基本介紹
- 中文名：蛋白質前體加工酶抑制劑研究
- 外文名：Protein precursor processing Inhibitors
- 學科專業 ：生物化學與分子生物學
- 論文作者：費浩
- 導師：戚正武院士指導
- 學位授予單位：中科院生化與細胞生物學研究所
中文摘要,外文摘要,
中文摘要
前體加工酶的結構與功能的相互聯繫：其翻譯後成熟過程中的分子機制；組織與細胞水平的表達分布；前體加工酶抑制劑的研究等。
本論文實驗部分的主題是前體加工酶抑制劑的研究。研究工作從兩個途徑展開，包括已知抑制劑的改造和天然抑制劑的尋找。
一）將已知的蛋白酶抑制劑改造為前體加工酶抑制劑。
（1）以枯草桿菌蛋白酶抑制劑eglin c為對象。本工作以定點突變方法在
野生型eglin c的抑制劑活性中心的P1，P2和P4位引入鹼性胺基酸殘基可以將其改造為很強的furin以及kexin抑制劑。同時根據枯草桿菌蛋白酶和eglin c複合物的晶體結構，計算機同源模建了前體加工酶與eglin c突變體結構之間的相互作用，並結合實驗數據得到以下結果：1）P1位引入的鹼性殘基是該抑制劑活力的前提。2）P4位鹼性殘基的引入可以極大的提高抑制劑活力約兩個數量級。3）P2位的鹼性殘基將有效提高抑制劑的活力，然而同時可以破壞抑制劑本身的穩定性。4）野生型P3位的疏水性殘基參與抑制劑活性環附近的疏水核心的構成。
（2）以胰蛋白酶抑制劑MBTI為對象。從天然中純化的綠豆Bowman-Birk抑制劑具有較強的對kexin的抑制活力。根據其已知的抑制劑蛋白質序列，選擇同源保守區域設計引物，採用RACE方法，從綠豆種子的總RNA中克隆了它的cDNA並進行了序列測定。已知的基因序列為今後的改造工作提供便利。
二）尋找天然中可能存在的前體加工酶的專一性抑制劑從豬腎臟中純化kexin的天然抑制劑。摸索並建立了一條高效純化路徑，對目標蛋白的純化倍數超過3000倍，從而得率達到20%。最終純化到的抑制組分是一等電點超過9.5的鹼性蛋白。測定了其N末端22個殘基的序列。該序列與非組蛋白HMG-17高度同源，後者含有5個易被kexin裂解的雙鹼性胺基酸位點。因此，該非組蛋白可與螢光底物強烈競爭。若將酶與非組蛋白長時間溫育，其抑制活力將最終喪失。
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...>> 詳細
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.
(2) Using mung bean trypsin inhibitor as a template. The Bowman-Birk inhibitor purified from the mung bean seeds displayed strong inhibitory activity toward kexin. Primers were designed according to the conserved regions of the determined amino acid sequence of the inhibitor. By the method of RACE (rapid amplification of cDNA ends) , the cDNA of mungbean trypsin inhibitor were cloned from the total RNA of mung bean seeds. The gene sequence of MBTI provides convenience to further protein engineering studies.
Approach two: To explore the possibility of naturally occurring inhibitor toward precursor convertase.
With establishment of an effective purification pathway, a basic protein with an isoelectric point over 9. 5 was purified over 3000 folds from porcine kidney. Its partial N-terminal sequence of 22 residues of this inhibitory component was determined, showing a high homology with nonhistone chromosomal protein HMG-17 in which there are five sites composed of dibasic residues, susceptible to be cleaved by kexin. As a result, this nonhistone protein can strongly compete with the fluorogenic substrate. However, this nonhistone protein will be degraded as a substrate by kexin if it is incubated with the enzyme for long time before adding the fluorogenic substrate, and subsequently loses its temporary inhibitory activity.
