Xiao Yongsheng, Ji Debin, Guo Lei, Wang Yinsheng
Department of Chemistry and ‡Environmental Toxicology Graduate Program, University of California , Riverside, California 92521-0403, United States.
Anal Chem. 2014 May 6;86(9):4550-8. doi: 10.1021/ac500588q. Epub 2014 Apr 11.
Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate ((S)GTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of (S)GTP to a GTP-binding protein, Rac1. However, it remains unclear whether (S)GTP can also bind to other cellular proteins. Here, we introduced an orthogonal approach, encompassing nucleotide-affinity profiling and nucleotide-binding competition assays, to characterize comprehensively (S)GTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of (S)GTP and GTP affinity probes, we identified 165 (S)GTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward (S)GTP and GTP, which allowed for the revelation of the relative binding affinities of the two nucleotides toward the nucleotide-binding motif sequence of proteins. Our results suggest that (S)GTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that (S)GTP binds to several cyclin-dependent kinases (CDKs), which may perturb the CDK-mediated phosphorylation and cell cycle progression. Together, this represents the first comprehensive characterization of (S)GTP-binding property for the entire human proteome. We reason that a similar strategy can be generally employed for the future characterization of the interaction of other modified nucleotides with the global proteome.
硫嘌呤类药物被广泛用作抗白血病药物和免疫抑制剂，6-硫鸟苷三磷酸（(S)GTP）是这些药物的主要代谢产物。最近的研究表明，硫嘌呤类药物可能部分通过(S)GTP与GTP结合蛋白Rac1结合发挥其细胞毒性作用。然而，(S)GTP是否也能与其他细胞蛋白结合仍不清楚。在此，我们引入了一种正交方法，包括核苷酸亲和谱分析和核苷酸结合竞争试验，以全面表征来自整个人类蛋白质组的(S)GTP结合蛋白及其特定结合位点。通过同时使用(S)GTP和GTP亲和探针，我们鉴定出165种参与几种不同生物学过程的(S)GTP结合蛋白。我们还检测了这些蛋白对(S)GTP和GTP的结合选择性，从而揭示了这两种核苷酸对蛋白质核苷酸结合基序序列的相对结合亲和力。我们的结果表明，(S)GTP主要靶向GTP酶，对多种异源三聚体G蛋白观察到强结合亲和力。我们还证明了(S)GTP与几种细胞周期蛋白依赖性激酶（CDK）结合，这可能会干扰CDK介导的磷酸化和细胞周期进程。总之，这代表了对整个人类蛋白质组(S)GTP结合特性的首次全面表征。我们认为，类似的策略通常可用于未来表征其他修饰核苷酸与整体蛋白质组的相互作用。
