Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Hydrogen bonding of 3-and 5-methyl-6-aminouracil with natural DNA bases

We have theoretically analyzed the hydrogen bonding of two artificial nucleobases (3- and 5-methyl-6-aminouracil) with the natural DNA bases using the generalized gradient approximation (GGA) of density functional theory at BP86/TZ2P level. The analysis of the monomers provides the possibility to distinguish the different active parts of molecules and the interactions with natural nucleobases have been determined. Another purpose of this work is to clarify the relative importance of electrostatic interaction vs. orbital interaction in the hydrogen bonds between the artificial base and the natural DNA base. At variance with widespread belief, the orbital interaction component in these hydrogen bonds is found to contribute about 40% of the attractive interactions and is thus of the same order of magnitude as the electrostatic component, which provides the remaining attraction. According to our theoretical results both candidates are potential artificial nucleobases for incorporation in DNA. © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2008