Redox regulation of surface protein thiols:: Identification of integrin α-4 as a molecular target by using redox proteomics
Redox regulation of surface protein thiols:: Identification of integrin α-4 as a molecular target by using redox proteomics
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DOI：
10.1073/pnas.2434516100
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发表时间：
2003-12-09
影响因子：
11.1
通讯作者：
Ghezzi, P
中科院分区：
文献类型：
--
作者：
Laragione, T;Bonetto, V;Ghezzi, P
硫醇影响多种细胞功能，这种作用被称为氧化还原调节。我们在这里显示，用0.1-5 mM N-乙酰基-L-半胱氨酸（NAC）处理细胞（1-2 h）可增加人外周血单核细胞中表面蛋白巯基的表达。这种效应与细胞谷胱甘肽（GSH）的变化无关，并且还观察到非GSH前体硫醇N-乙酰基-D-半胱氨酸或GSH本身，其不是细胞渗透性的，表明直接还原作用。NAC没有增加蛋白质SH在胞质溶胶中，表明他们已经最大限度地减少在正常，非应激，条件下。通过使用非渗透性的，生物素化的SH试剂，然后通过二维凝胶电泳和MS分析标记，我们确定了一些与MAC减少的膜相关的蛋白质。这些蛋白质包括：整合素α-4、肌球蛋白重链（非肌肉A型）、肌球蛋白轻链碱（非肌肉亚型）和β-肌动蛋白。MAC预处理增强了Jurkat细胞的整合素α-4依赖性纤连蛋白粘附和聚集，而不改变其荧光激活细胞分选仪的表达，表明表面二硫化物的减少可以影响蛋白质的功能。我们推测，NAC或其他巯基抗氧化剂的一些活动可能不仅是由于自由基清除或增加细胞内GSH和随后对转录因子的影响，但可以修改氧化还原状态的功能膜蛋白与exofacial SH关键的活动。
Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by MAC. These proteins include the following: integrin alpha-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and beta-actin. MAC pretreatment augmented integrin alpha-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.