Acetylation of the HIV Tat protein:: an in vitro study
Acetylation of the HIV Tat protein:: an in vitro study
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DOI：
10.1007/s00216-003-2058-z
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发表时间：
2003-08-01
影响因子：
4.3
通讯作者：
Schnölzer, M
中科院分区：
文献类型：
--
作者：
Dormeyer, W;Dorr, A;Schnölzer, M
在过去的几年中，赖氨酸乙酰化作为细胞信号级联中蛋白质的调节性翻译后修饰的理解有所增加。现在已知不仅组蛋白而且非组蛋白因子可以作为不同乙酰转移酶的底物。非组蛋白因子中的乙酰化赖氨酸残基通常使用放射性标记实验和合成肽的免疫化学分析来鉴定。在这项对人类免疫缺陷病毒1（HIV-1）达特蛋白的研究中，我们证明了基质辅助激光解吸/电离质谱、蛋白水解消化和Edman测序对于乙酰化位点定位的好处。我们证实，HIV-1达特蛋白在体外被乙酰转移酶p300在其RNA结合区50位的特异性赖氨酸残基乙酰化。此外，我们发现，达特半胱氨酸丰富的区域是乙酰化的多个半胱氨酸残基在酶的情况下。由于这种非酶促半胱氨酸乙酰化独立于周围的肽序列发生，我们认为乙酰化肽中半胱氨酸残基的存在是解释体外乙酰化测定的一个重要因素。
In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.