title: Fluorescence-based monitoring of PAD4 activity via a pro-fluorescence substrate analog.
abstract: Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.
authors.affiliation: Department of Chemistry, Lehigh University.
authors.forename: Mary J
authors.initials: MJ
authors.lastname: Sabulski
authors.affiliation: Department of Chemistry, Lehigh University.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Fura
authors.affiliation: Department of Chemistry, Lehigh University; map311@lehigh.edu.
authors.forename: Marcos M
authors.initials: MM
authors.lastname: Pires
mesh_terms: D000818:Animals; D002417:Cattle; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D006867:Hydrolases; D014357:Trypsin; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases