Department of Genomics and Molecular Medicine, CSIR-Institute of Genomics and Integrative Biology, New Delhi, Delhi, India.
Academy of Scientific & Innovative Research (AcSIR), New Delhi, Delhi, India.
Proteins. 2019 Aug;87(8):625-634. doi: 10.1002/prot.25681. Epub 2019 Apr 15.
An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys of albumin and homocysteine attached to lysine residues.
同型半胱氨酸是一种具有反应性巯基的氨基酸，其水平升高与多种复杂疾病有关，是心血管疾病的独立危险因素。循环同型半胱氨酸中，超过 80%与蛋白质结合。同型半胱氨酸通过巯基-二硫键交换反应与蛋白质中的半胱氨酸残基特异性结合，其机制已被报道。相比之下，同型半胱氨酸硫内酯是同型半胱氨酸的环状硫酯，被认为仅与赖氨酸残基的伯氨基结合，导致蛋白质的 N-同型半胱氨酸化，因此迄今为止，关于同型半胱氨酸硫内酯与蛋白质结合的研究仅集中在 N-同型半胱氨酸化上。尽管已知同型半胱氨酸硫内酯在生理 pH 下可以水解为同型半胱氨酸，但同型半胱氨酸硫内酯暴露于蛋白质时 S-同型半胱氨酸化的程度从未被研究过。在这项研究中，我们清楚地表明，同型半胱氨酸硫内酯的水解是 pH 依赖性的，在生理 pH 下，1 mM 同型半胱氨酸硫内酯在 24 小时内水解为约 0.71 mM 同型半胱氨酸。我们还使用白蛋白表明，HTL 与白蛋白孵育会导致更多的 S-同型半胱氨酸化（0.41 mol/mol 白蛋白），而不是 N-同型半胱氨酸化（0.14 mol/mol 白蛋白）。用 LC-MS 证实了同型半胱氨酸硫内酯处理后 HSA 上 Cys 的 S-同型半胱氨酸化。与早期的报告相反，我们的结果表明，与赖氨酸残基结合的同型半胱氨酸与附着在白蛋白上的半胱氨酸之间没有交叉对话。
