SGP-2 (CLUSTERIN) AND REPRODUCTION

聚簇素是培养的支持细胞分泌的主要蛋白。聚簇蛋白是由二硫键异单体组成的糖基化蛋白，在许多细胞中表达，但主要由上皮细胞表达。它被分泌到液-组织界面，在细胞应激条件下，如热休克或缺氧，分泌的基础水平大大增加。此外，它在睾丸中凋亡的生殖细胞中积累，但它主要由支持细胞合成，以应对附近细胞的阻碍死亡。聚簇蛋白的功能尚不清楚，但我们提出了一个由文献数据和我们自己的工作支持的假设，即聚簇蛋白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要的。这一功能将使聚簇蛋白在保持流体组织界面免受细胞碎片侵害方面发挥重要作用，并将对受压或受损或老化的组织产生影响。证据支持这样一种观点，即聚簇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。在这个提议中，我们提出了直接测试我们提出的聚类函数的实验。在第一个具体目标中，我们将彻底分析在正常和有毒条件下，聚簇蛋白基因敲除对男性生殖功能的影响。我们将研究聚簇蛋白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一些结构特性，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，并分析修饰的簇蛋白与定义的底物的结合。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。由于聚簇蛋白是细胞损伤和病理的标志，阐明其功能是非常重要的。

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.