Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase
Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase
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DOI：
10.1006/jmbi.1998.1864
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发表时间：
1998-07-03
影响因子：
5.6
通讯作者：
Bernhagen, J
中科院分区：
文献类型：
--
作者：
Kleemann, R;Kapurniotu, A;Bernhagen, J
巨噬细胞移动抑制因子（MIF）是一种在免疫和炎症反应中起关键作用的细胞因子，其作用的分子机制尚未确定。在这里，我们报告说，MIF可以作为一种酶表现出巯基蛋白氧化还原酶活性。使用MIF的十肽片段（MF 1）跨越保守的半胱氨酸序列基序Cys 57-Ala-Leu-Cys 60（CALC），Cys -> Ser突变体（C57 S MIF，COS MIF和C57 S/C60 S MIF）的人MIF（wtMIF），和减轻wtMIF，我们表明，这种活性是由CALC区域介导的，是重要的巨噬细胞激活特性的MIF。wtMIF和MF 1均被证明形成分子内二硫键。使用两种常见的氧化还原酶测定，MIF显示酶促催化胰岛素和2-羟乙基二硫化物（HED）的还原。wtMIF和突变体的远紫外圆二色光谱（CD）与变性研究的检查表明，取代或减少半胱氨酸残基的CALC导致降低的MIF的构象稳定性，但没有显着改变其整体构象。CALC区域的功能作用通过使突变体和烷基化wtMIF经受酶促测定来揭示。突变体C60 S没有任何酶活性，而突变体C57 S具有降低的活性。巯基修饰的wtMIF，在氧化条件下烷基化，被发现有充分的酶活性，而烷基化的wtMIF在还原条件下完全消除MIF介导的氧化还原活性。重要的是，通过在涉及巨噬细胞激活特性的MIF的免疫测定中检查突变体和烷基化MIF，获得了二硫键基序的进一步生理相关性。在该试验中，突变体C60 S基本上是无活性的，而突变体C57 S是部分有活性的，这共同表明MIF的至少一些类精氨酸生物活性依赖于半胱氨酸57和60的存在。总之，我们的研究结果表明：（a）MIF具有酶促氧化还原酶活性，（B）这种活性依赖于半胱氨酸残基57和60形成的催化中心的存在，和（c）某些MIF介导的免疫过程是由于半胱氨酸介导的氧化还原机制。(C)北京：科学出版社.
The molecular mechanism of action of macrophage migration inhibitory factor (MIF), a cytokine with a critical role in the immune and inflammatory response, has not yet Seen identified. Here we report that MIF can function as an enzyme exhibiting thiol-protein oxidoreductase activity. Using a decapeptide fragment of MIF (MF1) spanning the conserved cysteine sequence motif Cys57-Ala-Leu-Cys60 (CALC), Cys --> Ser mutants (C57S MIF, COS MIF, and C57S/C60S MIF) of human MIF ( wtMIF), and alleviated wtMIF, we show that this activity is mediated by the CALC region and is important for the macrophage-activating properties of MIF. Both wtMIF and MF1 were demonstrated to form an intramolecular disulfide bridge. Using two common oxidoreductase assays, MIF was shown to enzymatically catalyze the reduction of insulin and 2-hydroxyethyldisulfide (HED). Examination of wtMIF and the mutants by far-UV circular dichroism spectroscopy (CD) together with denaturation studies showed that substituting or reducing the cysteine residues of CALC led to a reduced conformational stability of MIF but did not significantly change its overall conformation. A functional role for the CALC region was revealed by subjecting the mutants and alkylated wtMIF to the enzymatic assays. Mutant C60S did not have any enzymatic activity while mutant C57S had a reduced activity. Thiol-modified wtMIF that was alkylated under oxidizing conditions was found to have full enzymatic activity, whereas alkylation of wtMIF under reducing conditions completely eliminated MIF-mediated redox activity. Importantly, further physiological relevance of the disulfide motif was obtained by examining the mutants and alkylated MIF in an immunological assay that involved the macrophage-activating properties of MIF. Ln this test, mutant C60S was essentially inactive and mutant C57S was partly active, indicating together that at least some of the cytokine-like biological activities of MIF are dependent on the presence of cysteine 57 and 60.Again, use of the alkylated MIF species confirmed the role of the cysteine motif for this MIF activity. Ln conclusion, our results argue (a) that MIF exhibits enzymatic oxidoreductase activity, (b) that this activity is dependent on the presence of the catalytic center that is formed by cysteine residues 57 and 60, and (c) that certain MIF-mediated immune processes are due to the cysteine-mediated redox mechanism. (C) 1998 Academic Press.