UBQuest is a probeless, tagless, proximity based assay for highly multiplexed formation of binary complexes of proteins with their cognate E3 ligases, deubiquitinases (DUBs), substrate-recognizing adapter proteins, and any other accessory proteins of UPS. The enriched complexes are purified and sequenced with mass spectrometry. This preparatory approach can then be used for matching any substrate in the UBQuest pool with its E3 ligase or DUB.
Our chemoproteomics platform, ubCAST, employs a proprietary ELISA assay for detecting proteostasis modulating compounds in high throughput formats. Compounds are screened against a protein fraction enriched in E3 ligases and other essential comonents of UPS comprising the ‘degradome’. A positive signal denotes alteration(s) to any of the ubiquitome components in the candidate cell type are detected.
The hit rate (number of positive signals in ELISA per 1,000 compounds tested) with our strategy are significantly higher when compared with contemporary approaches since any E3 ligase or protein complex in the cell may bind the compound and our assay has the ability to detect such changes without the using specific probes. Due to the high degree of multiplexing in the ubiquitome, we expect significantly greater coverage overall for compound screening projects, and fewer compounds may need to be screened for finding ‘hits’ than has hitherto been possible.
PINTACs are peptides which cause one or more proteins to be mono/polyubiquitinated when overexpressed in cells.
E-PINTACs target an E3 ligase and the ubiquitinated proteins are the authentic cellular substrates of the targeted E3 ligase. They are used for discovery, and recapitulate UBQuest data with respect to E3 ligase and substrate matching, the latter are the natural E3L-substrate pairs identified from cells.
S-PINTACs polyubiquitinate single proteins when targeting specific proteins. They are used for disrupting the function of individual candidate proteins, and can be developed for therapeutic purposes. Both types of PINTACs exhibit high levels of specificity and selectivity in vitro.
Once characterized, PINTAC peptides serve as capture moieties for characterizing the degrome signatures across cell types or samples in vitro, including clinical samples. Frequently, the degromes include proteins necessary for the E3 ligase to capture substrate for degradation. PINTACs identify ‘motifs’ recognized by an E3 ligases, which has applications in TPD research.
X-PINTACs are peptides which do not possess a ubiquitinating function, but may have other uses as discovery tools or targeting agents.