Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2

High-throughput caveolar proteomic signature profile for maternal binge alcohol consumption

“…Finally, the direct impact of the nitric oxide system on the fetal compartment is substantiated by reports that deficiency in nitric oxide production renders the developing fetal neuronal cells more vulnerable to the toxic effects of alcohol and that the nitric oxide-cGMP-PKG pathway has protective effect against alcohol-induced injury (Bonthius et al, 2003; 2008; 2009). Seventh , alcohol-induced dramatic reduction of USP2, the major deubiquitinating enzyme may provide mechanistic perspectives underlying caveolar protein depletion and/or degradation as USP2 depletion has been documented to significantly enhance protein degradation (Tirat et al, 2005). …”

The role of deubiquitinases in breast cancer

“…First, false positives are common, since AMC has an excitation wavelength in the UV range. Second, Ub-AMC is not hydrolyzed efficiently by the UBP/USP class of DUBs, weakening its application to some extent [114,115]. The FRET (fluorescence resonance energy transfer) assay, which selectively incorporates the FRET fluorescence donor (terbium) or acceptor (fluorochrome) onto ubiquitin, has been used to identify novel small-molecule inhibitors of AMSH [116].…”

Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins

“…In the case of the 15 aa AviTag TM sequence, synthetic oligonucleotides coding for this sequence were directly ligated into the ENTRY vector again by using the EcoRI/NotI restriction sites. The sequences for full-length Rad6B (Swissprot: P23567) and Ubc9 (Swissprot: P63279) were amplified by PCR as described in [14]. The inserts were ligated into the BamHI/NotI restriction sites of the corresponding Gateway ENTRY vector either carrying the SNAP-Tag TM for Rad6B or the BioEase TM and AviTag TM for Ubc9.…”

“…After ATP-dependent activation by the heterodimeric SUMO activating enzyme SAE1/SAE2, the formation of a thioester bond between the C-terminal COOH group of SUMO and the active site cysteine of Ubc9 was monitored by SDS-PAGE analysis under reducing and non-reducing conditions. Based on an inhouse protocol adapted to sumoylation [14], the reaction was run at room temperature for 10 min with a 10 l mixture consisting of 1 g SUMO activating enzyme SAE1/SAE2 [16], 6 g Ubc9 and 2 g SUMO in 50 mM HEPES pH 7.9, 50 mM NaCl, 10 mM MgCl 2 , 10 mM ATP and 0.1 mM DTT. The reaction was stopped by addition of 2 × NuPAGE sample buffer (Invitrogen) with or without 100 mM DTT.…”

“…Panel A depicts the reaction scheme between Ub, E1 and E2s. Panel B shows the SDS-PAGE analysis of activity experiments performed with reaction mixtures containing GST-E1 (∼150 kDa), Alexa488-Ub (∼9 kDa) made in-house[14] and labelled Rad6B-AGT or AGT/Rad6B (∼40 kDa). Lanes M: Novex Mark 12 molecular weight marker (Invitrogen), lanes 1 and 2: activity test of Rad6B-AGT-TAMRA with Alexa488-Ub, lanes 3 and 4: Rad6B-AGT-TAMRA reaction mixture without Alexa488-Ub, lanes 5 and 6: Alexa488-Ub reaction mixture without Rad6B-AGT-TAMRA, lanes 7 and 8: activity test of TAMRA-AGT-Rad6B with Alexa488-Ub, lanes 9 and 10: TAMRA-AGT-Rad6B reaction mixture without Alexa488-Ub.…”