【作者】 张群芳；

【导师】 张先恩； 喻子牛；

【作者基本信息】 华中农业大学 ， 微生物学， 2013， 博士

【摘要】 蛋白质的赖氨酸乙酰化是从细菌到真核生物都普遍存在的一种翻译后修饰。几十年来,研究者们发现赖氨酸乙酰化在真核生物细胞中发挥着许许多多的生物学功能。近几年,在细菌和古生菌中,也发现了这种翻译后修饰的广泛存在。尽管如此,其在原核细胞中的分布范围仍然不太清楚,这点也成为了开展原核系统中赖氨酸乙酰化修饰功能研究的障碍。蛋白质的赖氨酸乙酰化是一种可逆的修饰,主要由蛋白质乙酰转移酶和去乙酰化酶控制。在细菌中,研究最多的去乙酰化酶是与酵母中沉默信息调节蛋白Sir2同源的CobB蛋白。虽然近几年来有关CobB蛋白的功能研究取得了一些进展,但其底物和在细菌中的功能仍然不清楚。首先,我们使用蛋白质组芯片技术筛选到了十五种具有赖氨酸乙酰化修饰的蛋白质,其中的九种可以被大肠杆菌CobB催化去乙酰化,包括N-羟基芳香胺O-乙酰转移酶(NhoA)。我们使用Western blot验证了NhoA的确可以被CobB去乙酰化,并结合质谱分析技术鉴定出NhoA中三个具有可逆的乙酰化修饰的赖氨酸残基。定点突变试验显示了其中两个乙酰化赖氨酸的突变会导致NhoA乙酰化水平的降低。进一步的研究发现,NhoA的N-乙酰转移酶(NAT)活性和O-乙酰转移酶(OAT)活性都会受到这两个乙酰化赖氨酸残基突变的影响。并且,得到了直接的数据,证实CobB对NhoA的去乙酰化作用提高了NhoA在体外的NAT活性。为了进一步阐明体内的赖氨酸乙酰化修饰对NhoA活性的影响,我们对比了cobB基因敲除菌株与野生型菌株NhoA蛋白的乙酰化水平,以及对硝基芳香族化合物的诱变敏感性。结果显示,cobB基因的敲除会导致NhoA乙酰化水平的提高和OAT活性的降低。这些结果说明,可逆的赖氨酸乙酰化修饰在体外和体内条件下都能够调节NhoA的活性。并且,CobB在体外可以通过催化NhoA的去乙酰化提高其NAT活性,在体内,也很可能是通过相同的方式调节NhoA的OAT活性。综上所述,本研究中我们鉴定到了新的赖氨酸乙酰化修饰的蛋白质和去乙酰化酶CobB的底物,发现了赖氨酸乙酰化修饰和细菌中CobB蛋白的一个新功能,这对于原核生物中乙酰化修饰领域的研究具有十分重要的指导意义。更多还原

【Abstract】 Protein lysine acetylation is one of the most widespread protein post-translational modifications in both eukaryotes and bacteria. In recent decades, this post-translational modification has been shown to play key roles in many biological processes in eukaryotic cells. Recently, protein acetylation has also been found to be widespread in bacteria and archaea, however, the extent of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems.Protein acetylation may be controlled by protein acetyltransferases and deacetylases. In bacteria, the best known deacetylase is CobB, which is a homolog of eukaryotic Sir2. Though progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear.Using proteome microarray technology, fifteen potential lysine-acetylated proteins and nine potential substrates of Escherichia coli CobB were screened, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by Western blotting and mass spectrometry, and three acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of two of the acetylated lysines decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at these two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. And then direct data showed that deacetylation of NhoA by CobB increased its NAT activity in vitro. To elucidate the effects of reversible acetylation on NhoA activity in vivo, we tested the acetylation level of NhoA and mutagenicity of nitro aromatic compounds in a cobB-knockout strain. Results showed that, knockout of cobB leads to increased acetylation of NhoA and decreased OAT activity. These results suggest that reversible acetylation regulates the activity of NhoA both in vitro and in vivo, and that CobB could increase the NAT activity in vitro by deacetylating NhoA, it may affect the OAT activity of NhoA in vivo through the same way. Taken together, we identified new lysine acetylated proteins and substrates of deacetylase CobB, and explored a new function of lysine acetylation and bacterial cobB.更多还原