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  • 學位論文

探討酵母菌AMPK—Snf1蛋白表現量的調控機制

Regulation of Yeast AMPK Homolog Snf1 Protein Level

指導教授 : 羅翊禎

摘要


腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)為細胞中調控能量代謝的關鍵蛋白,廣泛存在於真核生物中,當其受到腺苷酸活化時,會促進細胞內的分解代謝作用產生能量;而AMPK活性的異常與很多疾病有關,如第二型糖尿病、帕金森氏症、阿茲海默症、癌症。酵母菌中的Snf1 (Sucrose non-fermenting 1)為AMPK的同源蛋白,而Snf1也與細胞內的能量代謝有關。當環境中缺乏葡萄糖時,Snf1會被活化,進而促進基因表現或下游蛋白質的活化,使酵母菌能夠利用環境中其他碳源生長。在先前實驗中發現某酵母菌菌株的Snf1蛋白表現量與正常酵母菌相比明顯較低,因此本實驗想要探討造成該菌株Snf1蛋白表現量下降的原因。利用全基因體定序(Whole genome sequencing)及細胞培養穩定同位素胺基酸標記(Stable Isotope Labeling with Amino acids in Cell culture, SILAC)尋找可能造成Snf1蛋白下降的突變基因或蛋白質,希望能發現目前尚未被找出的Snf1蛋白表現量的調控機制。實驗結果發現,Snf1蛋白表現量較低的菌株其SNF1基因上具有兩個點突變,但經由質體表現該突變蛋白後,Snf1蛋白表現量並無顯著下降的現象。另外,也發現標記Snf1蛋白的TAP (Tandem affinity purification)上存在著一個點突變,目前推測該突變可能是造成Snf1蛋白下降的原因。另一方面,SILAC的實驗結果發現大量表現Hsp104蛋白可能可以回復Snf1蛋白的表現量。因此,我們推論TAP上的點突變可能會去影響Snf1蛋白的結構,進而造成Snf1蛋白降解。

並列摘要


AMPK plays an important role in energy homeostasis, and it is also named as “energy sensor of the cell”. AMPK is highly conserved in eukaryotes, it stimulates catabolism to generate energy in cells upon activation. Abnormality in AMPK regulation may relate to many diseases, such as type 2 diabetes, neurodegenerative diseases, and cancer. The yeast homolog of AMPK Snf1 is required for maintaining energy homeostasis and stress resistance. The SNF1 protein kinase is consist of α, β, and γ subunits, when glucose limitation occurs, α subunit—Snf1 is activated by phosphorylation of T210, and then promotes gene expression to utilize other carbon sources. The phosphorylation level of Snf1 can be altered when mutations occur in SNF1. In this study, I investigated the underlining mechanisms that lead to the reduction of the Snf1 protein level. I used the whole genome sequencing and SILAC (Stable Isotope Labeling with Amino acids in Cell culture) analysis to search the possible gene mutation(s) or protein which may cause the diminishment of the Snf1 protein level. Surprisingly, I identified two specific mutations resided at the C terminus of SNF1 in the Snf1 lowly expressed strain, but my further analysis indicated that these two point mutations within the SNF1 did not induce significant reduction in the Snf1 protein level. I further discovered that the TAP which tagged at the C-terminal of SNF1 carried a novel mutation and I demonstrated that this mutation on TAP is likely responsible for the reduced Snf1 protein level. In addition, SILAC analysis showed that overexpression of Hsp104 may restore the Snf1-TAP protein level. Thus I propose that the newly identified variant of the Snf1-TAP may be structurally altered and lead to an instability and degradation.

並列關鍵字

AMPK yeast Snf1 whole genome sequencing SILAC

參考文獻


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