评估蛋白质酪氨酸硫酸化的新工具：酪氨酰蛋白质磺基转移酶（TPST）是RAF蛋白激酶抑制剂的新靶标。,Biochemical Journal

当前位置： X-MOL 学术 › Biochem. J. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

评估蛋白质酪氨酸硫酸化的新工具：酪氨酰蛋白质磺基转移酶（TPST）是RAF蛋白激酶抑制剂的新靶标。
Biochemical Journal ( IF 4.4 ) Pub Date : 2018-06-24 , DOI: 10.1042/bcj20180266
Dominic P Byrne ₁ , Yong Li ₁ , Pawin Ngamlert ₁ , Krithika Ramakrishnan ₁ , Claire E Eyers _{1,

2} , Carrow Wells ₃ , David H Drewry ₃ , William J Zuercher _{3,

4} , Neil G Berry ₅ , David G Fernig ₁ , Patrick A Eyers ₁

Affiliation

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与蛋白质的相互作用而闻名。酪氨酸硫酸盐化是由两种称为酪氨酰蛋白质磺基转移酶（TPST）1和2的高尔基驻留酶催化的，它们将辅酶PAPS（3'-磷酸腺苷5'-磷酸硫酸盐）中的硫酸盐转移至蛋白质底物中的上下文相关酪氨酸。缺乏定量的酪氨酸硫酸化测定法已经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，我们描述了针对TPST1和TPST2的基于非放射性迁移率的酶促测定方法的发展，通过该方法可以快速定量合成荧光肽的酪氨酸硫酸化。我们利用配体结合和抑制剂筛选来揭示TPST1和TPST2对不同类别的小分子（包括抗血管生成化合物苏拉明和激酶抑制剂rottlerin）的敏感性。通过筛选已发布的激酶抑制剂组，我们确定了基于羟吲哚的Ser / Thr激酶RAF（快速加速纤维肉瘤）抑制剂是TPST1和TPST2的低微摩尔抑制剂。有趣的是，以双BRAF / VEGFR2抑制剂RAF265为例的不相关的RAF抑制剂在体外也是TPST抑制剂。我们建议可以将靶标验证的蛋白激酶抑制剂作为一种更特异性的TPST抑制剂，以进行重新设计或重新设计，以帮助评估磺酰酪氨酰蛋白质组学。。最后，

"点击查看英文标题和摘要"

New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors.

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.

更新日期：2019-11-01

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南