Monitoring of Protein Arginine Deiminase Activity by Using Fluorescence Quenching: Multicolor Visualization of Citrullination

摘要

Epigenetics is driven by posttranslational modifications, such as methylation and acetylation of Arg and Lys residues. A less well publicized modification, namely conversion of Arg to citrulline (Cit), began to receive attention in the 1990s, when it was discovered that autoantibodies targeting citrulline-embedded epitopes are present in patients with rheumatoid arthritis. The Arg-to-Cit conversion in histones has now been correlated with the regulation of gene expression. PAD4, a member of the protein arginine deiminase (PAD) enzyme family that catalyze this transformation, is overex-pressed in rheumatoid arthritis, cancer, multiple sclerosis, and glaucoma.Only a few assays have been described for the PAD enzyme family and include colorimetric,coupled, SDS-PAGE gel,affinity probe fluorescent polarization, and protein array pproaches. We describe herein a PAD4 activity fluorescence-based sensing strategy that can be employed throughout the visible spectrum and into the near infrared. The flexibility inherent within this strategy offers the ability to simultaneously monitor the activity of PAD4 along with other enzymes that modify histones.