大腸桿菌ClpY蛋白質形成六元環關鍵胺基酸之研究
Other Title
The Study of Key Amino Acid on ClpY Hexamer Formation
Journal
臺灣農業化學與食品科學
Journal Volume
54
Journal Issue
2
Start Page
53
End Page
62
ISSN
1605-2471
Date Issued
2016
Author(s)
張祿高(Lu-Kao Chang)
Abstract
細菌具有蛋白質管理系統，包含蛋白酶(protease)，分解不適當或是已經毀損構型之蛋白質。主要的ATP依賴型蛋白酶(ATP-dependent protease)，具二功能區塊，解構酶(unfoldase)及胜肽酶(peptidase)，前者水解ATP將基質解構並傳送胜肽鏈給後者分解。大腸桿菌中，包含ClpAP、ClpXP、ClpYQ、LonA及FtsH等ATP依賴型蛋白酶，皆具形成多元環之特性，呈現桶狀結構。本研究以ClpY作為探討形成多元環的關鍵因素。前人研究顯示，第408個胺基酸[酪胺酸(Tyr)]對於ClpY六元環的形成具關鍵影響，突變蛋白質ClpY(Y408A)，酪胺酸轉換為丙胺酸，在ATP存在下，ClpY(Y408A) 形成六元環的能力都較野生型蛋白質為差。檢視ClpY結晶結構，推測Y408酪胺酸其較大的側基，可與鄰近胺基酸(R25，精氨酸)產生較多的凡得瓦爾力。因此，本研究選擇一系列不帶電的胺基酸，建構了側基由小到大的Y408點突變蛋白質。在胞外，以聯結試驗觀察ClpY及其點突變蛋白質在ATPγS存在下形成六元環的狀況；並利用ClpYQ之基質SulA，其作為抑制細胞分裂的特性，透過細菌存活程度，檢定胞內ClpY及Y408其點突變蛋白質等與ClpQ蛋白酶之活性。結果顯示，胞外與胞內的結果趨勢均隨ClpY (Y408)側基大小而改變。側基愈小的ClpY點突變蛋白質在胞外形成六元環的能力愈弱；而Y408所轉換的側基愈小，對細菌生長的限制愈大。因此，Y408是ClpY形成六元環的關鍵胺基酸。
Proteins play important roles in the cell. Protein quality control network maintain the physiology function of proteins in bacteria. There are porteases in the network. The proteases degrade the damaged proteins or not suitable ones. Degradation relies on ATP-dependent proteases, like ClpAP, ClpXP, ClpYQ, LonA and FtsH in the model microorganism Escherichia coli. Under physiology condition, all of them form homomultimer and then stack to form the barrel shape. This study exams the key amino acid in ClpY forming the hexamer. Before, it was known that ClpY Y408A, tyrosine substituted by alanine, strictly has an effect on the hexamer formation. In the crystal structure, Y408 is very close to the adjacent amino acid R25(arginine); therefore we speculate that the van der Waals force between ClpY R25 and Y408 might help ClpY hexamer formation. Here, the gradient size of point mutants on Y408 were constructed, according to their side chain size of the amino acid. In vitro, the purified mutant proteins crosslink by glutaraldehyde, were running on the sodium dodecyl sulfate agarose gel electrophoresis to see whether there is a 300 kDa hexamer cylinder or not. ClpY Y408W forms more hexamer than those of the wild-type, Y408L, Y408C and R25A/Y408A. In vivo, ClpY Y408 mutants were constructed on pBAD24, and were co-transformed with ClpQ and tagged-SulA, which is a substrate of Lon and ClpYQ and can inhibit cell division, in AC3112 (lon- clpY-Q^(--)) strain. The wild type ClpYQ can degrade SulA, then cell grow, otherwise the cell would become lethal. Cell viability reveals the Y408W growth equal to the wild type, and the sequential growth order as follows: Y408M, Y408L and Y408V/C/A. Briefly, the side chain of the amino acid of ClpY Y408 mutants has an effect on the function; gradually, once the side chain is bigger, the cell can grow better and the hexamer forms more. Taken together, the Y408 is the key amino acid for the ClpY hexamer formation.
Subjects
六圓環
分解基質
ATP-dependent protease
ATP依賴蛋白酶
Degradation of substrates
Hexameric ring
Type
journal article