CDC42乙酰化修饰在沙门菌感染宿主中的作用及机制研究

Acetylation of proteins plays an important role in regulating various aspects of cell life. Our previous work with SILAC quantitative mass spectrometry showed that Salmonella infection can cause significant changes in the acetylation levels of 90 proteins in monocyte cell line U937 cells. Among these host proteins, CDC42 is a small G protein belonging to the Rho family and involved in cell-to-cell adhesion, formation of cytoskeletal structures, and cell cycle regulation. It as a key molecule of the signal transduction pathways in host cells, can activate downstream proteins of CDC42 including PAKs, MLK3, N-WASP, and PI3Ks. The acetylation level of CDC42-K153 was reduced by 50% in the scenario of Salmonella infection. Crystal structure further shows K153 is involved in the interaction between CDC42 and its substrate p21-activated kinase 4 (PAK4). According to the results, we hypothesize that the Salmonella can mediate the binding of CDC42 to its substrates by regulating the acetylation of CDC42, thus affecting the activation of host signaling pathways and evading the host immune responses. We will use CRISPR technology combined with acetylation mimic mutations and other technologies to study the effect of acetylation of K153 on CDC42 activity, downstream signaling pathways, and bacterial infection outcome. The purpose of this application is to understand the role of CDC42 acetylation in anti-infection process, and the results may help understand the mechanism and significance of the interaction between pathogens and host cells at the post-translational modification level.

蛋白质乙酰化修饰对细胞生命活动具有重要调控作用。我们以人组织细胞淋巴瘤细胞系U937为模型，结合SILAC定量质谱分析技术，发现沙门菌感染导致U937细胞中包括与感染有关的CDC42等在内的90个蛋白质乙酰化水平发生显著改变。CDC42属于Rho家族小G蛋白，调控细胞骨架重排，与病原菌感染密切相关。沙门菌感染导致其K153乙酰化修饰水平降低了2倍，晶体结构显示K153参与CDC42与底物PAK4相互作用。因此我们推测沙门菌可通过调节乙酰化修饰介导CDC42与底物的结合，影响相关信号通路的活化，从而逃避宿主的免疫应答反应。因此本课题将利用CRISPR技术结合乙酰化模拟突变等技术研究K153乙酰化修饰对CDC42的活性、下游信号通路及沙门菌致病性的影响。目的是理解乙酰化修饰在CDC42抗感染过程中的作用，研究成果将有助于在蛋白质翻译后修饰水平理解病原菌与宿主细胞相互作用机制及意义。

细菌可以通过多种途径影响癌症的发生，但是细菌通过翻译后修饰对肿瘤的调控机制尚不清楚。CDC42属于RhoGTP家族成员，可以影响癌症中许多重要的信号通路。我们的研究证实了沙门菌感染可以通过去乙酰化酶SIRT2去乙酰化CDC42 K153，并且K153可被乙酰基转移酶p300/CBP乙酰化。CDC42 K153乙酰化降低可以显著抑制其与下游重要效应蛋白PAK4的结合，从而影响p38和JNK的磷酸化，这可能有助于肿瘤细胞抵抗凋亡。另外，K153乙酰化降低可能有助于增强结肠癌细胞的迁移、侵袭能力。免疫组化实验发现，在人结肠癌组织的CDC42 K153乙酰化水平降低普遍低于癌旁组织，且K153乙酰化水平可能提示预后不良。总之，我们的研究阐明了细菌通过调节CDC42的乙酰化来促进结直肠癌的发生的一种新机制。