Protein modification by acrolein: formation and stability of cysteine adducts.
Protein modification by acrolein: formation and stability of cysteine adducts.
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DOI：
10.1021/tx800465m
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发表时间：
2009-04
影响因子：
4.1
通讯作者：
Pierce, William M., Jr.
中科院分区：
文献类型：
--
作者：
Cai, Jian;Bhatnagar, Aruni;Pierce, William M., Jr.
无处不在的污染物和内源性代谢物丙烯醛的毒性部分是由于共价蛋白质修饰。丙烯醛通过Michael加成和席夫碱的形成很容易与蛋白质的亲核试剂反应。蛋白质中潜在的丙烯醛靶标包括半胱氨酸、组氨酸和赖氨酸残基的亲核侧链以及蛋白质的游离氨基末端。尽管半胱氨酸是丙烯醛活性最强的残基，但半胱氨酸-丙烯醛加合物在体外和体内都很难识别。在这项研究中，使用含有半胱氨酸、赖氨酸和组氨酸残基的模型多肽来检测丙烯醛的反应性。实验结果表明，丙烯醛与半胱氨酸残基通过Michael加成反应生成M+56Da加合物。然而，这些M+56加合物是不稳定的，即使加合物的自发解离很慢。进一步的研究表明，当丙烯醛与模型肽在生理pH和温度下孵育时，分子内Schiff碱形成的M+56加合物逐渐减少，而M+38加合物逐渐增加。与其他氨基酸残基(赖氨酸和组氨酸)侧链形成加合物的速度比半胱氨酸慢得多，需要更高的丙烯醛浓度。当半胱氨酸残基被碘乙酰胺反应封闭，并使用较高浓度的丙烯醛时，形成N-末端氨基或组氨酸残基的加合物，但没有检测到赖氨酸加合物。总而言之，这些数据表明，丙烯醛与蛋白质半胱氨酸残基发生强烈的反应，随着时间的推移，蛋白-丙烯醛Michael加合物的明显损失可能与新的(M+38)加合物的出现有关。这些发现对于在体内鉴定丙烯醛与蛋白质半胱氨酸残基的加合物具有重要意义。
The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues.
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