SPECIFICITY AND STUCTURE--THE KEX2 AND FURIN PROTEASES

从酵母到人类的真核生物分泌生物活性肽和蛋白质 在分泌后期通过蛋白水解从前体中切除 通路 酵母Kex 2蛋白酶，一种膜结合丝氨酸酶 与枯草杆菌蛋白酶有远亲关系，是第一种被证明可以 这种处理反应在体内。 我们鉴定了人弗林蛋白酶 一个Kex 2同源物提供了明确的证据， 在真核生物进化过程中的加工机制。 发现 脊椎动物和无脊椎动物中的其他Kex 2同源物表明， Kex 2家族的成员作用于不同的底物，包括 肽类激素前体，神经肽，生长因子，细胞 表面受体、血浆蛋白、凝血因子和病毒 包膜糖蛋白 因此，阐述了 基于Kex 2蛋白酶及其同源物的特异性，此外， 来提供关于后-- 翻译处理，应该证明在设计的巨大价值， 具有广泛药理学功能的抑制剂。 在这 应用，我们建议推进我们的Kex 2蛋白酶的研究， 将我们的分析扩展到人类弗林蛋白酶。 这项工作有三个目标： 1.阐明激素原特异性的基本依据 处理，我们提出了详细的研究底物识别Kex 2 蛋白酶 底物序列和高阶的贡献 将通过分析以下物质的反应来确定结构与特异性的关系： 纯化的Kex 2蛋白酶与模型肽底物和纯化的天然 前体 一种新的Kex 2蛋白酶突变的遗传选择方法 改变其特异性的基因将被用来绘制酶中的残基 这对于切割位点识别是至关重要的。 2.为了更广泛地了解Kex 2相关的加工酶，我们 建议对人类弗林蛋白酶的特异性进行平行研究， 普遍存在的组织分布表明参与了许多 生理上重要的加工反应。 弗林，我们有 设计了一个有效的表达系统，将纯化到同质。 纯化的酶的特异性将通过切割进行评价 模型肽底物和可能的生理底物， 胰岛素受体前体 3. Kex 2蛋白酶结构-功能研究的目的是确定 区分Kex 2家族特异性加工酶的特征 从它们进化上相关的降解枯草杆菌酶中分离出来。 我们建议阐明的分子内裂解的途径， N-末端前结构域。 此外，我们计划阐明的功能， Kex 2家族成员所特有的新的C-末端结构域， 我们已经发现其对于活性Kex 2蛋白酶的形成是必需的。

Eukaryotes from yeast to humans secrete bioactive peptides and proteins that are excised by proteolysis from precursors late in the secretory pathway. The yeast Kex2 protease, a membrane-bound serine enzyme distantly related to subtilisin, was the first enzyme proven to carry out such processing reactions in vivo. Our identification of human furin as a Kex2 homologue gave definitive evidence for conservation of the processing machinery throughout eukaryotic evolution. Discovery of additional Kex2 homologues in vertebrates and invertebrates suggests that members of the Kex2 family act on diverse substrates, including precursors of peptide hormones, neuropeptides, growth factors, cell surface receptors, plasma proteins, coagulation factors, and viral envelope glycoproteins. Thus, elaborating the nature and structural basis of the specificity of Kex2 protease and its homologues, in addition to providing basic information about a critical pathway of post-- translational processing, should prove of great value in the design of inhibitors having a wide range of pharmacological functions. In this application, we propose to advance our studies of Kex2 protease and to extend our analysis to human furin. The aims of this work are threefold: 1. To elucidate the fundamental basis of specificity in prohormone processing, we propose detailed studies of substrate recognition by Kex2 protease. The contributions of substrate sequence and higher order structure to specificity will be determined by analyzing the reaction of purified Kex2 protease with model peptide substrates and purified natural precursors. A novel genetic selection for mutations in Kex2 protease that alter its specificity will be used to map residues in the enzyme that are critical for cleavage site discrimination. 2. To obtain a broader understanding Kex2-related processing enzymes, we propose parallel studies of the specificity of human furin, the ubiquitous tissue distribution of which suggests involvement in numerous physiologically important processing reactions. Furin, for which we have devised an efficient expression system, will be purified to homogeneity. The specificity of the purified enzyme will be evaluated through cleavage of model peptide substrates and a likely physiological substrate, the insulin receptor precursor. 3. The goal of structure-function studies of Kex2 protease is to define features that distinguish the Kex2 family of specific processing enzymes from the degradative subtilases to which they are evolutionarily related. We propose to elucidate the pathway of intramolecular cleavage of the N-terminal pro-domain. Further, we plan to elucidate the function of a novel C-terminal domain that is unique to members of the Kex2 family and which we have found is essential for formation of active Kex2 protease.