Yersinia pestis acetyltransferase-mediated dual acetylation at the serine and lysine residues enhances the auto-ubiquitination of ubiquitin ligase MARCH8 in human cells

Lysine acetylation is known as a post translational modification (PTM) by histone acetyltransferases (HAT) that modifies histones and non-histone proteins to regulate gene expression. Serine acetylation, however, is reported in mammalian hosts by serine acetyltransferase of Yersinia pestis (YopJ) during infection. The protein target and cellular function of bacterial YopJ in mammalian systems are not fully addressed. Here we report dual acetylation at the serine and lysine residues by transiently expressed serine acetyltransferase YopJ mimicking Y. pestis infection in HeLa cells. Using shotgun proteomics followed by label-free quantification, we demonstrate an increase of dual acetylation in YopJ transfected human cells, including 10 Ser- (YopJ/non-YopJ 1.3-fold, p = 0.02) and 8 Lys- (YopJ/non-YopJ 3.5-fold, p = 0.00003) acetylation sites. Specifically, YopJ expression augments acetylation of membrane-associated E3 ubiquitin ligase MARCH8 at the serine residue Sac44, Sac71 and Sac253, and the lysine residue Kac247 and Kac252. YopJ-mediated Ser- and Lys-acetylation of MARCH8 is further confirmed by Western blotting using the specific antibodies against MARCH8 Sac71 and pan-acetyl lysine. Functional study demonstrates that YopJ-mediated Ser- and Lys-acetylation affects the auto-ubiquitination of MARCH8. The mutant C172A of YopJ previously shown to abolish the acetyltransferase activity also reduces Ser- and Lys-acetylation and diminishes the auto-ubiquitination of MARCH8. In support, MARCH8 is indeed acetylated at serine and lysine <i>in vitro</i> by purified YopJ but the activity is reduced by the C172A mutant in YopJ. Our study provides evidence that bacterial serine acetyltransferase YopJ mediates Ser- and Lys-acetylation and affects auto-ubiquitination of ubiquitin ligase MARCH8 in human cells.

赖氨酸乙酰化（Lysine acetylation）是一类由组蛋白乙酰转移酶（histone acetyltransferases, HAT）介导的翻译后修饰（post translational modification, PTM），可通过修饰组蛋白与非组蛋白来调控基因表达。然而，有研究报道，在感染过程中，鼠疫耶尔森菌（Yersinia pestis）的丝氨酸乙酰转移酶YopJ可在哺乳动物宿主中介导丝氨酸乙酰化。目前，细菌YopJ在哺乳动物系统中的蛋白靶点与细胞功能尚未完全阐明。本研究通过在HeLa细胞中瞬时表达丝氨酸乙酰转移酶YopJ以模拟鼠疫耶尔森菌感染过程，发现其可在丝氨酸与赖氨酸残基上介导双重乙酰化修饰。采用鸟枪法蛋白质组学（shotgun proteomics）结合无标记定量（label-free quantification）技术，我们证实转染YopJ的人源细胞中双重乙酰化水平显著升高，共鉴定到10个丝氨酸乙酰化位点（YopJ组/对照组比值为1.3倍，p=0.02）与8个赖氨酸乙酰化位点（YopJ组/对照组比值为3.5倍，p=0.00003）。具体而言，YopJ的表达可增强膜相关E3泛素连接酶MARCH8的丝氨酸残基Sac44、Sac71与Sac253，以及赖氨酸残基Kac247与Kac252的乙酰化修饰水平。我们进一步通过针对MARCH8 Sac71的特异性抗体与泛乙酰赖氨酸抗体进行蛋白质印迹（Western blotting）实验，证实了YopJ介导的MARCH8丝氨酸与赖氨酸乙酰化修饰。功能研究表明，YopJ介导的MARCH8丝氨酸与赖氨酸乙酰化修饰可影响其自身泛素化（auto-ubiquitination）过程。此前被证实可消除乙酰转移酶活性的YopJ突变体C172A，同样可降低MARCH8的丝氨酸与赖氨酸乙酰化水平，并削弱其自身泛素化过程。进一步的体外（in vitro）实验证实，纯化的YopJ确实可对MARCH8的丝氨酸与赖氨酸残基进行乙酰化修饰，而C172A突变体则会削弱该乙酰化活性。本研究证实，细菌丝氨酸乙酰转移酶YopJ可在人源细胞中介导MARCH8的丝氨酸与赖氨酸乙酰化修饰，并影响其自身泛素化过程。