New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.
New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.
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DOI：
10.1042/bcj20180266
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发表时间：
2018-08-14
期刊：
影响因子：
--
通讯作者：
Eyers PA
中科院分区：
文献类型：
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作者：
Byrne DP;Li Y;Ngamlert P;Ramakrishnan K;Eyers CE;Wells C;Drewry DH;Zuercher WJ;Berry NG;Fernig DG;Eyers PA
蛋白质酪氨酸硫酸化是一种翻译后修饰，以调节细胞外蛋白质-蛋白质相互作用而闻名。酪氨酸硫酸化由两种高尔基体驻留酶催化，称为酪氨酸蛋白磺基转移酶（TPSTs）1和2，其将硫酸盐从辅因子PAPS（3′-磷酸腺苷5′-磷酸硫酸盐）转移到蛋白质底物中的上下文依赖性酪氨酸。缺乏定量酪氨酸硫酸化测定阻碍了用于鉴定酪氨酸硫酸化的小分子抑制剂的化学生物学方法的发展。在本文中，我们描述了一种非放射性的流动性为基础的酶法测定TPST 1和TPST 2，通过它的酪氨酸硫酸化的合成荧光肽可以快速定量的发展。我们利用配体结合和抑制剂筛选来揭示TPST 1和TPST 2对不同类型的小分子的敏感性，包括抗血管生成化合物苏拉明和激酶抑制剂rottlerin。通过筛选已发表的激酶抑制剂集，我们确定了基于羟吲哚的丝氨酸/苏氨酸激酶RAF（快速加速纤维肉瘤）抑制剂作为TPST 1和TPST 2的低微摩尔抑制剂。有趣的是，不相关的RAF抑制剂，例如双重BRAF/VEGFR 2抑制剂RAF 265，在体外也是TPST抑制剂。我们建议，目标验证的蛋白激酶抑制剂可以重新利用，或重新设计，作为更具体的TPST抑制剂，以帮助评估磺基酪氨酰蛋白质组。最后，我们推测细胞酪氨酸硫酸化的机械抑制可能与暴露于阴离子TPST配体和RAF蛋白激酶抑制剂的细胞中观察到的一些表型有关。
Protein tyrosine sulfation is a post-translational modification best known for regulating extracellular protein–protein interactions. Tyrosine sulfation is catalysed by two Golgi-resident enzymes termed tyrosylprotein sulfotransferases (TPSTs) 1 and 2, which transfer sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulfation assays has hampered the development of chemical biology approaches for the identification of small-molecule inhibitors of tyrosine sulfation. In the present paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulfation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and TPST2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set, we identified oxindole-based inhibitors of the Ser/Thr kinase RAF (rapidly accelerated fibrosarcoma) as low-micromolar inhibitors of TPST1 and TPST2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulfotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulfation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.