Site-Specific Acetyl Lysine Antibodies Reveal Differential Regulation of Histone Acetylation upon Kinase Inhibition
Site-Specific Acetyl Lysine Antibodies Reveal Differential Regulation of Histone Acetylation upon Kinase Inhibition
复制标题
位点特异性乙酰赖氨酸抗体揭示了激酶抑制后组蛋白乙酰化的差异调节
DOI：
10.1007/s12013-016-0777-y
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发表时间：
2017-03-01
影响因子：
2.6
通讯作者：
Xu, Guoqiang
中科院分区：
文献类型：
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作者：
Chen, Shi;Chen, Suping;Xu, Guoqiang
赖氨酸乙酰化修饰的蛋白质具有多种生物学功能。基于质谱的蛋白质组学方法已经在细胞和组织中鉴定出数千个赖氨酸乙酰化位点。然而，这些乙酰化位点的功能研究受到缺乏识别特异性修饰位点的抗体的限制。在这里，我们产生了55个位点特异性乙酰赖氨酸抗体，用于检测细胞裂解物中的这种修饰，并评估了这些抗体的质量。基于免疫印迹分析，我们发现邻近修饰位点的氨基酸序列的性质影响位点特异性乙酰赖氨酸抗体的特异性。在修饰位点附近具有带电荷的、亲水的、小的或柔性侧链的氨基酸增加获得高质量位点特异性乙酰赖氨酸抗体的可能性。这一结果可以提供有价值的见解，微调的氨基酸序列的表位的产生位点特异性乙酰赖氨酸抗体。使用位点特异性乙酰赖氨酸抗体，我们进一步发现组蛋白3在四个赖氨酸残基的乙酰化受到激酶抑制剂的差异调节。这一结果证明了这些抗体在研究赖氨酸乙酰化可能参与的新信号通路中的潜在应用。
Lysine acetylation regulates diverse biological functions for the modified proteins. Mass spectrometry-based proteomic approaches have identified thousands of lysine acetylation sites in cells and tissues. However, functional studies of these acetylation sites were limited by the lack of antibodies recognizing the specific modification sites. Here, we generated 55 site-specific acetyl lysine antibodies for the detection of this modification in cell lysates and evaluated the quality of these antibodies. Based on the immunoblotting analyses, we found that the nature of amino acid sequences adjacent to the modification sites affected the specificity of the site-specific acetyl lysine antibodies. Amino acids with charged, hydrophilic, small, or flexible side chains adjacent to the modification sites increase the likelihood of obtaining high quality sitespecific acetyl lysine antibodies. This result may provide valuable insights in fine-tuning the amino acid sequences of the epitopes for the generation of site-specific acetyl lysine antibodies. Using the site-specific acetyl lysine antibodies, we further discovered that acetylation of histone 3 at four lysine residues was differentially regulated by kinase inhibitors. This result demonstrates the potential application of these antibodies in the study of new signaling pathways that lysine acetylation may participate in.