A novel fluorescent assay for ubiquitin isopeptide bond cleavage

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 non-physiological ubiquitin conjugates. The most widely used is Ub-AMC. In this assay the C- terminus of ubiquitin is fused to a small fluorophore. Upon cleavage by an ubiquitin isopeptidase there is an increase in 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 only a viable option if a small number of samples are being tested. For instance in order to screen small molecules or natural products for inhibitors of isopeptidases SDS-PAGE and western blotting are unacceptable methods. For these reasons we propose to develop a novel assay for measuring ubiquitin 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. Briefly, C-terminus of wild type ubiquitin is conjugated to two different ubiquitins, which contain only lysine 48 or lysine 63, available for isopeptide bond formation and fluorescent labeling. In vitro conjugation wild type ubiquitin, via an isopeptide bond to another ubiquitin results in di-ubiquitin that will contain optimized internally quenched fluorescent pairs. We will establish physiological role of the novel di-ubiquitin substrates by demonstrating cleavage with lysine 48 or 63 isopeptide bond selective de-ubiquitylases. The generation of high throughput assays for quantifying ubiquitin isopeptidase activity using physiological substrates represents a major advancement in the study of these crucial cellular enzymes. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin plays important roles in many cellular processes. In the last decade there has been an explosive growth in the field of ubiquitin research. The enzymes that remove ubiquitin from target proteins are very important drug targets. There is a need for better assays to measure the activity of the enzymes, which are highly specific and physiologically relevant. The development of assays using physiological substrates represents a major advancement in the study of this important group of cellular enzymes and the topic of this proposal.