SPECIFICITY AND STRUCTURE--THE KEX2 AND FURIN PROTEASES

在分泌区室和在细胞膜上的特异性蛋白水解加工 细胞表面是许多细胞成熟或修饰所必需的 真核细胞中的跨膜和分泌蛋白。 的研究 酵母Kex 2蛋白酶，其切割α-交配因子前体 和其他蛋白质在反式高尔基体中的配对碱性残基位点 网络（TGN），导致发现了一个新的枯草杆菌蛋白酶家族- 在真核生物中保守的相关前蛋白加工酶， 真菌到无脊椎动物和脊椎动物。 这些酶处理 调节分泌途径中的激素原和神经肽， 组成性分泌途径中的多种前蛋白。 第一 已知人Kex 2同源物弗林蛋白酶涉及许多 生理学/医学上重要的过程： 生长因子及其受体，凝血因子，病毒 糖蛋白和细菌毒素。 在以前的资助工作 这段时间给了我们一个全面的，定量的Kex 2视图 弗林蛋白酶的特异性和分析工具。 我们还 开发了强大的遗传工具来操纵Kex 2特异性。 最后，我们对一类新型酵母进行了分析 加工酶的期望，与Kex 2一样， 这些酶同系物在真核生物中具有普遍的重要性。 目前的研究目标如下：1）以往的研究为我们提供了一个 对Kex 2特异性的定量理解和一套工具， 包括纯化的弗林蛋白酶，用其分析弗林蛋白酶特异性， 详细 这里提出的实验将扩展我们对 特异性在这个重要的酶家族的机制水平， 回答关于特异性如何“分配”的基本问题 在催化循环中。 （二） 在上一个赠款期间，开发了定量方法， 体内Kex 2特异性分析和突变体的遗传选择 表现出改变的特异性的Kex 2蛋白酶的形式。 剥削 这些方法结合突变体的生化分析， 酶，旨在提供一个全面的了解， 酶特异性的结构基础。 3)整体利益 关于前蛋白质加工的问题让我们确定了一个细胞表面 酵母中的“分泌酶”活性。 我们已经证明，这种活动取决于 GPI锚定细胞表面新家族成员 乙酰基蛋白酶。 特异性和生理学分析 这些酶的功能将导致更深入的了解 真核细胞中发生的一系列加工反应。

Specific proteolytic processing in secretory compartments and at the cell surface is required for maturation or modification of numerous transmembrane and secreted proteins in eukaryotic cells. Studies of the yeast Kex2 protease, which cleaves the alpha-mating factor precursor and other proteins at paired basic residues sites in the trans Golgi network (TGN), led to the discovery of a novel family of subtilisin- related proprotein processing enzymes conserved in eukaryote from fungi to invertebrates and vertebrates. These enzymes process prohormones and neuropeptides in the regulated secretory pathway and a variety of proproteins in the constitutive secretory pathway. The first known human Kex2 homologue, furin, is implicated in many physiology/medically important processes: maturation of precursors of growth factors and growth factor receptor, coagulation factors, viral glycoproteins and bacterial toxins. Work in the previous funding period has given us a comprehensive, quantitative view of Kex2 specificity and insights and tools for analysis of furin. We have also developed powerful genetic tools for manipulating Kex2 specificity. Finally, we have undertaken the analysis of a novel class of yeast processing enzymes with the expectation that, as with Kex2, the homologs of these enzymes will be of general importance in eukaryote. Current aims as follows: 1) Previous studies have given us a quantitative understanding of Kex2 specificity and a set of tools, including purified furin, with which to analyze furin specificity in detail. Experiments proposed here will extend our understanding of specificity in this important family of enzymes to the mechanistic level, answering fundamental questions about how specificity is 'apportioned' in the catalytic cycle. 2) In the previous grant period, methods were developed for quantitative analysis of Kex2 specificity in vivo and for genetic selection of mutant forms of Kex2 protease that exhibit altered specificity. Exploitation of these methods, combined with biochemical analysis of the mutant enzymes, is intended to provide a comprehensive understanding of the structural basis of specificity in the enzyme. 3) Interest in general questions about proprotein processing led us to identify a cell surface 'secretase' activity in yeast. We have shown that this activity depends on members of an emerging family of GPI-anchored cell surface aspartyl proteases. Analysis of the specificity and physiological function of these enzymes will lead to a deeper understanding of the range of processing reactions that occur in eukaryotic cells.