title: Comprehensive characterization of (S)GTP-binding proteins by orthogonal quantitative (S)GTP-affinity profiling and (S)GTP/GTP competition assays.
abstract: 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.
authors.affiliation: Department of Chemistry and ‡Environmental Toxicology Graduate Program, University of California , Riverside, California 92521-0403, United States.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Xiao
authors.forename: Debin
authors.initials: D
authors.lastname: Ji
authors.forename: Lei
authors.initials: L
authors.lastname: Guo
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000345:Affinity Labels; D001667:Binding, Competitive; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D020543:Proteome
chemical_list: D000345:Affinity Labels; D020543:Proteome; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins