Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation.
Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation.
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工程半胱氨酸的翻译后修饰对取代半胱氨酸可及性方法的影响：谷胱甘肽化的证据。
DOI：
10.1152/ajpcell.00350.2016
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Goldman,IDavid
中科院分区：
文献类型：
--
作者：
Zhao,Rongbao;Najmi,Mitra;Aluri,Srinivas;Goldman,IDavid
取代半胱氨酸可及性方法（SCAM）被广泛用于研究通道、受体和转运蛋白的结构和功能。在其通常的应用中，将半胱氨酸残基引入缺乏天然半胱氨酸的蛋白质中，然后评估该残基对水性隔室的可及性。隐含的，并且通常假设的是，如果半胱氨酸取代的残基不能与巯基试剂反应，则其不暴露于细胞外区室或水转位途径内。我们在这里证明，在Hela细胞系中，质子偶联叶酸转运蛋白（PCFT，SLC 46 A1）的一些半胱氨酸取代的残基是不可接近的2-（（生物素）氨基）乙基甲硫基磺酸酯的谷胱甘肽生物素化谷胱甘肽乙酯在氧化剂的情况下。在某些情况下，还发现了涉及半胱氨酸取代残基的分子内二硫键形成。这些翻译后修饰限制了半胱氨酸残基对巯基反应试剂的可及性，并可能对SCAM的解释产生深远影响，但可能不会改变功能。当使用后修饰的残基作为参考细胞外对照时，在Western印迹上检测所需的高水平暴露导致错误检测否则不可接近的细胞内半胱氨酸取代的残基。这些数据表明，在应用SCAM，当半胱氨酸取代的残基似乎不容易巯基反应试剂，翻译后修饰的可能性应被排除。这些数据解释了评估中的差异，并证实了PCFT的第一个细胞内环的定位。
The substituted cysteine accessibility method (SCAM) is widely used to study the structure and function of channels, receptors and transporters. In its usual application, a cysteine residue is introduced into a protein which lacks native cysteines following which the accessibility of the residue to the aqueous compartment is assessed. Implicit, and generally assumed, is that if the cysteine-substituted residue is not available to react with sulfhydryl reagents it is not exposed to the extracellular compartment or within the aqueous translocation pathway. We demonstrate here, in a Hela-derived cell line, that some cysteine-substituted residues of the proton-coupled folate transporter (PCFT, SLC46A1) that are inaccessible to 2-((biotinoyl)amino)ethyl methanethiosulfonate are glutathionylated by biotinylated glutathione ethyl ester in the absence of an oxidizing agent. Intramolecular disulfide formation involving cysteine-substituted residues was also identified in some instances. These posttranslational modifications limit the accessibility of the cysteine residues to sulfhydryl-reactive reagents and can have a profound impact on the interpretation of SCAM but may not alter function. When a posttranslationally modified residue is used as a reference extracellular control, the high level of exposure required for detection on Western blot results in erroneous detection of otherwise inaccessible intracellular cysteine-substituted residues. The data indicate that in the application of SCAM, when a cysteine-substituted residue does not appear to be accessible to sulfhydryl-reactive reagents, the possibility of a posttranslational modification should be excluded. The data explain the discrepancies in the assessment, and confirm the localization, of the first intracellular loop of PCFT.