title: Engineering pre-SUMO4 as efficient substrate of SENP2.
abstract: SUMOylation, one of the most important protein post-translational modifications, plays critical roles in a variety of physiological and pathological processes. SENP (Sentrin/SUMO-specific protease), a family of SUMO-specific proteases, is responsible for the processing of pre-SUMO and removal of SUMO from conjugated substrates. SUMO4, the latest discovered member in the SUMO family, has been found as a type 1 diabetes susceptibility gene and its maturation is not understood so far. Despite the 14 amino acid differences between pre-SUMO4 and SUMO2, pre-SUMO4 is not processed by SENP2 but pre-SUMO2 does. A novel interdisciplinary approach involving computational modeling and a FRET-based protease assay was taken to engineer pre-SUMO4 as a substrate of SENP2. Given the difference in net charge between pre-SUMO4 and pre-SUMO2, the computational framework analysis of electrostatic similarities of proteins was applied to determine the contribution of each ionizable amino acid in a model of SENP2-(pre-SUMO4) binding, and to propose pre-SUMO4 mutations. The specificities of the SENP2 toward different pre-SUMO4 mutants were determined using a quantitative FRET assay by characterizing the catalytic efficiencies (kcat/KM). A single amino acid mutation made pre-SUMO4 amenable to SENP2 processing and a combination of two amino acid mutations made it highly accessible as SENP2 substrate. The combination of the two approaches provides a powerful protein engineering tool for future SUMOylation studies.
authors.affiliation: Department of Bioengineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Chris A
authors.initials: CA
authors.lastname: Kieslich
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Morikis
authors.forename: Jiayu
authors.initials: J
authors.lastname: Liao
mesh_terms: D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011499:Protein Processing, Post-Translational; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055672:Static Electricity
chemical_list: C117909:SUMO2 protein, human; C489647:SUMO4 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C501833:SENP2 protein, human
keywords: Computational modeling of electrostatic similarities; FRET-based kinetics determination; SUMO4/2; SUMOylation; protease substrate engineering