大腸桿菌熱休克蛋白酶ClpYQ分解基質之研究

Abstract

大腸桿菌ClpYQ (HslUV) 蛋白酶為ATP 依賴型蛋白酶的一種，由49 kDa的ClpY 及19 kDa 的ClpQ 組合而成。這類蛋白酶在細胞中可降解不正常堆積的蛋白質或將折疊錯誤的蛋白質恢復正常構形，避免細胞受損。SulA 為一細胞分裂抑制者，當細胞遭遇自由基傷害或是曝露於UV 光下，造成DNA 受損，會誘發SulA 蛋白表現，抑制細胞分裂。目前已知ClpY 是辨識SulA C-端第20 - 30個具高疏水性的胺基酸片段，若將此片段之胺基酸點突變為親水性胺基酸，會降低SulA 與ClpY 之間的交互作用。然而，若將SulA C-端末20 個胺基酸去除，會使SulA 失去活性且成為一聚集之蛋白質。此外，RcsA 亦被認為是ClpYQ 之基質，但RcsA 是如何被ClpY 辨識，目前仍不清楚。 本研究以大腸桿菌表現蛋白質，利用西方墨點法測試ClpY 突變蛋白分解SulA ΔC20、SulA*F143A 的能力，發現ClpY 對於SulA*F143A 突變蛋白及失活且結構已打開之SulAΔC20 突變蛋白的辨識情形與辨識野生型SulA 蛋白相類似。也發現ClpY 第91 個胺基酸Tyrosine 之hydroxyl-group 對於基質的傳送，亦扮演了重要角色。以酵母菌雙雜交系統測試SulA C-端第20 - 30 個胺基酸點突變蛋白與SulA 之間的交互作用，發現這個區域點突變之活性，與能否形成dimer相關。以β-galactosidase 測試RcsA 蛋白於不同溫度下之活性與被ClpYQ 降解的情形，發現RcsA 蛋白的活性隨溫度上升而下降，且β-galactosidase units 值皆因ClpYQ 蛋白酶的出現而下降。之後再利用西方墨點法直接偵測RcsA 蛋白的累積量，在不同溫度下RcsA 的累積量皆相似，誘導ClpYQ 後，累積量皆有下降的趨勢，故RcsA 亦為ClpYQ 之基質。此外也發現，ClpY 對於SulA 及RcsA 之辨識是具相類似的機制。

ClpYQ (HslUV) is an ATP-dependent protease from Escherichia coli, composed of the 49 kDa ClpY and 19 kDa ClpQ. ClpQ peptidase. The degradation of the abnormal proteins or the misfolded proteins is accomplished by this protease to avoid cell damage. When the cells are exposed to UV light or damaged by free radical, the SOS response induced a cell division inhibitor, SulA. ClpY recognizes its C-terminal 129 - 149 hydrophobic amino acids. However, SulA becomes inactive and aggregated while it lacks the last 20 amino acids. Also it was observed that HA-SulA*F143A was slowly degraded by ClpYQ. In addition, RcsA, as one of its substrates, is not clear for its direct targeted by ClpYQ. In this study, Western blot analysis was used to determine how ClpY is to recognize SulA ΔC20, and SulA*F143A. As results, ClpY recognizes SulAΔC20 and SulA*F143A in a similar way as it did to the wild-type SulA. We showed here that the C129 - 149 region of SulA is related to the monomer or dimer formation. The aromatic ring of ClpY91 (tyrosine,Tyr) is, however, known necessary for the translocation of substrates. In addition, through our study, we also showed that the hydroxyl-group of ClpY91 (Tyr) plays an important role in translocating of the substrates to ClpQ. From an alternative approach, we adopted the assays of β-galactosidase activity of cpsB::lacZ with pTH18kr-rcsA with or without ClpYQ. We found that the activity of RcsA decreases with an increased temperature, and the β-galactosidase levels decrease when ClpYQ is present. Through Western blot analysis, it was shown that the accumulation of RcsA was equal under the different temperature. However, it was reduced when expressing the ClpYQ protease. Therefore, RcsA is one of the substrates for ClpYQ. In addition, we also demonstrated that ClpY recognizes RcsA in a similar way as it did toward SulA.