SPECIFICITY AND STRUCTURE--THE KEX2 AND FURIN PROTEASES

在分泌室和在 细胞表面是许多细胞成熟或修饰所必需的 真核细胞中的跨膜蛋白和分泌蛋白。对中国传统文化的研究 酵母菌Kex2蛋白酶，裂解α-交配因子前体 和其他在反式高尔基体成对碱性残基位置的蛋白质 网络(TGN)，导致了一个新的枯草杆菌蛋白家族的发现。 真核生物中保守的相关蛋白原加工酶 真菌到无脊椎动物和脊椎动物。这些酶的作用是 调节分泌途径中的前激素和神经肽 构成分泌途径中的多种原蛋白。第一 已知的人类Kex2同系物，Furin，与许多 重要的生理/医学过程：血管紧张素转换酶前体的成熟 生长因子和生长因子受体，凝血因子，病毒 糖蛋白和细菌毒素。在以前的资金中工作 给了我们一个全面的、定量的关于Kex2的看法 分析呋喃的专一性、洞察力和工具。我们还有 开发了强大的基因工具来操纵Kex2的特异性。 最后，我们对一类新的酵母菌进行了分析 处理酶的期望是，就像Kex2一样， 这些酶的同源物在真核生物中具有普遍的重要性。 目前的目标如下：1)以前的研究给我们提供了一个 定量了解Kex2的特异性和一套工具， 包括纯化的呋喃，用它来分析呋喃的特异性 细节。这里提出的实验将扩大我们对 在这个重要的酶家族中的专一性体现在机械层面上， 回答有关具体情况如何“分配”的基本问题 在催化循环中。2) 在之前的赠款期间，开发了用于量化的方法 Kex2的体内特异性分析及其突变体的遗传选择 表现出改变的特异性的Kex2蛋白酶的形式。开发利用 这些方法，结合对突变体的生化分析 ，旨在提供对酶的全面理解 酶的专一性的结构基础。3)一般权益 关于蛋白原加工的问题让我们识别了细胞表面 酵母中的‘分泌酶’活性。我们已经证明，这一活动取决于 关于一个新兴的GPI锚定细胞表面家族的成员 天冬氨酸蛋白酶。专一性和生理性的分析 这些酶的功能将使我们更深入地了解 真核细胞中发生的加工反应的范围。

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.