RESUMO
Small ubiquitin-like proteins (SUMO) are recently discovered post-translational modifiers that regulate protein functions and intracellular trafficking. In this study, we are describing two chemoluminescence-based assays, one for SUMOylation and another one for SUMO-mediated protein-protein interactions. These assays can be used to characterize the activity and kinetics of the enzymes that catalyze SUMOylation, and in high-throughput screening for inhibitors of SUMOylation and SUMO-dependent protein-protein interactions. These novel assays represent the most sensitive assays for ubiquitin-like systems published to date. Similar strategies can be used to develop assays for other ubiquitin-like modification systems.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo , Cinética , Medições Luminescentes , Ligação Proteica , Sensibilidade e EspecificidadeRESUMO
Protein kinases are directly implicated in many human diseases; therefore, kinase inhibitors show great promises as new therapeutic drugs. In an effort to facilitate the screening and the characterization of kinase inhibitors, a novel application of the AlphaScreen technology was developed to monitor JNK activity from (1) purified kinase preparations and (2) endogenous kinase from cell lysates preactivated with different cytokines. The authors confirmed that both adenosine triphosphate (ATP) competitive as well as peptide-based JNK inhibitors were able to block the activity of both recombinant and HepG2 endogenous JNK activity. Using the same luminescence technique adapted for binding studies, the authors characterized peptide inhibitor mechanisms by measuring the binding affinity of the inhibitors for JNK. Because of the versatility of the technology, this cell-based JNK kinase assay could be adapted to other kinases and would represent a powerful tool to evaluate endogenous kinase activity and test a large number of potential inhibitors in a more physiologically relevant environment.