Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.
PLoS One. 2012;7(6):e39408. doi: 10.1371/journal.pone.0039408. Epub 2012 Jun 25.
Thimet oligopeptidase (EP24.15) is a cysteine-rich metallopeptidase containing fifteen Cys residues and no intra-protein disulfide bonds. Previous work on this enzyme revealed that the oxidative oligomerization of EP24.15 is triggered by S-glutathiolation at physiological GSSG levels (10-50 µM) via a mechanism based on thiol-disulfide exchange. In the present work, our aim was to identify EP24.15 Cys residues that are prone to S-glutathiolation and to determine which structural features in the cysteinyl bulk are responsible for the formation of mixed disulfides through the reaction with GSSG and, in this particular case, the Cys residues within EP24.15 that favor either S-glutathiolation or inter-protein thiol-disulfide exchange. These studies were conducted by in silico structural analyses and simulations as well as site-specific mutation. S-glutathiolation was determined by mass spectrometric analyses and western blotting with anti-glutathione antibody. The results indicated that the stabilization of a thiolate sulfhydryl and the solvent accessibility of the cysteines are necessary for S-thiolation. The Solvent Access Surface analysis of the Cys residues prone to glutathione modification showed that the S-glutathiolated Cys residues are located inside pockets where the sulfur atom comes into contact with the solvent and that the positively charged amino acids are directed toward these Cys residues. The simulation of a covalent glutathione docking onto the same Cys residues allowed for perfect glutathione posing. A mutation of the Arg residue 263 that forms a saline bridge to the Cys residue 175 significantly decreased the overall S-glutathiolation and oligomerization of EP24.15. The present results show for the first time the structural requirements for protein S-glutathiolation by GSSG and are consistent with our previous hypothesis that EP24.15 oligomerization is dependent on the electron transfer from specific protonated Cys residues of one molecule to previously S-glutathionylated Cys residues of another one.
组织蛋白酶抑制剂 24.15（Thimet oligopeptidase，EP24.15）是一种富含半胱氨酸的金属肽酶，包含 15 个半胱氨酸残基，没有蛋白内二硫键。之前对该酶的研究表明，在生理 GSSG 水平（10-50 μM）下，通过基于巯基-二硫键交换的机制，EP24.15 的氧化寡聚化是由 S-谷胱甘肽化触发的。在本工作中，我们的目的是鉴定易发生 S-谷胱甘肽化的 EP24.15 半胱氨酸残基，并确定胱氨酸残基中的哪些结构特征负责通过与 GSSG 反应形成混合二硫键，在这种特殊情况下，EP24.15 中的半胱氨酸残基有利于 S-谷胱甘肽化或蛋白间巯基-二硫键交换。这些研究通过计算机结构分析和模拟以及定点突变进行。通过质谱分析和用抗谷胱甘肽抗体进行的 Western blot 确定 S-谷胱甘肽化。结果表明，硫醇巯基的稳定和半胱氨酸的溶剂可及性是 S-硫代化所必需的。对半胱氨酸易于谷胱甘肽修饰的溶剂可及表面分析表明，S-谷胱甘肽化的半胱氨酸残基位于硫原子与溶剂接触的口袋内，带正电荷的氨基酸指向这些半胱氨酸残基。将共价谷胱甘肽对接模拟到相同的半胱氨酸残基上，可以完美地模拟谷胱甘肽的构象。突变与半胱氨酸残基 175 形成盐桥的精氨酸残基 263 显著降低了 EP24.15 的总体 S-谷胱甘肽化和寡聚化。本研究首次显示了 GSSG 对蛋白质 S-谷胱甘肽化的结构要求，并与我们之前的假设一致，即 EP24.15 寡聚化依赖于特定质子化的 Cys 残基从一个分子向另一个分子中先前 S-谷胱甘肽化的 Cys 残基的电子转移。
