高鹽太古生物在溫度、鹽及氧氣逆境下化學伴護因子對分子伴護蛋白基因表現的影響

Abstract

細胞內的環境會受胞外環境的波動變化而在瞬間劇烈的改變，使胞內蛋白因逆境失去正常的功能及結構。分子伴護蛋白系統是生物細胞用來修復失活蛋白的最佳工具，ClpB、DnaK/DnaJ/GrpE（KJE）、GroEL/ES這些分子伴護蛋白系統主要的功能是能將逆境下產生的聚集蛋白進行去凝集化，協助失活蛋白重新折疊後能恢復正常功能。當生物面臨高滲透壓逆境時，會藉由自體生合成或由外界攝取的方式在細胞內累積相容質來對抗逆境，相容質因具有協助蛋白維持正常構形，及協助未折疊多胜肽鏈折疊的功能，被稱為化學伴護因子。高鹽甲烷太古生物Methanohalophilus portucalensis FDF1生長的鹽濃度在1.2到2.9 M間，適應高鹽環境的機制主要為自體生合成β-glutamine、Nε-acetyl-β-lysine與glycine betaine，或自外界攝取glycine betaine及其前驅物，也具有分子伴護蛋白基因clpB及groEL/S，基因表現量受到鹽度及溫度的影響。本研究以北方墨點法分析，clpB基因會在M. portucalensis FDF1生長初期、對數期、及平穩期表現，groELS基因則僅在生長初期與對數期表現，推測GroEL/ES在M. portucalensis FDF1快速生長時協助胞內蛋白結構的穩定，而ClpB在細胞生長時皆會表現。ClpB和GroEL/ES的基因都受高溫熱休克誘導表現，clpB基因表現量在高溫逆境41及45℃時分別增加9.7與8.7倍；groELS基因表現量則分別增加6.7及9.3倍，但clpB與groELS基因表現量皆不受低溫逆境20與29℃的影響。低鹽逆境0.9 M及高鹽逆境3.3 M下，clpB基因表現量分別增加2.04與1.64倍，顯示clpB基因受鹽逆境滲透壓力誘導表現；groELS基因表現量則不受到高鹽逆境3.3、2.7 M及低鹽逆境1.5 M的影響，但在低鹽逆境0.9 M時基因表現量增加1.96倍；推測groELS並不受鹽濃度改變所形成的滲透逆境誘導表現，但胞外鹽濃度太低造成此高鹽生物的新合成蛋白的折疊不正常會誘導groELS基因表現。於鹽及溫度逆境時，在培養液中添加0.5 mM的glycine betaine使3.3 M高鹽逆境時clpB基因表現量下降，高溫逆境45℃時clpB與groELS基因表現量下降，推測M. portucalensis FDF1自培養液攝取glycine betiane作為化學伴護因子，使胞內蛋白結構穩定，便不需大量轉錄、轉譯製造ClpB與GroEL/ES蛋白。絕對厭氧的M. portucalensis FDF1面對氧氣逆境時，clpB基因轉錄量會下降，groELS基因轉錄量則在曝氧20分鐘後開始下降，推測M. portucalensis FDF1因接觸氧氣而逐漸死亡。

Environmental fluctuations usually affect protein structures and functions in cell. By use of molecular chaperone systems, native form proteins are maintained. ClpB, GroEL/ES, and DnaK, DnaJ, GrpE (KJE) chaperone systems are involved in protein disaggregation process. These chaperones liberate the polypeptide from aggregates and facilitate the polypeptide folding correctly. Under high osmotic stress, organism accumulates osmolytes to adapt the stress. Osmolytes are also called chemical chaperones. Not only chemical chaperones help proteins maintain native form, assist refolding of unfolding polypeptide. Methanohalophilus portucalensis FDF1 can grow over a range of external NaCl concentration from 1.2 to 2.9 M. It can de novo synthesize glycine betaine, β-glutamine, and Nε-acetyl-β-lysine as omolytes. It can also transport glycine betaine or precursor of it in cell. M. portucalensis FDF1 has clpB, groEL and groES genes, induced by salt and high temperature stress. At different growth phases, clpB gene transcribed at the lag, exponential and stationary phases and groELS gene only transcribed at the lag and exponential phases. These results suggested that ClpB and GroEL/ES control protein quality during the cell growth. Under hyper salt (3.3 M) stress and hypo salt (0.9 M) stress, clpB transcript levels increased 1.64 and 2.04 fold, respectively. GroELS transcript level increased 1.96 fold under hypo salt (0.9 M) stress, but not in hyper salt (3.3 M) stress. These results suggested that groELS gene expression was not induced by salt and osmotic stress. However, the low salt concentrations for high salt lover might lead to the increasing proteins folding and aggregation which induced the groELS gene expression. Under heat stress, clpB transcript levels increased 9.7 or 8.7 fold under 41 or 45℃ and groELS transcript levels increased 6.7 or 9.3 fold, and both genes have no effect on cold stress. With the addition of glycine betaine, clpB transcript levels decreased both at hyper salt and heat stress and groELS transcript levels decreased under heat stress. These effects indicated glycine betaine may function as chemical chaperone to protect the protein.