SIRT2调控MEK乙酰化修饰及其在创伤性脑损伤后神经损伤修复作用和机制的研究

Lysine acetylation，an important post-translational modification (PTM) to modulate protein structure and function, plays a crucial role in the neural repair after traumatic brain injury (TBI). We previously found that SIRT2 knock out (KO) increased TBI-induced neuron apoptosis and decreased phosphorylation of ERK1/2, resulting in a significant deterioration in neurologic deficits, compared with the wild type (WT) group. The mechanism of SIRT2 mediated neuroprotection, however, remains elusive. We postulated that SIRT2 activates mitogen-activated protein kinase kinase (MEK) by deacetylation prevents TBI induced neural injury. To verify this hypothesis, we test the level of MEK acetylation and assay its activity after treatment with deacetylase inhibitor. To further determine which lysine residuse plays a major role in the regulation of MEK, we mutate each of putative acetylation sites, acquired from sequence alignments from diverse species, to arginine (R) or glutamine (Q) and assay their activity individually. A siRNA library is also generated to search for potential lysine acetyltransgerase (KATs) for MEK. Co-immunoprecipitations is undertaken to elucidate whether endogenous MEK interacted with SIRT2. The project will not only observe TBI induced acetylation and phosphorylation of MEK/ERK in SIRT2 KO mouse and primary neuron, using stretch induced cell injury or controlled cortical injury model, but also evaluate the effects of SIRT2 KO on neuronal cell injury and behavioral changes following TBI. Our study aim to uncover a previously unknown mechanism by which SIRT2 mediated deacetylation and activation of MEK stimulate ERK signal pathway to protect neuronal cells, and provide new treatment strategies for neurological function restoration after TBI.

蛋白质乙酰化修饰在创伤性脑损伤（TBI）神经修复中可能具有重要作用。课题组前期发现，SIRT2基因敲除明显增加TBI后损伤半暗区神经元凋亡，降低与神经元存活相关的磷酸化ERK1/2水平，加重神经功能障碍。但是SIRT2介导的神经保护作用机制尚未阐明。我们推测，SIRT2通过去乙酰化修饰MEK激活ERK信号通路从而保护TBI后神经损伤。为验证此假说，本项目首先过表达野生型或模拟乙酰化突变MEK蛋白后检测其乙酰化水平和催化活性；然后构建siRNA文库筛选MEK的乙酰化酶，免疫共沉淀检测SIRT2与MEK的相互关系。进一步通过神经元牵张损伤模型和小鼠控制性皮层损伤模型，分别从细胞和器官水平观察SIRT2基因敲除后MEK/ERK信号通路的乙酰化和磷酸化及下游基因表达变化，并评估TBI后神经损伤和神经功能改变。期望揭示SIRT2介导的MEK乙酰化修饰调控机制，为TBI神经保护提供新的治疗靶点和策略。

创伤性颅脑损伤是青年人主要致死病因，具有高发病率和高致死致残率的特点，至今的临床救治仍是巨大的医学挑战。本课题中，我们成功建立、繁殖、鉴定、筛选出野生型和SIRT2基因敲除纯合子C57BL/6小鼠，构建了MEK、SIRT2、ERK1/2质粒和模拟MEK不同位点乙酰化突变（Q突变）和非乙酰化突变（R突变）的质粒K175R K362R K175Q K362Q，同时在活体水平建立了控制性皮层损伤模型和离体条件下建立机械性牵张损伤模型，对SIRT2基因对创伤性颅脑损伤的作用进行了一系列研究。我们发现，SIRT2基因可以去乙酰化MEK，MEK乙酰化水平下降，MEK活性增强，p-ERK水平增高。在活体水平，SIRT2基因敲除小鼠在脑建立皮层损伤模型后，减轻脑外伤发生后神经元的凋亡、血脑屏障的破坏、脑水肿以及神经功能障；与此同时，在离体水平，原代神经元细胞建立机械性牵张损伤模型后，SIRT2减少了凋亡，并可能是通过影响MKP3通路实现。本课题为SIRT2基因作为临床治疗创伤性颅脑损伤的靶点提供了理论依据和前期基础。