A coupled fluorescence-based assay for the detection of protein arginine N-methyltransferase 6 (PRMT6) enzymatic activity

Anal Biochem. 2018 Apr 15;547:7-13. doi: 10.1016/j.ab.2018.01.023. Epub 2018 Feb 2.

Abstract

The protein arginine N-methyltransferase 6 (PRMT6) is overexpressed in a variety of different cancer types and plays a role in human immunodeficiency virus (HIV) infections. Furthermore, the PRMT6 activity might also influence the pathogenesis of neurodegenerative, inflammatory, and cardiovascular diseases, whereby it becomes an interesting target for drug development. Previously reported activity assays for PRMT6 activity are either expensive, time-consuming or use radioactive substrates. To overcome these challenges, we developed a coupled fluorescence-based activity assay using recombinant PRMT6 expressed in E. coli. In the first step of the assay, the fluorogenic substrate Nα-Benzoyl-L-arginine-7-amido-4-methylcoumarin (Bz-Arg-AMC) is methylated by PRMT6, while in a second step the remaining un-methylated substrate is cleaved by trypsin, producing the fluorescent 7-amino-4-methylcoumarin.

Keywords: Coupled enzyme assay; Epigenetics; Fluorescence; Protein arginine N-methyltransferase 6 (PRMT6).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence
  • Humans
  • Nuclear Proteins / analysis*
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Peptides / chemistry*
  • Protein-Arginine N-Methyltransferases / analysis*
  • Protein-Arginine N-Methyltransferases / chemistry
  • Protein-Arginine N-Methyltransferases / genetics
  • Recombinant Proteins / analysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics

Substances

  • Nuclear Proteins
  • Peptides
  • Recombinant Proteins
  • PRMT6 protein, human
  • Protein-Arginine N-Methyltransferases