高鹽甲烷太古生物第一類型與第二類型分子伴護因子之特性分析

Abstract

分子伴護蛋白系統 (molecular chaperone) 普遍存在於原核生物與真核生物之間，能夠在細胞面臨外界逆境下，協助多胜肽摺疊與組裝並幫助未摺疊、部分摺疊、錯誤摺疊的蛋白摺疊成正確構型及修復、降解聚集蛋白來避免細胞受到損害。分子伴護因子 (chaperonin) 屬於分子伴護蛋白系統所獨立出來的一群，在形成雙環結構與確保蛋白摺疊過程中的正確性及失活蛋白的再摺疊都需要ATP的參與。分子伴護因子可分為二種類型，第一類型存在於細菌及真核生物的粒線體與葉綠體中；第二類型則存在太古生物與真核生物細胞質內。近年在高鹽甲烷太古生物Methanohalophilus portucalensis FDF1T 中也發現第一類型分子伴護因子groEL與groES的存在，並由轉錄分析顯示其會受到熱逆境與鹽逆境的誘導表現。接著將MpgroEL與MpgroELS構築於groEL基因缺陷大腸桿菌突變株BB4565中，由結果顯示在突變株當中表現MpGroEL和MpGroES能夠協助突變株在高溫逆境下提高存活率。此外，異源表現的MpGroEL ATPase活性測試得到每毫克蛋白於每分鐘能夠釋放出0.03 nmole的無機磷酸根，推測MpGroEL具有自體水解ATP的能力。額外添加化學伴護因子glycine betaine (0.3 M)，能夠提升MpGroEL七倍的ATP水解速率，推測glycine betaine具有穩定MpGroEL蛋白結構並幫助調節其ATP水解能力。本篇研究的結果證實M. portucalensis FDF1T除了具有與細菌親源較接近的GroEL與GroES，能互補細菌GroEL和GroES的功能之外，並藉由基因體分析發現亦有第二類型分子伴護因子thermosome的序列，於三級結構分析發現thermosome特有之結構helical protrusion，因此得知在FDF1T中同時具備二種類型的分子伴護因子，應有助於高鹽甲烷太古生物適應高鹽高溫的曬鹽場極端環境。

Molecular chaperones are ubiquitous among Three Domains of living organisms. They can help correct folding and assembling of unfolded, misfolded or nascent polypeptides and reactivate aggregated proteins which were damaged by stress. Chaperonins are ubiquitous class of proteins belonged to the molecular chaperones, form double ring complexes that mediate the folding of nascent and denatured proteins in an ATP-dependent manner. The chaperonins are divided into two groups: Group I chaperonin, usually found in bacteria, mitochondria and chloroplasts from eukarya, and Group II chaperonin, occurring in archaea and the eukaryotic cytosol. Lately, genes encoded for group I chaperonin GroEL and GroES in halophilic methanogen were found. The group I chaperonin GroEL usually cooperate with GroES to prevent stress-induced protein aggregation. Transcriptional analysis indicated that groEL and groES genes from Methanohalophilus portucalensis FDF1T were up regulated by heat and salt stresses. The expression of MpGroEL and MpGroELS in groEL-null mutant strain E. coli BB4565 showed higher survival rate which accumulated less aggregated proteins than host E. coli BB4565 under high temperature environment (43�C). Furthermore, in vitro ATPase activity assays were carried out by heterologously expressed and purified MpGroEL. Specific activity of MpGroEL was 0.03 nmole Pi released per mg protein in per minute and could be enhanced to 7 folds with chemical chaperone glycine betaine (0.3 M). This study revealed the chaperonin function of MpGroELS in halophilic methanogenic archaea. In addition, the osmolyte betaine plays an important role in regulating the ATPase activity of MpGroEL. These results confirmed that MpGroEL and MpGroELS can complement the function of bacterial GroELS. Additionally, three group II chaperonin thermosome were found in the chaperonin system of M. portucalensis FDF1T and revealed it contains both group I chaperonin GroEL/ES and group II chaperonin thermosome. The co-existence of group I and II chaperonin in the halophilic methanogen may release the cellular damage from salt and temperature stresses to halophilic methanogen under solar saltern environment.