Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones
Selective inactivation of glutaredoxin by sporidesmin and other epidithiopiperazinediones
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DOI：
10.1021/bi060440o
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发表时间：
2006-07-25
期刊：
影响因子：
2.9
通讯作者：
Mieyal, John J.
中科院分区：
文献类型：
--
作者：
Srinivasan, Usha;Bala, Aveenash;Mieyal, John J.
Glutaredoxin（巯基转移酶）是一种巯基二硫氧化还原酶，具有高效和特异性的蛋白质- ssg去谷胱甘肽化催化作用，因此与细胞蛋白质的巯基二硫状态的稳态调节有关。Sporidesmin是一种epidithiopiopperazine -2,5-dione （ETP）真菌毒素，可能通过氧化改变关键蛋白上的半胱氨酸残基来破坏细胞功能。在目前的研究中，孢子素以时间和浓度依赖的方式灭活人戊二醛。在可比条件下，其他硫-二硫氧化还原酶，谷胱甘肽还原酶，硫氧还蛋白和硫氧还蛋白还原酶不受孢菌素的影响。戊二氧还蛋白的失活需要酶的还原（二硫醇）形式、孢子素的氧化（分子内二硫化物）形式和分子氧。只有在尿素存在的情况下，二硫苏糖醇才能使失活的戊二氧还蛋白重新活化，然后去除变性剂，这表明酶的失活涉及一个构象上不可接近的二硫键。谷氨酰胺还蛋白的各种半胱氨酸-丝氨酸突变体对孢子素的失活具有抗性，这表明这种失活反应特异性地涉及人谷氨酰胺还蛋白中5种半胱氨酸残基中的至少两种。各种附硫代哌嗪-2,5-二酮对戊二氧嘧啶失活的相对能力表明，除附硫代二氧哌嗪部分外，至少需要一个苯基取代基才能具有抑制活性。修饰蛋白的质谱分析与分子间二硫化物的形成一致，每个glutaredoxin含有一个内合毒素，但从检测产物中消除了两个硫原子。我们认为，最初的反应是在毒素硫和半胱氨酸22之间的戊二醇还毒素活性位点。这项研究暗示了选择性修饰靶蛋白的巯基在一些细胞毒性作用的ETP真菌毒素及其合成类似物。
Glutaredoxin (thioltransferase) is a thiol-disulfide oxidoreductase that displays efficient and specific catalysis of protein-SSG deglutathionylation and is thereby implicated in homeostatic regulation of the thiol-disulfide status of cellular proteins. Sporidesmin is an epidithiopiperazine-2,5-dione (ETP) fungal toxin that disrupts cellular functions likely via oxidative alteration of cysteine residues on key proteins. In the current study sporidesmin inactivated human glutaredoxin in a time- and concentration-dependent manner. Under comparable conditions other thiol-disulfide oxidoreductase enzymes, glutathione reductase, thioredoxin, and thioredoxin reductase, were unaffected by sporidesmin. Inactivation of glutaredoxin required the reduced (dithiol) form of the enzyme, the oxidized (intramolecular disulfide) form of sporidesmin, and molecular oxygen. The inactivated glutaredoxin could be reactivated by dithiothreitol only in the presence of urea, followed by removal of the denaturant, indicating that inactivation of the enzyme involves a conformationally inaccessible disulfide bond(s). Various cysteine-to-serine mutants of glutaredoxin were resistant to inactivation by sporidesmin, suggesting that the inactivation reaction specifically involves at least two of the five cysteine residues in human glutaredoxin. The relative ability of various epidithiopiperazine-2,5-diones to inactivate glutaredoxin indicated that at least one phenyl substituent was required in addition to the epidithiodioxopiperazine moiety for inhibitory activity. Mass spectrometry of the modified protein is consistent with formation of intermolecular disulfides, containing one adducted toxin per glutaredoxin but with elimination of two sulfur atoms from the detected product. We suggest that the initial reaction is between the toxin sulfurs and cysteine 22 in the glutaredoxin active site. This study implicates selective modification of sulfhydryls of target proteins in some of the cytotoxic effects of the ETP fungal toxins and their synthetic analogues.