大肠杆菌赖氨酸乙酰基转移酶Pka的逆境调控机制及特性研究

Lysine acetylation is a well-established post-translational modification widely conserved and distributed in bacteria. Although multiple regulatory roles have been proved, little is known about its regulation. It has been shown previously that the Escherichia coli protein lysine acetyltransferase Pka is down-regulated by stress conditions, such as cold shock, starvation and entry into stationary phase. As a result, RNase R, an important exoribonuclease that participates in the degradation of structured RNAs in Escherichia coli, is acetylated on lys544 in exponential phase but not in stationary phase. This modification results in tighter binding of tmRNA and SmpB and subsequent proteolytic degradation of the RNase by HslUV and Lon proteases. What was not understood, however, is how the regulation of Pka by stress is accomplished. We found that the cellular concentration of Pka is controlled at transcriptional level using real-time RT-PCR analysis. After forward genetics screening, we isolated the sopd1 (suppressor of pka down-regulation 1) mutant in which the expression level of Pka in stationary phase is much elevated. Interestingly, Western blot analysis showed that at least five bands of acetylated protein disappeared in pka mutant while the majority was unchanged, suggesting Pka specifically acts on some proteins in the cells. We aim at exploring the molecular mechanism of Pka regulation by Sopd1 in the current project with approaches of genetics, biochemistry, molecular biology and bioinformatics. We also intend to identify Pka specific substrates through mass spectrometry analysis as well as to characterize its enzymatic properties. Results of this study will not only greatly advance our understanding of how this post-translational modification system enables microbes to adapt to and survive in stress environments, but also lay the foundation for the design of new drgus that could kill or cripple pathogenic bacteria.

赖氨酸乙酰化是细菌中一种广泛存在的蛋白翻译后修饰方式，参与调节许多生物学过程，但有关其自身调控的研究还很少。在我们以前的研究中发现大肠杆菌赖氨酸乙酰基转移酶Pka的表达受到逆境如冷胁迫以及进入稳定生长期等的下调，但是到目前为止，Pka是如何受到调控的还不清楚。我们在前期实验中发现Pka的调控发生在转录水平上。通过遗传学筛选，我们得到了一个突变体sopd1，该突变体中Pka在逆境下的表达水平与野生型相比大大提高。此外，Western blot分析发现在pka突变体中至少5个乙酰化蛋白条带缺失，而大部分蛋白没有明显变化，表明Pka在体内特异性的乙酰化一些蛋白。本申请项目旨在探索逆境条件下Sopd1调控Pka表达的分子机制，鉴定Pka特异性的底物，并阐明Pka的酶学性质。本研究的结果将有助于理解蛋白赖氨酸乙酰化修饰在微生物适应环境变化及在逆境中存活的作用，以及设计抗菌药物控制病原细菌的发生。

赖氨酸乙酰化是细菌中一种广泛存在的蛋白翻译后修饰方式，参与调节许多生物学过程，但其自身的调控机制以及在生物逆境反应过程中的作用并不清楚。本项目研究发现一个功能未知的蛋白Sopd1能够结合到乙酰基转移酶Pka的启动子区域抑制其转录。把Sopd1的螺旋-转角-螺旋结构去掉或者把pka启动子的核心元件突变之后，Sopd1不再调控pka表达。当细胞遭遇低温等逆境胁迫之后，Sopd1上调表达，导致pka的表达量极大降低。质谱分析发现，当把pka敲除掉之后，RNase II、RNase R等13个蛋白的乙酰化水平显著提高，表明Pka可能特异性的催化这些蛋白的乙酰化修饰。RNaseⅡ第501位赖氨酸存在乙酰化修饰，这一修饰由Pka与去乙酰化酶CobB共同调控。由于K501位于RNaseⅡ的催化活性中心，这一修饰会降低RNaseⅡ结合底物的能力并最终影响该酶的活性。当细胞遭遇逆境如营养胁迫后，RNaseⅡ的乙酰化水平升高，活性降低，从而使得细菌能够更好的生存。RNase R第544位赖氨酸的乙酰化修饰使得它能够结合到核糖体上降解含有回文结构（REP序列）的mRNA。REP序列对于调节蛋白翻译非常重要。当REP序列与上游基因终止密码子的距离小于或者等于15个碱基时，其能够抑制翻译。这是因为在翻译过程中，当距离过短时，REP序列会阻止核糖体的前进，使得翻译不能正常终止，并由核酸酶RNase R以及蛋白酶分别对该mRNA以及其多肽产物进行降解。以nrdAB为例，当细胞遭遇逆境如紫外照射后，RNA解旋酶的表达量上升，该酶能够对REP序列进行解链，解除REP序列的负调控作用。生物信息学分析显示大肠杆菌中大约50%的REP序列与上游基因终止密码子的间距小于或者等于15个碱基，因此这一调控机制可能在细菌中普遍存在。这些研究结果表明乙酰化修饰在生物逆境反应过程中起重要作用。