Comprehensive Characterization of SGTP-Binding Proteins by Orthogonal Quantitative SGTP-Affinity Profiling and SGTP/GTP Competition Assays

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• 作者：Yongsheng Xiao ; Debin Ji ; Lei Guo ; Yinsheng Wang
• 刊名：Analytical Chemistry
• 出版年：2014
• 出版时间：May 6, 2014
• 年：2014
• 卷：86
• 期：9
• 页码：4550-4558
• 全文大小：464K
• 年卷期：v.86,no.9(May 6, 2014)
• ISSN：1520-6882

文摘

Thiopurine drugs are widely used as antileukemic drugs and immunosuppressive agents, and 6-thioguanosine triphosphate (^SGTP) is a major metabolite for these drugs. Recent studies have suggested that thiopurine drugs may exert their cytotoxic effects partly through binding of ^SGTP to a GTP-binding protein, Rac1. However, it remains unclear whether ^SGTP 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 ^SGTP-binding proteins along with the specific binding sites from the entire human proteome. With the simultaneous use of ^SGTP and GTP affinity probes, we identified 165 ^SGTP-binding proteins that are involved in several different biological processes. We also examined the binding selectivities of these proteins toward ^SGTP 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 ^SGTP mainly targets GTPases, with strong binding affinities observed for multiple heterotrimeric G proteins. We also demonstrated that ^SGTP 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 ^SGTP-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.