Shetty Vivekananda, Spellman Daniel S, Neubert Thomas A
Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, New York 10016, USA.
J Am Soc Mass Spectrom. 2007 Aug;18(8):1544-51. doi: 10.1016/j.jasms.2007.05.013. Epub 2007 May 24.
Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation.
半胱氨酸亚磺酸（Cys-SOH）是活性氧诱导的许多蛋白质（如过氧化物酶和酪氨酸磷酸酶）氧化反应中难以捉摸的中间体。有人提出Cys-SOH在催化和信号传导功能中起着至关重要的作用。半胱氨酸亚磺酸盐（Cys-SO(2)H）和半胱氨酸磺酸盐（Cys-SO(3)H）的形成与基质金属蛋白酶-7（MMP-7）的激活有关，而硫醇氧化为半胱氨酸亚磺酸盐与MMP-7的自溶裂解有关。我们研究了合成肽PRCGVPDVA中半胱氨酸亚磺酸的形成，该肽是MMP-7和其他基质金属蛋白酶的半胱氨酸开关结构域。我们通过与芬顿反应（Fe(+2)/H(2)O(2)）产生的羟基自由基反应，制备了含有半胱氨酸亚磺酸的肽PRC(SOH)GVPDVA。我们通过串联质谱和精确质量测量实验对这种修饰肽进行了表征。此外，我们使用7-氯-4-硝基苯并-2-恶唑-1,3-二唑（NBD-Cl）试剂与PRC(SOH)GVPDVA形成加合物，为PRC(SOH)GVPDVA在溶液中的活性提供额外证据。我们还基于串联质谱和精确质量测量实验，对分子内半胱氨酸亚磺酰胺交联产物PRC[S(O)N]GVPDVA进行了表征。这些结果有助于理解传统上与MMP激活相关的蛋白水解裂解机制。
