Deubiquitylase, deSUMOylase, and deISGylase activity microarrays for assay of substrate preference and functional modifiers.
Microarray-based proteomics expanded the information potential of DNA arrays to the level of protein translation and interaction, but so far, not much beyond. Although enzymatic activity from immobilized proteins has been reliably studied using surface plasmon resonance, a microarray of catalytically competent enzymes would facilitate high throughput, parallel study of their function. The ability to localize activity from soluble substrates has frustrated development of such an array. Here, we report the novel use of previously developed, highly specific suicide substrates for three families of enzymes: deubiquitylases, deSUMOylases, and deISGylases. We show specificity of each family to its cognate substrate, and demonstrate utility of the array in a secondary screen of small molecule inhibitors.
Division of Research & Development, LifeSensors, Inc., 271 Great Valley Parkway, Malvern, PA 19355, USA. loch@lifesensors.com
Christian M
CM
Loch
Charles L
CL
Cuccherini
Craig A
CA
Leach
James E
JE
Strickler
D010450:Endopeptidases / Q000378:metabolism*; D057075:Enzyme Assays; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004800:Enzymes, Immobilized / Q000378:metabolism; D004952:Esters / Q000378:metabolism*; D006801:Humans; D040081:Protein Array Analysis / Q000379:methods*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000494:pharmacology; D025841:Small Ubiquitin-Related Modifier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013194:Staining and Labeling; D013379:Substrate Specificity / Q000187:drug effects; D014452:Ubiquitins / Q000378:metabolism*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D004952:Esters; D054852:Small Molecule Libraries; D025841:Small Ubiquitin-Related Modifier Proteins; D014452:Ubiquitins; D010450:Endopeptidases