利用酵母菌雙雜交系統，研究大腸桿菌中Lon蛋白酶自身聚合和基質辨識的位置以及篩選基因庫中可能存在的其他基質

Study of E. coli Lon protease with its oligomerization, substrate recognition, and screen of genomic library for its candidate substrate in yeast two-hybrid system

江鎮宇(Chen-Yu Chiang)

指導教授 ： 吳蕙芬

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2005年)

https://doi.org/10.6342/NTU.2005.00093

摘要

Lon蛋白酶是一種 ATP-dependent protease，普遍地存在於各種原核生物及真核生物粒線體中。同時Lon在演化上也具有高度的保留性，由此可見其在生物體內的重要。至於 ATP-dependent protease 在功能方面，它能夠分解細胞內不正常累積的蛋白質，或是將折疊錯誤的蛋白質回復成正常構形。最近有相關的研究指出，位於大腸桿菌中的 Lon C 端的P domain(proteolytic domain)，主要是負責蛋白酶間的聚合 (oligomerization)，而辨識基質的部份則位於N端 (N domain)。但在 Brevibacillus thermoruber 中，則有人認為負責聚合的位置在Lon的N端。雖然在本研究中，是使用大腸桿菌 K12 作為研究的材料。但由於序列保留性高的關係，在其他菌種的資料也值得作為參考。
在大腸桿菌中，有已知 Lon 的主要的基質為－ SulA、RcsA，而Lon 蛋白酶本身辨識基質的確切位置卻仍不甚了解。在本論文中，首先將 Lon 和已知的基質以酵母菌雙雜合系統做測試，結果發現 Lon 和 SulA 有交互作用。爲了找出 Lon 蛋白酶辨識 SulA 的區域以及本身聚合的位置，於是將Lon蛋白酶及其 N 區塊、P 區塊及基質於此系統當中表現，配合前人的研究以找出可能蛋白－蛋白的位置，針對特定的區塊作隨機性的突變。隨後以 SulA 篩選出含有對於辨識基質有影響突變點的轉殖菌株－ Lon30、Lon63 以及 Lon66。藉由 β-galactosidase assay 及 MMS (Methyl Methanesulfonate) test 的測試，發現在胺基酸序列 S307R 突變的位置，對於Lon 蛋白酶辨識 SulA 有較明顯的影響，佐證了之前的推測。另外，有研究指出 SulA 蛋白末端的八個胺基酸對於 Lon 的辨識有很大的影響，其中又以最後的ㄧ個胺基酸Histidine為最，不過在 SulA deletion 的實驗當中發現，僅僅去除掉 C 端15個胺基酸還是看的到蛋白質之間的交互作用，而要去除 C 端40個胺基酸才會對辨識有顯著的影響。此外，在酵母菌雙雜合系統中，以Lon蛋白酶篩選整個大腸桿菌的基因庫之研究，發現在挑到的蛋白質基因序列當中，都沒有任何有相似性的共識序列，因此推論 Lon 蛋白酶可能藉由特定的結合位置，對很多種不同序列的小分子蛋白作用。

關鍵字

酵母菌雙雜合系統 ； ATP-dependent protease ； Lon 蛋白酶

並列摘要

Lon is an ATP-dependent protease found in most organisms. The DNA sequence of Lon is highly conserved in evolution. Lon protease of Escherichia coli regulates a diverse set of physiological responses including cell division, capsule production, plasmid stability, and phage replication. Lon protease and members of Clp family of molecular chaperons and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domain. Recently the crystal structure of the proteolytic domain (P domain) of Escherichia coli Lon has been solved. The proteolytic domain (P domain) has a unique fold and assembles into hexameric rings, and the substrate recognition site locates in the N domain. But in the Brevibacillus thermoruber, N domain assembles the Lon protease to become an active oligomer.
Several substrates of Lon protease have been known in Escherichia coli－including SulA and RcsA. However, the precise position of substrate-recognizing site in Lon protease is still not clear yet. In this study, an interaction between Lon protease and SulA protein was demonstrated by yeast two-hybrid system. For finding the SulA-recognizing site and the Lon-Lon oligomerizing position in Lon, protein-protein interactions between Lon 、N domain、P domain and SulA were also tested here. From our observations and previous data, we hypothesize that SulA recognition site of Lon is located at N domain. Lon mutants (30Lon, 63Lon, 66Lon) for alteration of substrate binding were found by screening the mutagenized Lon proteins. Based on the results of the β-galactosidase assay and MMS (Methyl Methanesulfonate) test in Escherichia coli, we showed that Lon mutant S307R reduces the SulA-binding affinity. It has been reported that the C-terminal eight residues of SulA is recognized by Lon protease, and the last one amino acid Histidine is specifically necessary. However in our studies, the protein-protein interaction can yet be detected when we deleted 15 residues from the C-terminal of SulA protein. Only the last 40 residues deleted from the C-terminal of SulA showed an influence on the recognition and binding affinity with Lon. Finally, fish to the conserved regions of Lon specific-substrates, respectively screening the E.coli genomic library has been done in this study. By using the above similar strategy, several small molecules interacted with Lon have been found. However, the results indicate that no conserved region interacted with Lon has been found among all the isolated proteins. We suggest that Lon protease may use an unique site for binding with the small proteins that we identified.

並列關鍵字

yeast two-hybrid system ； ATP 依賴蛋白酶Lon protease

參考文獻

Aertsen, A. & Michiels, C. W. (2005). SulA-dependent hypersensitivity to high pressure and hyperfilamentation after high-pressure treatment of Escherichia coli lon mutants. Res Microbiol 156, 233-237.

Botos, I., Melnikov, E. E., Cherry, S. & other authors (2004a). Crystal structure of the AAA+ alpha domain of E. coli Lon protease at 1.9A resolution. J Struct Biol 146, 113-122.

Botos, I., Melnikov, E. E., Cherry, S. & other authors (2004b). The catalytic domain of Escherichia coli Lon protease has a unique fold and a Ser-Lys dyad in the active site. J Biol Chem 279, 8140-8148.

Chandu, D. & Nandi, D. (2004). Comparative genomics and functional roles of the ATP-dependent proteases Lon and Clp during cytosolic protein degradation. Res Microbiol 155, 710-719.

Christensen, S. K., Maenhaut-Michel, G., Mine, N., Gottesman, S., Gerdes, K. & Van Melderen, L. (2004). Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: involvement of the yefM-yoeB toxin-antitoxin system. Mol Microbiol 51, 1705-1717.