總體的找出熱量限制時老化訊息傳遞路徑中的磷酸化位點與功能

Abstract

老化是一種細胞逐漸喪失生理功能的複雜過程，過去研究指出熱量限制能有效地延長各物種的平均壽命、延緩老化，其重要調控機制已被報導是藉由TOR/Sch9及 Ras/PKA等訊息傳遞路徑所調控，藉此適應外在環境營養的改變，延長細胞壽命，然而確切的調控機制目前還不清楚。由於熱量限制會影響許多磷酸化蛋白的訊息傳遞，所以我們想探討在熱量限制的酵母菌中是如何藉由磷酸化或去磷酸化來調控細胞的壽命。本篇研究分別將酵母菌培養在含有不同濃度葡萄糖(2%，0.5%)的培養基中，並用蛋白質體學分析找出可能是經由熱量限制導致磷酸化調控變異的蛋白。結果顯示在熱量限制的情況下，酵母菌有318個蛋白會被磷酸化，425個蛋白會被去磷酸化，接著利用Gene ontology及GeneMANIA的分析，並結合SGD phenotype，GeneAge資料庫，我們一共選擇了22個蛋白做後續的分析，透過單點或雙點點突變去模擬磷酸化狀態的改變，觀察在不同的壓力下其生長情形是否會被影響，結果顯示這些磷酸化位點皆不會影響到其功能，因此我們未來還需要做更進一步的分析。

Aging often characterized as a complex process accompanied by progressive loss of physiological functions. Previous studies reveal that calorie restriction (CR) can effectively extend the lifespan of various organisms, delay the aging process. The critical mechanisms of CR have been reported by the regulation of TOR/Sch9 and Ras/PKA signaling pathways. While the exact mechanism still remains elusive, due to CR affecting many signaling pathways through phosphorylation, we aim to investigate how yeast cells mediate the lifespan by phosphorylation under CR. In this study, yeast cells were grown in medium containing 2% or 0.5% glucose, respectively. Then we employed the quantitative proteomics to elucidate which proteins may be regulated. There were 318 proteins been phosphorylated, and 425 proteins been dephosphorylated under CR. Next, we analyzed these proteins by Gene ontology and GeneMANIA, and combined with SGD phenotype and GeneAge database. In total, 22 candidates were selected for further investigation. By generating phospho-mimic or phospho-abolishing mutants to verify growth under different types of stress. Results showed that the single point or multiple site mutations did not affect the function of identified proteins. Consequently, further analysis are needed in the future.