Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin‐binding entities

Abstract

Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and IjBa, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new 'molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.

Efficient monitoring of protein ubiquitylation levels using TUBEs‐based microarrays

“…As previously proposed, the presence of multiple UBA domains had a synergistic effect on their interaction with tetra-ubiquitin chains [14] which is more pronounced for long contact times (Fig. 1) compared with the shorter ones ( Figs S1 and S2).…”

“…For this reason, methods for ubiquitin protection and enrichment are essential for their analysis. Several methods have been developed in the last years to isolate and study ubiquitylated proteins, including the expression of tagged ubiquitin molecules, the development of specific antiubiquitin antibodies or the use of tandem ubiquitin-binding entities (TUBEs) [13,14]. TUBEs show two very useful properties: ubiquitylated substrates are protected from deubiquitylating enzymes and also from proteasome-mediated proteolysis, leading to an improved capture of ubiquitylated proteins.…”

Arabidopsis ubiquitin ligase MIEL1 mediates degradation of the transcription factor MYB30 weakening plant defence

“…We next purified poly-ubiquitinated proteins from Col-0 wildtype and MYB30-TAP expressing Arabidopsis using a tandem Ub-binding entities (TUBEs) agarose resin. This resin specifically recognizes tetra-Ub and allows purification of poly-ubiquitinated proteins in native conditions 13 . In this assay, a smear corresponding to purified poly-ubiquinated proteins was detected using an anti-Ub antibody and, as expected, the detected signal was stronger in the presence of MG132 in both Col-0 and MYB30 OE plants (Fig.…”

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

“…To validate this observation, the TUBE-agarose beads 49 were used to pull down ubiquitinated proteins in protein extracts derived from WTA or D7E myotube cultures. Protein extracts were prepared in the presence of PR619, a specific DUB inhibitor.…”