作用是必需的。这种功能将使聚集蛋白对于保持液体-组织界面免受细胞碎片的影响非常重要，并且会对受压、受损或老化的组织产生影响。 证据支持这样的观点：凝聚素的生物学功能是其与疏水性大分子和分子复合物紧密结合的能力的结果。 在这个提案中，我们提出了直接测试我们提出的 clusterin 函数的实验。 在第一个具体目标中，我们将彻底分析正常和有毒条件下敲除凝聚素基因对男性生殖功能的影响。 我们将研究凝聚素与多种靶蛋白结合的基础。 在第二个具体目标中，我们将分析簇蛋白的许多结构特性，并尝试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，并分析修饰的凝聚素与特定底物的结合。 我们将通过有限的蛋白水解、CD 光谱以及荧光和交联来分析结构。 由于簇蛋白显然是细胞损伤和病理学的标志物，因此阐明其功能非常重要。

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.