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

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。

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.