Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.
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 IkappaBalpha, 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.
Hjerpe
Roland
R
Proteomics Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, Building 801A, Derio 48160, Spain.
Aillet
Fabienne
F
Lopitz-Otsoa
Fernando
F
Lang
Valerie
V
England
Patrick
P
Rodriguez
Manuel S
MS
D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007700:Kinetics; D000072000:NF-KappaB Inhibitor alpha; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry; D020349:Surface Plasmon Resonance; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry; D025801:Ubiquitin / Q000737:chemistry*
D015853:Cysteine Proteinase Inhibitors; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D000072000:NF-KappaB Inhibitor alpha; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D046988:Proteasome Endopeptidase Complex