糖酵解酶赖氨酸ε-氨基乙酰化对宰后猪肌肉糖酵解的调控作用及机理

The high incidence of PSE in pork, turkey and chicken causes a huge economic loss to the animal slaughter and meat processing industry, which has been recognized for decades, but remains unsolved. This is mainly due to the lack of our understanding in the biochemical mechanisms of postmortem glycolysis. Recently, it was reported that protein acetylation regulates glycolysis from bacteria to human, but the mechanism is unclear. In the proposal, we aim to define the mechanism by which protein acetylation regulates postmortem glycolysis and the signaling pathway that antemortem stress plays on postmortem glycolysis through protein acetylation. We will use pigs as model in combination with mice and in vitro cell culture. With the employment of high through-put proteomics, protein acetylation in postmortem muscle will be profiled first. Glycolytic enzymes that play rate-limiting role in postmortem muscle glycolysis by acetylation and deacetylation will be determined. The changes in structure and function of glycolytic enzymes after acetylation will be studied. The upstream HAT and HDAC that catalyze the acetylation and deacetylation of glycolytic enzyme will be defined. Finally, the signaling pathway by which antemortem stress regulates the acetylation/deacetylation of glycolytic enzymes and postmortem gycolysis will be revealed. Our study will provide new insight into the biochemistry of postmortem glycolysis, which will be helpful for the effective control of PSE meat.

PSE肉带来的巨大经济损失早已引起人们的高度重视，但一直未得到彻底解决，究其根本原因是对宰后肌肉糖酵解的生化机制缺乏全面、深入的了解。最近研究发现蛋白质的乙酰化对糖酵解具有重要的调控作用，但其机理尚不清楚。本项目以猪为主要研究对象，同时结合小鼠模型和细胞培养，利用高通量乙酰蛋白质组学、免疫共沉淀、蛋白质印迹、基因超表达和干扰、蛋白质芯片、酶促动力学等技术，分析宰后肌肉蛋白质乙酰化谱的变化，确证蛋白质乙酰化对糖酵解的调控作用；通过分离、鉴定乙酰化差异糖酵解酶，确证其乙酰化位点，筛选通过乙酰化对糖酵解起关键控制作用的酶；研究乙酰化对糖酵解酶结构和功能的影响，揭示乙酰化调控糖酵解的分子机制；筛选并确证催化糖酵解酶乙酰化和去乙酰化的上游HAT和HDAC；揭示应激通过PKA- HDAC / HAT信号通路调控宰后肌肉糖酵解的分子机理，创新肌肉代谢理论，为有效预防PSE肉的发生提供新的思路。

白肌肉（PSE肉）是猪和禽类中常发生的一种劣质肉，每年给畜禽屠宰工业带来巨大损失，对其分子机制的揭示，可为预防和控制PSE肉的发生提供理论基础。本项目以猪为主要研究对象，同时结合小鼠模型和细胞培养，利用高通量乙酰蛋白质组学、免疫共沉淀、蛋白质印迹、基因超表达和干扰等细胞和分子生物学技术对糖酵解酶赖氨酸ε-氨基乙酰化在宰后肌肉糖酵解和肉品质形成过程中的作用进行了研究，研究结果证实了蛋白质赖氨酸ε-氨基乙酰修饰对宰后肌肉糖酵解和肉品质的形成具有重要的调节作用，并参与了宰前应激对宰后肌肉糖酵解和肉品质的影响过程。此外，乙酰蛋白质组学分析揭示蛋白质ε-氨基乙酰化还可能通过细胞凋亡、尸僵、钙信号通路、嘌呤代谢等多条途径/机制调节宰后骨骼肌向肉的转变过程和最终肉品质的形成，包括肉的保水性、嫩度、风味、色泽等。通过细胞代谢和酶活性分析筛选出，并确证了丙酮酸激酶是宰后肌肉糖酵解的一个关键调控酶，乙酰化修饰对其活性具有重要的调控作用，宰前应激可通过调控丙酮酸激酶的乙酰化修饰，从而调控宰后肌肉糖酵解和肉品质，乙酰化修饰调控丙酮酸激酶活性的具体机制还在研究中，并取得了一些创新性的研究结果。

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