UbiTest: Detection of polyubiquitinated proteins using TUBE based screening platforms
by Gauthami S. Jalagadugula
The ubiquitin proteasome system (UPS) is a highly regulated mechanism that controls many cellular processes through protein ubiquitination and degradation. Ubiquitin is conjugated via its C-terminus to lysine residues on the target proteins as a monomer or as a polymer chain. Ubiquitin chains are formed via ubiquitin’s first methionine (M1), giving rise to a linear chain, or via its seven lysine residues (K6,K11, K27, K29, K33, K48, K63) leading to lysine specific chains. K48 and K63-linked ubiquitin chains are the most predominant forms of polyubiquitination in cells. Ubiquitination is a reversible process in which deubiquitinating enzymes (DUBs) remove the ubiquitins from the target proteins and rescue them from proteasomal degradation.
Determining the extent and type of polyubiquitination on target proteins is important to understand their ubiquitin mediated regulation in normal cellular processes as well as in UPS-associated disease states. Traditionally, these are monitored either through mass spectrometry or via immunoprecipitation using anti-ubiquitin antibodies followed by immunoblotting. However, the substrate antibody often interacts differently with the poly-ubiquitinated forms of the substrate in the immuno-blotting step due to epitope masking, reduced affinity, or changes in selectivity caused by the protein’s polyubiquitination.
Overview of the UbiTest Assay
Conceptual schematic of the UbiTest assay: Ubiquitylated proteins are enriched and isolated by Magnetic TUBEs, then eluted from the TUBEs. The isolated polyubiquitylated eluate is then split into two fractions: one fraction is left untreated, while the second fraction is digested with a broad-spectrum DUB to remove ubiquitin chains. Both fractions are analyzed by immunoblot using an antibody for the POI. The removal of the ubiquitin chains from the polyubiquitylated POI results in the simple visualization of a single, unmodified band of POI in the immunoblot.
A more definitive method for demonstrating protein ubiquitination is coupling immunoprecipitation with deubiquitination using a broad spectrum deubiquitinase (DUB) prior to immunoblot analysis. An increased signal for the protein of interest (POI) after DUB treatment is a clear indication that the protein was ubiquitinated even if there was no clear reactivity in the untreated sample. LifeSensors’ UbiTest assay utilizes TUBEs (Tandem Ubiquitin Binding Entities) to pull-down poly-ubiquitinated proteins to avoid potential problems arising from changes in immunoreactivity of the POI. TUBEs are engineered tandem ubiquitin-binding domains with dissociation constants for poly-ubiquitin in the nanomolar range. LifeSensors’ pan-selective TUBEs have been demonstrated to bind to all eight ubiquitin linkage types. To determine the linkage types of polyubiquitins on target proteins, LifeSensors has developed a more specific method that utilizes linkage-specific DUBs prior to immunoblot analysis. An increased signal for the band corresponding to the unmodified POI after a K48/K63-specific DUB treatment indicates that the protein is K48/K63 poly-ubiquitinated.
This technology has a variety of applications including: monitoring of cellular protein ubiquitination (shown below), E3 ligase validation, detection of linkage specific polyubiquination, evaluation of PROTAC and Molecular Glue mediated protein ubiquitination and degradation, as well as neurodegenerative disease biomarker discovery.
(A) Ubiquitination and deubiquitination of USP7 in Jurkat cells treated with DMSO or BTZ (200 nM, proteosome inhibitor) or BTZ (200 nM) + drug (USP7 inhibitor (10 uM). Lane 1: ubiquitinated USP7 in DMSO treated cells; Lane 2: increase in the USP7 due to deubiquitination by DUB; Lane 3: ubiquitinated USP7 in BTZ treated cells; Lane 4: deubiquitinated USP7 in BTZ treated cells showing higher expression; Lane 5: USP7 with its inhibitor and BTZ; Lane 6: Increased level of USP7 with inhibitor+BTZ after DUB treatment. (B) shows densitometric quantitation of the bands shown in A with DUB treatment (lanes 2,4 and 6). (C) shows a corresponding blot on polyubiquitinated protein from cells with different treatments. DUB untreated lanes showed polyubiquitinated proteins migrated at high molecular weight (Lanes 1, 3 and 5). Lanes 2, 4 and 6: DUB treated lanes with no smears of proteins.
For further reading, the complete application note can be found on any of the UbiTest product pages, as well as included here.
For further inquires as to whether the UbiTest technology is right for your specific application, please reach out to us at Info@lifesensors.com.