Protein Lysine Crotonylation Analysis Reveals an Important Role in the Regulation of Aeromonas hydrophila Maltose Transport

Protein lysine crotonylation (Kcr) has been reported
to play a
role in the regulation of prokaryotic cell metabolism. However, its
distribution and functional significance remain largely unexplored.
In this study, a global proteomic landscape of Kcr in Aeromonas hydrophila was mapped, identifying 4424
Kcr sites on 1248 proteins using LC-MS/MS. Gene ontology analysis
revealed that Kcr-modified proteins are primarily enriched in cellular
and primary metabolic processes, stress responses, macromolecule biosynthesis,
and transmembrane transport. Moreover, Kcr proteins are involved in
diverse metabolic pathways, such as the pentose phosphate pathway,
TCA cycle, and methane metabolism. Functional validation assays revealed
that the maltodextrin-binding protein (MalE) at the K57 site, as well
as the maltose/maltodextrin import ATP-binding protein (MalK) at the
K11 and K181 sites, positively regulated maltose transport, whereas
the K223 site on MalE had a negative regulatory effect. Bioinformatics
analysis further demonstrated that the different Kcr status at the
K11 site of MalK may positively regulate the cavity volume within
the MalFGK2 complex, which may be involved in the regulation of bacterial
maltose transport. Overall, our findings indicate the important role
of Kcr modification in the nutrient transport system, providing insights
into the regulatory roles of bacterial protein post-translational
modifications.

蛋白质赖氨酸巴豆酰化（Protein lysine crotonylation, Kcr）已有研究表明其在原核细胞代谢调控中发挥作用。然而，其分布特征与功能意义在很大程度上仍有待深入探究。本研究针对嗜水气单胞菌（Aeromonas hydrophila）中的Kcr开展了全局蛋白质组学图谱绘制，借助液相色谱-串联质谱（Liquid Chromatography-Tandem Mass Spectrometry, LC-MS/MS）技术，在1248个蛋白质上共鉴定到4424个Kcr修饰位点。基因本体（Gene Ontology, GO）富集分析结果显示，Kcr修饰蛋白主要富集于细胞代谢与初级代谢过程、应激反应、大分子生物合成以及跨膜转运等生物学过程。此外，Kcr修饰蛋白参与了多种代谢通路，包括磷酸戊糖途径、三羧酸（Tricarboxylic Acid, TCA）循环以及甲烷代谢等。功能验证实验显示，位于K57位点的麦芽糖结合蛋白（maltodextrin-binding protein, MalE）以及位于K11和K181位点的麦芽糖/麦芽糖糊精转运ATP结合蛋白（maltose/maltodextrin import ATP-binding protein, MalK）可正向调控麦芽糖转运，而MalE上的K223位点则发挥负向调控作用。生物信息学分析进一步证实，MalK的K11位点的不同Kcr修饰状态可正向调控MalFGK2复合物的内腔体积，这可能参与细菌麦芽糖转运的调控过程。综上，本研究结果证实了Kcr修饰在营养转运系统中的重要作用，为探究细菌蛋白质翻译后修饰的调控功能提供了新的研究视角。