Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry.
Deconjugation of ubiquitin from cellular proteins is catalyzed by the deubiquitin hydrolase (DUB) family of enzymes and is an important component of the ubiquitin regulatory system affecting cellular function beyond simple maintenance of monomeric pools of ubiquitin. Specific deconjugation of ubiquitinated substrates has been described, but substrate recognition is poorly understood. To determine whether specificity may be conferred by recognition of a primary cognate sequence, the substrate preferences of two DUBs, UCH-L3 and isopeptidase T (IsoT), were profiled using a positional scanning branched peptide library. The sequence of the library was based on K48-branched diubiquitin, RLXXXXK(GGRLRLVL)QLEDGR, where X denotes a diversified position in the library (P1' '-P4' ' numbered from K48). Hydrolysis of the branched peptide was indicative of DUB activity and was detected and quantified by mass spectrometry. IsoT was active toward the library but demonstrated little preference for the diversified positions. In contrast, UCH-L3 exhibited minor amino acid preferences at P2' ' and P4' ' and a 10-fold preference for the basic residues Arg and Lys at P3' '. Kinetic analysis of substrates with optimized and suboptimized sequences (as defined by the library profile) confirmed the preference at P3' '. Substrate inhibition of UCH-L3 but not IsoT was noted for the optimized sequence at concentrations greater than 5 microM and with an IC(50) of 12.2 microM; the inhibition was determined to be competition with Ub-AMC (ubiquitin C-terminal 7-amido-4-methylcoumarin).[1]References
- Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry. Mason, D.E., Ek, J., Peters, E.C., Harris, J.L. Biochemistry (2004) [Pubmed]
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