THE CRES GENE IN REPRODUCTION

我们研究的长期目标是确定CRES(胱抑素相关附睾生精蛋白)蛋白在生殖功能中的作用。基因和蛋白质结构研究表明，CRES蛋白是半胱氨酸蛋白酶抑制剂超家族半胱氨酸半胱氨酸蛋白2家族中的一个新成员。然而，一些证据表明，CRES可能不像经典的胱抑素那样发挥作用。首先，CRES蛋白缺少抑制半胱氨酸蛋白酶的三个共同位点中的两个。其次，我们的体外酶分析表明，与半胱氨酸蛋白酶C相比，CRES蛋白不抑制半胱氨酸蛋白酶木瓜酶、组织蛋白酶B、豆蛋白或半胱氨酸蛋白酶。最后，与广泛表达的2家族半胱氨酸氨基转移酶不同，CRES蛋白的表达是独特的，并且局限于特定生殖组织中蛋白分解活性较高的部位，包括附睾头近端、垂体前叶促性腺激素和精子顶体。除了定位于高蛋白水解酶活性的部位外，我们最近的体外酶分析表明，CRES蛋白对被称为前激素转换酶的一类高底物特异性丝氨酸蛋白酶家族的成员具有抑制作用，这是非常有力的证据，表明CRES蛋白是一种蛋白酶抑制剂。这个最近发现的蛋白质家族在包括神经内分泌和生殖系统在内的各种器官系统的前激素和前蛋白处理中发挥着关键作用。综上所述，我们的初步研究提供了强有力的证据，表明CRES蛋白是一种具有广泛生物学意义的重要蛋白酶家族的潜在抑制物，因此有必要对CRES蛋白进行进一步的研究。由于附睾体和精子顶体是前体蛋白被加工成成熟形式的活性部位，在体外，CRES抑制蛋白水解酶在蛋白质加工中的生物学作用，我们推测CRES蛋白是一种新的激素原转换酶抑制剂，它调节精子顶体和附睾体中的蛋白水解性加工事件，从而对受精和精子成熟起重要作用。我们将从以下几个方面对我们的假设进行验证：1)通过体外酶分析和免疫共沉淀实验验证CRES蛋白作为转换酶抑制因子的作用。作为转换酶抑制剂的CRES的功能分析将从检测转换酶的潜在靶点开始；2)检测顶体CRES蛋白在小鼠精子功能中的作用；以及3)通过基因敲除研究检测CRES蛋白在体内的生物学功能。

The long-range objective of our studies is to determine the role of the CRES (cystatin-related epididymal spermatogenic) protein in reproductive function. Gene and protein structure studies show that the CRES protein is a new member of the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, several lines of evidence suggest that CRES may not function as a classic cystatin. Firstly, CRES protein lacks two of three consensus sites important for inhibition of cysteine proteases. Secondly, our in vitro enzyme assays show that, in contrast to cystatin C, CRES protein does not inhibit the cysteine proteases papain, cathepsin B, legumain, or caspase. Finally, unlike the ubiquitous expression of the family 2 cystatins, CRES protein expression is unique and is restricted to sites of high proteolytic activity within specific reproductive tissues including the proximal caput epididymis, anterior pituitary gonadotropes, and sperm acrosomes. Besides being localized to sites of high proteolytic activity, very strong evidence that CRES protein is a protease inhibitor is our recent in vitro enzyme assays showing that CRES protein is inhibitory against members of a family of highly substrate specific serine proteases known as the prohormone convertases. This recently discovered family of proteins play critical roles in prohormone and proprotein processing in a variety of organ systems including the neuroendocrine and reproductive systems. Taken together, our preliminary studies provide strong evidence that CRES protein is a potential inhibitor of an important family of proteases with widespread biological significance and therefore further studies on CRES protein are warranted. Because the epididymis and sperm acrosome are active sites where precursor proteins are processed to their mature forms and in vitro, CRES inhibits proteases with confirmed biological roles in protein processing, we hypothesize that CRES protein is a novel prohormone convertase inhibitor which regulates proteolytic processing events in the sperm acrosome and epididymis and thus is important for fertilization and sperm maturation. We will test our hypothesis in the following specific aims: 1) examine CRES protein as a convertase inhibitor by in vitro enzyme assays and co- immunoprecipitation experiments. Functional analyses of CRES as a convertase inhibitor will be initiated by examining potential targets of convertases; 2) examine acrosomal CRES protein in mouse sperm function; and 3) examine the biological function of CRES protein in vivo by gene knock-out studies.