Physiological substrates in a high throughput isopeptidase assay

DESCRIPTION (provided by applicant): As the prominence of the ubiquitin and ubiquitin-like pathways increases, the need for assays to measure the activity of the enzymes involved in these pathways grows. Currently the only high throughput methods for measuring ubiquitin and ubiquitin-like isopeptidase activity rely on nonphysiological ubiquitin conjugates. The most widely used is Ub-AMC. In this assay the C-terminus of ubiquitin is fused to a small fluorophore. Cleavage by an ubiquitin isopeptidase increases fluorescence. This assay format does not represent a physiological event, which may explain why many isopeptidases are unable to cleave this conjugate. Although it is possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by western blotting this is a viable option only if a small number of samples is tested. To screen small molecules or natural products for inhibitors of isopeptidases, SDSPAGE and western blotting are unacceptable methods. For these reasons it is proposed to develop a novel assay to measure ubiquitin and ubiquitin-like isopeptidase activity with physiological substrates. This assay will be amenable to high throughput screening and will not suffer from the limitations shared by current ubiquitin isopeptidase assays. In brief, the assay utilizes ubiquitin binding domains to capture either ubiquitin chains or ubiquitylated substrates. Cleavage of ubiquitin chains or ubiquitin from the substrate will result in reduced ubiquitin binding to the ubiquitin binding domain and a decrease in relative fluorescent intensity when measured with a FITC-conjugated antibody recognizing ubiquitin. Assaying deSUMOylase activity depends on the recent discovery that E2-25k, an ubiquitin E2 enzyme capable of forming ubiquitin chains, is inhibited by SUMOylation. E2-25k will be SUMOylated in vitro and its ability to form chains measured using the ubiquitin binding domain strategy described above. The presence of a deSUMOylase will increase the activity of E2-25k which will be detected by the increase in ubiquitin chains. To determine the commercial feasibility of these assays Progenra will work with LifeSensors to perform beta testing of the assays by academic and industry researchers. This will yield valuable information about the assay design and product literature which will be used to market the assay. The generation of high throughput assays for quantifying ubiquitin and ubiquitin-like isopeptidase activity using physiological substrates represents a major advancement in the study of these crucial cellular enzymes. PUBLIC HEALTH RELEVANCE: Enzymes of the ubiquitin pathway, which modifies and regulates nearly all cellular proteins with the 76 amino acid tag ubiquitin, have become highly interesting therapeutic targets. In particular, De-ubiquitylating enzymes (DUBs), which remove ubiquitin tags from proteins, are associated with nearly all disease classes. Inhibitors of various DUBs are expected to be therapeutic. It is not possible to find inhibitors of these critical enzymes until truly physiological assays can be devised for use in screening. The present application proposes to do this by employing specific protein sequences, called ubiquitin binding domains, to capture ubiquitin chains or ubiquitylated substrates of the DUBs. Cleavage of ubiquitin chains or ubiquitin from the substrate will result in reduced ubiquitin binding, which can be quantified by a fluorescent antibody, and molecules that block this reduction in fluorescence will be considered to be inhibitors of the particular DUB. To determine the commercial feasibility of these newly developed assays, they will be tested by various laboratories in both academia and industry. Information from these field tests will be used to market the assay.