Proteomic Analysis Reveals Differentially Regulated Protein Acetylation in Human Amyotrophic Lateral Sclerosis Spinal Cord

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease that primarily affects motor neurons in the brain and spinal cord. Histone deacetylase (HDAC) inhibitors have neuroprotective effects potentially useful for the treatment of neurodegenerative diseases including ALS; however, the molecular mechanisms underlying their potential efficacy is not well understood. Here we report that protein acetylation in urea-soluble proteins is differently regulated in post-mortem ALS spinal cord. Two-dimensional electrophoresis (2-DE) analysis reveals several protein clusters with similar molecular weight but different charge status. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identifies glial fibrillary acidic protein (GFAP) as the dominant component in the protein clusters. Further analysis indicates six heavily acetylated lysine residues at positions 89, 153, 189, 218, 259 and 331 of GFAP. Immunoprecipitation followed by Western blotting confirms that the larger form of GFAP fragments are acetylated and upregulated in ALS spinal cord. Further studies demonstrate that acetylation of the proteins additional to GFAP is differently regulated, suggesting that acetylation and/or deacetylation play an important role in pathogenesis of ALS.肌萎缩侧索硬化症（Amyotrophic lateral sclerosis, ALS）是一种进行性致死性神经退行性疾病，主要累及大脑与脊髓中的运动神经元。组蛋白去乙酰化酶（Histone deacetylase, HDAC）抑制剂具备神经保护作用，有望用于包括ALS在内的多种神经退行性疾病的治疗，但其潜在疗效的分子机制尚未得到充分阐明。本研究报道，尿素可溶性蛋白的乙酰化修饰在ALS患者死后脊髓组织中存在差异调控模式。二维凝胶电泳（Two-dimensional electrophoresis, 2-DE）分析结果显示，存在若干分子量相近但电荷状态存在差异的蛋白簇。液相色谱-串联质谱（Liquid chromatography and tandem mass spectrometry, LC-MS/MS）鉴定确认，胶质纤维酸性蛋白（Glial fibrillary acidic protein, GFAP）是该蛋白簇的核心组成成分。进一步分析表明，GFAP的89、153、189、218、259及331位赖氨酸残基存在高度乙酰化修饰。免疫沉淀（Immunoprecipitation）联合蛋白质免疫印迹（Western blotting）实验证实，ALS患者脊髓组织中较大分子量的GFAP片段发生了乙酰化修饰且表达水平显著上调。后续研究还发现，除GFAP外的其他蛋白质的乙酰化修饰同样存在差异调控，提示蛋白质乙酰化和/或去乙酰化过程在ALS的发病机制中发挥关键作用。