利用化學探針尋找參與在老化過程中的絲氨酸水解酶

Identification and Characterization of Serine Hydrolases in Senescence Using Chemical-based Proteomic Approach

胡容綺(Rong-Chi Hu)

指導教授 ： 林敬哲

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/NTU201603370

摘要

人體中大部分的正常體細胞無法無限制地進行細胞分裂，並最終會進入不可逆的生長停止階段，稱之為細胞老化。雖然目前已經有研究發現端粒縮短(telomere shortening)為誘發老化的主因，但仍有許多調控和維持細胞老化過程的未知因子。絲氨酸水解酶在人體蛋白質中佔了約1%的含量，且許多絲氨酸水解酶參與在重要的生理作用中，除此之外，許多絲氨酸水解酶被發現會影響細胞老化，所以本研究的目的為尋找會參與在老化過程的絲氨酸水解酶並進一步研究其特性。本研究利用以活性為基礎的化學蛋白質體分析方法，來鑑定在老化過程中活性有改變的絲氨酸水解酶。在本研究中，利用人類肺纖維母細胞IMR90作為研究細胞老化的平台，並以擁有ethyl-benzylphosphonofluoridates反應端的化學探針LCL08027來標定具有活性的絲氨酸水解酶。透過比較年輕和老化細胞中標定蛋白的差異，鑑定到一些絲氨酸水解酶在老化過程中會有活性的改變，其中tripeptidyl peptidase 2 (TPPII)和protein phosphatase methylesterase 1 (PPME1)的活性會隨著老化而降低。進一步利用small hairpin RNAs發現，當抑制TPPII和PPME1的基因表現時會誘導年輕的IMR90細胞進入老化，證實了這兩個絲氨酸水解酶會參與在老化過程中。總結以上，本研究成功利用以活性探針為基礎的蛋白質體分析法去鑑定到尚未被證明會被參與在老化過程的絲氨酸水解酶。

關鍵字

細胞老化 ； 絲氨酸水解酶 ； 化學探針

並列摘要

Most of normal human somatic cells cannot divide indefinitely and eventually enter a state of irreversible proliferative arrest termed replicative senescence. Although previous researches have shown that telomere shortening is the major reason for inducing senescence in human somatic cells, factors that mediate and maintain senescence are largely unknown. Serine hydrolases is a large enzyme family which constitutes about 1% of the human proteome and many of them are involved in important physiological processes. In addition, many serine hydrolases have been shown to affect cellular senescence. Thus, the goal of this research is to identify and characterize novel serine hydrolases that participate in senescence. An activity-probe based chemical proteomic approach was applied to identify serine hydrolases with their activities been altered during senescence. Here the human lung fibroblast IMR-90 cell was used as a model in this research. Chemical probe LCL8027, which has an ethyl-benzylphosphonofluoridates reactive group, is used to label active serine hydrolases. Through comparative labeling of both young and old cells, several serine hydrolases were identified that showed alteration of labeling activities during senescence. Among them, both the activities of tripeptidyl peptidase 2 (TPPII) and protein phosphatase methylesterase 1 (PPME1) were found to be decreased during senescence. Further analyses showed that knocking down TPPII or PPME1 using small hairpin RNAs induced young IMR90 cells into senescence, suggesting a role of these two serine hydrolases in senescence. Together, this study successfully applied an activity probe-based proteomic analysis to identify novel serine hydrolases that might be involved in cellular senescence.

並列關鍵字

cellular senescence ； serine hydrolases ； chemical-based proteomic approach ； activity-based probe

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