分析腺嘌呤生合成激酶在細胞老化中的角色

Abstract

正常的人體細胞因為端粒縮短所以不能無限分裂，這種現象稱為細胞老化。端粒縮短會誘導 DNA 損傷反應以阻止細胞週期停滯。DNA 損傷途徑主要由一系列蛋白質的磷酸化和去磷酸化介導。激酶是可以進行多種基質磷酸化的酵素，包括蛋白質和小分子代謝物。為了找出參與在細胞老化中的激酶，我們的實驗室應用基於活性的蛋白質分析 (ABPP)策略來篩選其活性在年輕和老化細胞中發生改變的激酶。在該分析中使用選擇性標記活性激酶的化學探針LCL16001。在本篇研究中，我們首先分析了質譜數據，並選擇了三種激酶，ADK (adenosine kinase)、AK4 (adenylate kinase 4)和 AK5 (adenylate kinase 5)進行進一步分析。這三種激酶參與腺嘌呤生物合成反應，發現它們的活性在老化細胞中升高。使用基於 LCL16001 的 ABPP 方法，我證實了 ADK、AK4 和 AK5 的激酶活性在老化細胞中升高。有趣的是，只有AK4 在老化細胞中顯示出蛋白表現有增加，老化細胞中 ADK 和 AK5 蛋白表現則無。我同時也發現 AK4 的升高不是由於老化細胞中轉錄因子 p53 的激活。為了測試這三種激酶在老化中的作用，我通過腺病毒感染過度表達這些基因，發現它們都沒有誘導細胞的老化。由於激酶活性在細胞中受到嚴格調節，因此實驗可能需要這些基因constitutively-active 突變。有趣的是，通過short hairpin RNAs 降低這三個基因中任一個的表達都會導致正常人成纖維細胞的老化。因此，雖然尚不清楚ADK、 AK4 和 AK5 如何參與衰老，但它們對於防止細胞老化是必要的。

Due to telomere shortening, normal human somatic cells cannot divide infinitely. This phenomenon is called cellular senescence. Telomere shortening induces DNA damage response to arrest cell cycle arrest. DNA damage pathway is mainly mediated by a series of phosphorylation and de-phosphorylation of proteins. Kinases are enzymes that carry out phosphorylation of a variety of substrates including proteins and small molecule metabolites. In order to identify kinases that are involved in cellular senescence, our lab applied activity-based protein profiling (ABPP) strategy to screen for kinases whose activities are altered in proliferating and senescent cells. A chemical probe, LCL16001, that selectively labels active kinases was used in this analysis. In this study, we first analyzed the mass spectrometry data and selected three kinases, ADK (adenosine kinase), AK4 (adenylate kinase 4) and AK5 (adenylate kinase 5) for further analysis. These three kinases are involved in adenine biosynthesis process and their activities were found to be elevated in senescent cells. Using a LCL16001-based activity assay system, I confirmed the kinase activities of ADK, AK4 and AK5 are elevated in senescent cells. Interestingly, only AK4 showed increased protein level in senescent cells. The ADK and AK5 protein levels were not significantly increased in senescent cells. I also found the elevation of AK4 was not due to the activation of transcription factor p53 in senescent cells. To test the role of these three kinases in senescence, I overexpressed these genes by adenovirus infection and found that none of them induced senescence in normal human fibroblasts. Since the kinase activities are tightly regulated in cells, the constitutively-active mutant of these genes might be required for the experiment. Interestingly, knocking down the expression of either one of these three genes by short hairpin RNAs resulted in senescence of normal human fibroblasts. Thus, although it is not clear how ADK, AK4 and AK5 are involved in senescence, they are required to prevent cells from senescence.