THE CRES GENE IN REPRODUCTION

我们研究的长期目标是确定克雷斯（胱抑素相关的附睾精子发生）蛋白在生殖功能中的作用。基因和蛋白质结构研究表明，克雷斯蛋白是半胱氨酸蛋白酶抑制剂cystatin超家族2 cystatin家族的新成员。然而，一些证据表明，克雷斯可能不作为一个典型的半胱氨酸蛋白酶抑制剂的功能。首先，克雷斯蛋白缺乏对抑制半胱氨酸蛋白酶重要的三个共有位点中的两个。其次，我们的体外酶测定显示，与胱抑素C相反，克雷斯蛋白不抑制半胱氨酸蛋白酶木瓜蛋白酶、组织蛋白酶B、豆荚蛋白酶或胱天蛋白酶。最后，与家族2半胱氨酸蛋白酶抑制剂的普遍表达不同，克雷斯蛋白表达是独特的，并且限于特定生殖组织内的高蛋白水解活性位点，包括近端附睾头、垂体前叶促性腺激素和精子顶体。除了定位于高蛋白水解活性的位点之外，克雷斯蛋白是蛋白酶抑制剂的非常有力的证据是我们最近的体外酶测定，其显示克雷斯蛋白对称为激素原转化酶的高度底物特异性丝氨酸蛋白酶家族的成员具有抑制作用。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多种器官系统中的激素原和蛋白原加工中起关键作用。综上所述，我们的初步研究提供了强有力的证据表明，克雷斯蛋白是一个潜在的抑制剂的一个重要的家庭的蛋白酶具有广泛的生物学意义，因此，进一步研究克雷斯蛋白是必要的。由于附睾和精子顶体是前体蛋白被加工成其成熟形式的活性位点，并且在体外，克雷斯抑制蛋白酶在蛋白加工中具有确认的生物学作用，我们假设克雷斯蛋白是一种新型激素原转化酶抑制剂，其调节精子顶体和附睾中的蛋白水解加工事件，因此对受精和精子成熟很重要。我们将在以下具体目标中检验我们的假设：1）通过体外酶测定和免疫共沉淀实验来检验克雷斯蛋白作为转化酶抑制剂。将通过检查转化酶的潜在靶点启动克雷斯作为转化酶抑制剂的功能分析; 2）检查小鼠精子功能中的顶体克雷斯蛋白; 3）通过基因敲除研究检查体内克雷斯蛋白的生物学功能。

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.