中文摘要

许多有生物活性的蛋白质与多肽，是由它们的前体通过前体加工酶的加工水解而获得最终活性的。这些前体加工酶成为生物体内重要的生理病理过程的调控者。

前体加工酶的结构与功能的相互联系：其翻译后成熟过程中的分子机制；组织与细胞水平的表达分布；前体加工酶抑制剂的研究等。

本论文实验部分的主题是前体加工酶抑制剂的研究。研究工作从两个途径展开，包括已知抑制剂的改造和天然抑制剂的寻找。

一）将已知的蛋白酶抑制剂改造为前体加工酶抑制剂。

（1）以枯草杆菌蛋白酶抑制剂eglin c为对象。本工作以定点突变方法在

野生型eglin c的抑制剂活性中心的P1，P2和P4位引入碱性氨基酸残基可以将其改造为很强的furin以及kexin抑制剂。同时根据枯草杆菌蛋白酶和eglin c复合物的晶体结构，计算机同源模建了前体加工酶与eglin c突变体结构之间的相互作用，并结合实验数据得到以下结果：1）P1位引入的碱性残基是该抑制剂活力的前提。2）P4位碱性残基的引入可以极大的提高抑制剂活力约两个数量级。3）P2位的碱性残基将有效提高抑制剂的活力，然而同时可以破坏抑制剂本身的稳定性。4）野生型P3位的疏水性残基参与抑制剂活性环附近的疏水核心的构成。

（2）以胰蛋白酶抑制剂MBTI为对象。从天然中纯化的绿豆Bowman-Birk抑制剂具有较强的对kexin的抑制活力。根据其已知的抑制剂蛋白质序列，选择同源保守区域设计引物，采用RACE方法，从绿豆种子的总RNA中克隆了它的cDNA并进行了序列测定。已知的基因序列为今后的改造工作提供便利。

二）寻找天然中可能存在的前体加工酶的专一性抑制剂从猪肾脏中纯化kexin的天然抑制剂。摸索并建立了一条高效纯化路径，对目标蛋白的纯化倍数超过3000倍，从而得率达到20%。最终纯化到的抑制组分是一等电点超过9.5的碱性蛋白。测定了其N末端22个残基的序列。该序列与非组蛋白HMG-17高度同源，后者含有5个易被kexin裂解的双碱性氨基酸位点。因此，该非组蛋白可与荧光底物强烈竞争。若将酶与非组蛋白长时间温育，其抑制活力将最终丧失。

外文摘要

Abstract

Biologically active proteins and peptides are often generated by intracellular limited proteolysis of inactive precursors. These precursor convertases become key regulators in many physiologically and pathologically important processes.

The review section of the thesis will deal with some recent progresses and developments in the study of precursor convertases (PCs) and their inhibitors. It will include the following aspects: the structure an...>> 详细

Abstract

Biologically active proteins and peptides are often generated by intracellular limited proteolysis of inactive precursors. These precursor convertases become key regulators in many physiologically and pathologically important processes.

The review section of the thesis will deal with some recent progresses and developments in the study of precursor convertases (PCs) and their inhibitors. It will include the following aspects: the structure and function relationship of PCs, the molecular mechanisms underlying the post-translational maturation, the tissue and cellular distribution of PCs, the study on the inhibitors of PCs.

In the experimental section of the thesis, the studies on the inhibitors of precursor convertases will be introduced and discussed. Two approaches were developed and applied in this study.

Approach one: To modify an existing protease inhibitor to be an inhibitor for precursor convertases.

(1) Using the subtilisin inhibitor eglin c as a template. Substitution of residues at each position 〓 and 〓 of eglin c with a basic residue using protein engineering could make eglin c a very strong inhibitor for both furin and even kexin. Based on the known crystal structures of subtilisin and eglin c, the interaction between the enzyme and inhibitor was modeled, and their involved residues were predicted which gave a good explanation to the experimental results: 1) A basic residue Lys or Arg at 〓 site is prerequisite for the inhibitor. 2) The second mutation with basic residue at 〓 site drastically increase the inhibitory activity by two orders of magnitude. 3) A basic residue at 〓 site is favorable for the binding to the enzyme, but unfavorable for the stability of the inhibitor, resulting in a temporary inhibition. 4) A wild type hydrophobic residue at 〓 site is preferred for the formation of a hydrophobic core in eglin c.