title: Development of a Plate-Based Screening Assay to Investigate the Substrate Specificity of the PRMT Family of Enzymes.
abstract: There are nine protein arginine methyltransferases (PRMTs 1-9) expressed in humans that vary in both subcellular localization and substrate specificity. The variation in substrate specificity between isozymes leads to competing effects that result in either activation or repression of tumor suppressor genes. Current methods used to study substrate specificity for these enzymes utilize radioisotopic labeling of substrates, mass spectrometry analysis of complex samples, or coupled assays that monitor cofactor degradation. Herein, we report the development of a rapid, nonradioactive, and sensitive method for screening multiple peptides in parallel to gain insight into the substrate specificity of PRMT enzymes. Our assay provides a major advantage over other high-throughput screening assays (e.g., ELISA, AlphaScreen chemiluminescence) by eliminating the need for purification of individual peptides and provides a timesaving, cost-effective alternative to the traditional PRMT assays. A one-bead one-compound (OBOC) peptide library was synthesized and subsequently screened against PRMT1 in a 96-well plate. This screen resulted in identification of a novel PRMT1 substrate with kinetic parameters similar to histone H4-21 (e.g., the best-known PRMT1 peptide substrate).
authors.affiliation: Department of Chemistry, University of North Florida , Jacksonville, Florida 32224-7699, United States.
authors.forename: Hao C
authors.initials: HC
authors.lastname: Nguyen
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts , Worcester, Massachusetts 01655, United States.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Department of Chemistry, University of North Florida , Jacksonville, Florida 32224-7699, United States.
authors.forename: Andrew
authors.initials: A
authors.lastname: Salsburg
authors.affiliation: Department of Chemistry, University of North Florida , Jacksonville, Florida 32224-7699, United States.
authors.forename: Bryan
authors.initials: B
authors.lastname: Knuckley
mesh_terms: D000595:Amino Acid Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D057166:High-Throughput Screening Assays; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D049449:Luminescence; D008969:Molecular Sequence Data; D019151:Peptide Library; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D006657:Histones; D007527:Isoenzymes; D019151:Peptide Library; D011484:Protein-Arginine N-Methyltransferases
keywords: arginine; high-throughput; histone; one-bead one-compound; protein arginine methyltransferase; substrate screening