THE CRES GENE IN REPRODUCTION

我们研究的长期目标是确定CRES（胱抑素相关的附睾生精蛋白）在生殖功能中的作用。基因和蛋白结构研究表明，CRES蛋白是半胱氨酸蛋白酶抑制剂胱抑素超家族2胱抑素家族的新成员。然而，一些证据表明，CRES可能不像典型的胱抑素那样起作用。首先，CRES蛋白缺乏抑制半胱氨酸蛋白酶的三个重要位点中的两个。其次，我们的体外酶分析表明，与胱抑素C相反，CRES蛋白不会抑制半胱氨酸蛋白酶木瓜蛋白酶、组织蛋白酶B、豆类蛋白酶或半胱天冬酶。最后，与2家族胱抑素的普遍表达不同，CRES蛋白的表达是独特的，并且仅限于特定生殖组织内的高蛋白水解活性位点，包括附睾近端、垂体前叶促性腺激素和精子顶体。除了定位于高蛋白水解活性的位点外，我们最近的体外酶分析表明，CRES蛋白是一种蛋白酶抑制剂，这是非常有力的证据，表明CRES蛋白对被称为激素原转化酶的高度底物特异性丝氨酸蛋白酶家族成员具有抑制作用。这个最近发现的蛋白家族在包括神经内分泌和生殖系统在内的多种器官系统的激素原和蛋白原加工中起着关键作用。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剂，因此对CRES蛋白的进一步研究是有必要的。由于附睾和精子顶体是前体蛋白加工成成熟形态的活性位点，在体外实验中，CRES抑制的蛋白酶在蛋白质加工中具有明确的生物学作用，因此我们假设CRES蛋白是一种新的激素原转化酶抑制剂，它调节精子顶体和附睾的蛋白质水解加工事件，因此对受精和精子成熟很重要。我们将在以下具体目标中验证我们的假设：1)通过体外酶测定和共免疫沉淀实验来检验CRES蛋白作为转化酶抑制剂的作用。CRES作为转化酶抑制剂的功能分析将通过检测转化酶的潜在靶点来启动；2)检测顶体CRES蛋白对小鼠精子功能的影响；3)通过基因敲除研究来检测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.