Naturally occurring cancer-associated mutations disrupt oligomerization and activity of protein arginine methyltransferase 1 (PRMT1)
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101336
ISSN1083-351X
AutoresOwen M. Price, Abhishek Thakur, Ariana Ortolano, Arianna Towne, Caroline Velez, Orlando Acevedo, Joan M. Hevel,
Tópico(s)Epigenetics and DNA Methylation
ResumoProtein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences. Protein arginine methylation is a posttranslational modification catalyzed by the protein arginine methyltransferase (PRMT) enzyme family. Dysregulated protein arginine methylation is linked to cancer and a variety of other human diseases. PRMT1 is the predominant PRMT isoform in mammalian cells and acts in pathways regulating transcription, DNA repair, apoptosis, and cell proliferation. PRMT1 dimer formation, which is required for methyltransferase activity, is mediated by interactions between a structure called the dimerization arm on one monomer and a surface of the Rossman Fold of the other monomer. Given the link between PRMT1 dysregulation and disease and the link between PRMT1 dimerization and activity, we searched the Catalogue of Somatic Mutations in Cancer (COSMIC) database to identify potential inactivating mutations occurring in the PRMT1 dimerization arm. We identified three mutations that correspond to W215L, Y220N, and M224V substitutions in human PRMT1V2 (isoform 1) (W197L, Y202N, M206V in rat PRMT1V1). Using a combination of site-directed mutagenesis, analytical ultracentrifugation, native PAGE, and activity assays, we found that these conservative substitutions surprisingly disrupt oligomer formation and substantially impair both S-adenosyl-L-methionine (AdoMet) binding and methyltransferase activity. Molecular dynamics simulations suggest that these substitutions introduce novel interactions within the dimerization arm that lock it in a conformation not conducive to dimer formation. These findings provide a clear, if putative, rationale for the contribution of these mutations to impaired arginine methylation in cells and corresponding health consequences. Protein arginine methylation is a set of widespread posttranslational modifications that have been reported on a large number of both histone and nonhistone substrates (1Larsen S.C. Sylvestersen K.B. Mund A. Lyon D. Mullari M. Madsen M.V. Daniel J.A. Jensen L.J. Nielsen M.L. 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We found three mutations resulting in a change to the protein sequence, with one resulting in a W215L substitution, one in a Y220N substitution, and one in an M224V substitution (Fig. 1A). As a first step in determining if these mutations might be functionally relevant, we performed a sequence alignment of all type I PRMT paralogs in humans and an alignment of PRMT1 homologs across several species (Fig. 1, B and C). A Consurf (51Ashkenazy H. Abadi S. Martz E. Chay O. Mayrose I. Pupko T. Ben-Tal N. ConSurf 2016: An improved methodology to estimate and visualize evolutionary conservation in macromolecules.Nucleic Acids Res. 2016; 44: W344-350Crossref PubMed Scopus (1050) Google Scholar, 52Celniker G. Nimrod G. Ashkenazy H. Glaser F. Martz E. Mayrose I. Pupko T. Ben-Tal N. ConSurf: Using evolutionary data to raise testable hypotheses about protein function.Isr. J. Chem. 2013; 53: 199-206Crossref Scopus (302) Google Scholar, 53Ashkenazy H. Erez E. Martz E. Pupko T. Ben-Tal N. 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Additionally, structures of available PRMTs indicate that the dimerization arm varies considerably in length and in the angle at which it protrudes from the core. However, we found relatively good conservation of W215, Y220, and M224 compared with the rest of the dimerization arm (Fig. 1, B and C). We hypothesized that these residues form important interactions that mediate dimer formation and that their mutations would disrupt both oligomerization and activity of PRMT1. We tested this hypothesis using R. norvegicus PRMT1 (rPRMT1), which differs in protein sequence from the comparable human splice variant by a single residue; Y179 in the H. sapiens PRMT1 (hPRMT1) Rossman Fold is ∼30 Å distal to the dimer interface and is a histidine in rPRMT1. The conformation of the dimerization arm in rPRMT1 and hPRMT1 crystal structures (Fig. 1D) shows an RMSD of 0.5 Å when the two arms are aligned, and previous work has shown that the methyltransferase rates measured from hPRMT1 and rPRMT1 are similar for most substrates (56Osborne T.C. Obianyo O. Zhang X. Cheng X. Thompson P.R. Protein arginine methyltransferase 1: Positively charged residues in substrate peptides distal to the site of methylation are important for substrate binding and catalysis.Biochemistry. 2007; 46: 13370-13381Crossref PubMed Scopus (110) Google Scholar). Finally, we have previously studied rPRMT1 using both experimental and computational methods and have accumulated a strong foundation for comparison studies (7Price O.M. Hevel J.M. Toward understanding molecular recognition between PRMTs and their substrates.Curr. Protein Pept. Sci. 2020; 21: 713-724Crossref PubMed Scopus (2) Google Scholar, 11Morales Y. Cáceres T. May K. Hevel J.M. 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