探討酵母菌去甲基酶Rph1在世代壽命中之功能與調控機制

Functional and mechanistic studies of the H3K36 demethylase Rph1 during the chronological life span in Saccharomyces cerevisiae

林暐瀚(Wei-Han Lin)

指導教授 ： 羅椀升

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603541

摘要

對生物體來說，老化是一個必然會發生的生理過程，而老化被認為是造成疾病與死亡的風險因子之一。老化發生的機制仍有許多未解之謎，而在過往的十年中，其相關的研究已經發展至分子生物的層次，因此，以單一細胞做為一個獨立個體的出芽酵母提供了一個極好的模型來研究高等真核生物的老化機制。在本篇論文中，我利用酵母菌的世代壽命 (chronological lifespan, CLS) 模型來進行老化的研究。由於染色體的動態變化被認為是老化過程中重要的調控方式之一，因此本篇論文之研究方向主要是想探討酵母菌的H3K36去甲基酶Rph1在老化過程中扮演的角色。
在第一部分中，我使用內源性表達Rph1之細胞作為研究材料。首先，本研究發現Rph1對於老化的酵母菌之熱耐受性是十分重要的，而且其功能性區域zing-finger與JmjN對於熱耐受性皆是不可或缺的，但失去Rph1去甲基酶活性的細胞則不會對於其熱耐受性有明顯的影響。此外，Rph1可能會參與細胞週期的調控，並對於細胞進入與離開靜止期(stationary phase)有其重要的功能。再者，本研究亦發現酵母菌會藉由調控Rph1磷酸化與去磷酸化等轉譯後修飾的方式來控制其在不同時期之功能，而此調控機制可能是受到Rim15相關的訊號傳遞路徑來調控的。除了轉譯後修飾之外，本研究也發現在老化過程中，細胞會藉由調控Rph1信使RNA的轉錄與Rph1蛋白質的量來控制Rph1的功能。而在第二部分中，本研究發現過度表達Rph1會造成細胞延長其生長遲滯期(lag phase)，而異位表達Rph1則會提高細胞在老化時期的熱耐受性。綜合上述結果，本研究指出H3K36去甲基酶Rph1參與在酵母菌細胞進入老化的進程以及老化細胞回到適當環境後重新啟動生長等重要的過程。

關鍵字

世代壽命 ； 組蛋白去甲基酶 ； Rph1 ； 老化

並列摘要

Aging is an inevitable biological process leading to death and remains as a long-standing mystery in biology. In the last decade, research of aging has been advanced extensively into the molecular level. The single-celled Saccharomyces cerevisiae (budding yeast) is an excellent model organism to study the molecular and cellular events of chronological lifespan (CLS) in eukaryotes. Chromatin dynamics has been appreciated as one of the master modulators in aging process. Therefore, my thesis research focuses on the mechanistic roles of H3K36 demethylase Rph1 in regulation of the CLS in S. cerevisiae.
First, I investigated the functional roles of Rph1 expressed by its own promoter in response to stress condition during chronological aging. I showed that Rph1 is required for thermotolerance in aging yeast. The functional domains of Rph1, zing finger (ZF) and JmjN, but not the catalytic domain for demethylase activity, contribute to the thermotolerance activity in aging cells. In addition, Rph1 is likely involved in cell cycle progression, quiescence entry and exit. Furthermore, Rph1 is hyper-phosphorylation upon diauxic-shift and hypo-phosphorylation upon re-feeding and such reversible protein phosphorylation of Rph1 is Rim15-dependent during CLS. In the second part of my work, I found that constitutively expressed and overexpressed Rph1 prolong the lag phase of earlier cell growth. Furthermore, the higher expressed Rph1 enhanced the thermotolerance of aging yeast in stationary phase, suggesting that the expression of Rph1 is highly regulated and consequently to control cell growth in chronological aging. Taken together, my work reveals the functional roles of Rph1 in regulation of yeast CLS and further demonstrates the post-translational modifications contribute to modulate Rph1 on aging and/or rejuvenation process.

並列關鍵字

chronological life span ； H3K36 demethylase ； Rph1 ； aging

參考文獻

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