siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9

Abstract

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation. Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation. In eukaryotic cells, one of the most common post-translational modifications (PTMs) is protein methylation, that is, the transfer of a methyl group from SAM to lysine and arginine residues in the target protein (1Hyun K. Jeon J. Park K. Kim J. Writing, erasing and reading histone lysine methylations.Exp. Mol. Med. 2017; 49e324Crossref PubMed Scopus (358) Google Scholar, 2Bhat K.P. Umit Kaniskan H. Jin J. Gozani O. Epigenetics and beyond: Targeting writers of protein lysine methylation to treat disease.Nat. Rev. Drug Discov. 2021; 20: 265-286Crossref PubMed Scopus (16) Google Scholar, 3Blanc R.S. Richard S. Arginine methylation: The coming of age.Mol. Cell. 2017; 65: 8-24Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 4Jarrold J. Davies C.C. PRMTs and arginine methylation: Cancer's best-kept secret?.Trends Mol. Med. 2019; 25: 993-1009Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Lysine and arginine methylation consist of three distinct forms: monomethylation, dimethylation, and trimethylation of lysine and monomethylation, disymmetric methylation, and diasymmetric methylation of arginine. The functional significance of protein methylation has been extensively studied in histones, whose methylation plays a fundamental role in regulating gene expression and chromatin state (5Husmann D. Gozani O. Histone lysine methyltransferases in biology and disease.Nat. Struct. Mol. Biol. 2019; 26: 880-889Crossref PubMed Scopus (70) Google Scholar, 6Zhang J. Jing L. Li M. He L. Guo Z. Regulation of histone arginine methylation/demethylation by methylase and demethylase (Review).Mol. Med. Rep. 2019; 19: 3963-3971PubMed Google Scholar). Recently, however, a growing body of evidence has established that methylation also occurs on nonhistone proteins and is involved in a widespread phenomenon that regulates diverse biological processes, including protein synthesis, mRNA splicing, and signal transduction (7Levy D. Lysine methylation signaling of non-histone proteins in the nucleus.Cell Mol. Life Sci. 2019; 76: 2873-2883Crossref PubMed Scopus (18) Google Scholar, 8Guccione E. Richard S. The regulation, functions and clinical relevance of arginine methylation.Nat. Rev. Mol. Cell Biol. 2019; 20: 642-657Crossref PubMed Scopus (123) Google Scholar). In addition to lysine and arginine, protein methylation of atypical residues, such as glutamine and histidine, is considered to have profound biochemical and physiological significance in eukaryotes (9Kwiatkowski S. Drozak J. Protein histidine methylation.Curr. Protein Pept. Sci. 2020; 21: 675-689Crossref PubMed Scopus (5) Google Scholar, 10Heurgue-Hamard V. Champ S. Mora L. Merkulova-Rainon T. Kisselev L.L. Buckingham R.H. The glutamine residue of the conserved GGQ motif in Saccharomyces cerevisiae release factor eRF1 is methylated by the product of the YDR140w gene.J. Biol. Chem. 2005; 280: 2439-2445Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Histidine can potentially be methylated on the nitrogen in either position 1 (Nπ) or 3 (Nτ) of the imidazole ring, producing the isomers Nπ-methylhistidine (also referred to as 1-methylhistidine) or Nτ-methylhistidine (3-methylhistidine), respectively (9Kwiatkowski S. Drozak J. Protein histidine methylation.Curr. Protein Pept. Sci. 2020; 21: 675-689Crossref PubMed Scopus (5) Google Scholar). Protein histidine methylation was first discovered as Nτ-methylhistidine in the constituents of actin and myosin from muscle proteins half a century ago (11Huszar G. Elzinga M. Homologous methylated and nonmethylated histidine peptides in skeletal and cardiac myosins.J. Biol. Chem. 1972; 247: 745-753Abstract Full Text PDF PubMed Google Scholar, 12Johnson P. Harris C.I. Perry S.V. 3-methylhistidine in actin and other muscle proteins.Biochem. J. 1967; 105: 361-370Crossref PubMed Scopus (145) Google Scholar). After that, histidine methylation has been known for many years, but so far, there are only a few reports regarding histidine-methylated proteins: yeast ribosomal protein Rpl3 that carries an Nτ-methylhistidine (13Webb K.J. Zurita-Lopez C.I. Al-Hadid Q. Laganowsky A. Young B.D. Lipson R.S. Souda P. Faull K.F. Whitelegge J.P. Clarke S.G. A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase.J. Biol. Chem. 2010; 285: 37598-37606Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), mammalian myosin light chain kinase 2 (MLCK2) (14Meyer H.E. Mayr G.W. N pi-methylhistidine in myosin-light-chain kinase.Biol. Chem. Hoppe Seyler. 1987; 368: 1607-1611Crossref PubMed Scopus (12) Google Scholar), and proinflammatory protein S100A9 (15Raftery M.J. Harrison C.A. Alewood P. Jones A. Geczy C.L. Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: Characterization of post-translational modifications and zinc binding.Biochem. J. 1996; 316: 285-293Crossref PubMed Scopus (71) Google Scholar), both of which carry an Nπ-methylhistidine. However, a recent high-throughput proteomics analysis has successfully captured hundreds of proteins methylated at histidine residue in human cells, indicating that protein histidine methylation may be more prevalent type of PTM than predicted (16Ning Z. Star A.T. Mierzwa A. Lanouette S. Mayne J. Couture J.F. Figeys D. A charge-suppressing strategy for probing protein methylation.Chem. Commun. (Camb). 2016; 52: 5474-5477Crossref PubMed Google Scholar). The biggest obstacle to understanding the biological significance of histidine methylation is the lack of information about enzymes that catalyze methylhistidine formation. At present, however, the two distinct histidine-specific protein methyltransferases (MTases) have been identified in yeast and mice. The first one is yeast histidine protein MTase 1 that belongs to the seven-β-strand MTases responsible for the Nτ-methylation of His-243 in Rpl3 (13Webb K.J. Zurita-Lopez C.I. Al-Hadid Q. Laganowsky A. Young B.D. Lipson R.S. Souda P. Faull K.F. Whitelegge J.P. Clarke S.G. A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase.J. Biol. Chem. 2010; 285: 37598-37606Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), thereby contributing to the assembly of the large ribosomal subunit and translational elongation fidelity (17Al-Hadid Q. Roy K. Munroe W. Dzialo M.C. Chanfreau G.F. Clarke S.G. Histidine methylation of yeast ribosomal protein Rpl3p is required for proper 60S subunit assembly.Mol. Cell Biol. 2014; 34: 2903-2916Crossref PubMed Scopus (25) Google Scholar, 18Al-Hadid Q. Roy K. Chanfreau G. Clarke S.G. Methylation of yeast ribosomal protein Rpl3 promotes translational elongation fidelity.RNA. 2016; 22: 489-498Crossref PubMed Scopus (14) Google Scholar). The second one is mouse SETD3 that belongs to the SET domain MTases responsible for the Nτ-methylation of His-73 in the actin and regulates contraction in uterine smooth muscle (19Wilkinson A.W. Diep J. Dai S. Liu S. Ooi Y.S. Song D. Li T.M. Horton J.R. Zhang X. Liu C. Trivedi D.V. Ruppel K.M. Vilches-Moure J.G. Casey K.M. Mak J. et al.SETD3 is an actin histidine methyltransferase that prevents primary dystocia.Nature. 2019; 565: 372-376Crossref PubMed Scopus (49) Google Scholar, 20Kwiatkowski S. Seliga A.K. Vertommen D. Terreri M. Ishikawa T. Grabowska I. Tiebe M. Teleman A.A. Jagielski A.K. Veiga-da-Cunha M. Drozak J. SETD3 protein is the actin-specific histidine N-methyltransferase.Elife. 2018; 7e37921Crossref PubMed Scopus (38) Google Scholar). It should be noted that both enzymes are histidine Nτ-MTases, and there are no reports on enzymes that catalyze histidine Nπ-methylation so far. In this study, we established a new method that allows discrimination of the methylhistidine isomers of cellular proteins by using LC–MS/MS and combined it with an unbiased siRNA screen to identify molecules specifically required for histidine Nπ-methylation of S100A9. We found that mammalian seven-β-strand MTase METTL9 introduces Nπ-methylhistidine in S100A9, but not MLCK2, in vivo and in vitro. We also observed that METTL9 is localized predominantly to the endoplasmic reticulum (ER), binding to and methylating S100A9. Furthermore, we demonstrated that METTL9-mediated methylation of S100A9 at His-107 did not affect its heterodimer formation with S100A8, whereas histidine methylation of recombinant S100A9 by METTL9 substantially its binding affinity to zinc. Thus, our findings that METTL9 is a histidine in the and to histidine Nπ-methylation of S100A9, its zinc-binding We that findings have for not only the of protein histidine methylation but also the biological of Histidine methylation can on the distal or proximal nitrogen atom of its imidazole ring, producing Nτ-methylhistidine or respectively identify the protein histidine MTase that catalyzes Nπ-methylhistidine in we a novel method to either the or histidine methylation of cellular a protein of is as in human and with affinity by acid and analysis of the amino by coupled to the of this we two mammalian and S100A9, both of which are known to Nπ-methylhistidine residues (14Meyer H.E. Mayr G.W. N pi-methylhistidine in myosin-light-chain kinase.Biol. Chem. Hoppe Seyler. 1987; 368: 1607-1611Crossref PubMed Scopus (12) Google Scholar, M.J. Harrison C.A. Alewood P. Jones A. Geczy C.L. Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: Characterization of post-translational modifications and zinc binding.Biochem. J. 1996; 316: 285-293Crossref PubMed Scopus (71) Google Scholar), and the protein of mouse a at the of whereas no was observed for Nτ-methylhistidine observed in mouse S100A9 but it should be noted that the signal of Nπ-methylhistidine was with that of protein suggest the of a protein histidine that S100A9 in and to identify the using an siRNA screening this we a protein as a target for screening of its methylation than We siRNA the MTase on the to yeast and mouse in with siRNA with expression and proteins by the only METTL9 in a in the of Nπ-methylhistidine of S100A9, the of METTL9 gene expression and In of either SETD3 or a human of yeast no in the Nπ-methylhistidine of S100A9 In this our screening identified METTL9 as a novel protein histidine MTase that specifically catalyzes the Nπ-methylhistidine formation of S100A9 in METTL9 S100A9, we an in methylation by the of the group of recombinant S100A9. We the methylation signal of by only with and the signal was S100A9 we identified the METTL9 gene as the responsible for histidine Nπ-methylation of S100A9, we the of S100A9 methylation and mice. We found that mouse S100A9, to Nπ-methylhistidine residue (15Raftery M.J. Harrison C.A. Alewood P. Jones A. Geczy C.L. Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: Characterization of post-translational modifications and zinc binding.Biochem. J. 1996; 316: 285-293Crossref PubMed Scopus (71) Google Scholar), is a more than human whereas the of METTL9 are Thus, we the of mouse METTL9 in the methylation of mouse S100A9. we an of METTL9 by of and in the motif to as and that the to S100A9 mouse S100A9 has been to be methylated at His-107 (15Raftery M.J. Harrison C.A. Alewood P. Jones A. Geczy C.L. Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: Characterization of post-translational modifications and zinc binding.Biochem. J. 1996; 316: 285-293Crossref PubMed Scopus (71) Google Scholar), we His-107 of S100A9 with and that this METTL9-mediated methylation to the of we proteins from and in an in methylation In to S100A9, was not methylated by that S100A9 is a target of METTL9 evidence for histidine of METTL9 in we LC–MS/MS to recombinant S100A9 which with or in the of with the of the siRNA screening METTL9 Nπ-methylhistidine in S100A9 in our established METTL9 as a protein histidine that S100A9 at In addition to the MTase motif as METTL9 a signal that the protein to the of the I. Protein in the endoplasmic Biol. PubMed Scopus Google Scholar). We observed the of METTL9 by for a mouse METTL9 with a for the we found that was localized in the in both and and however, a the signal a but to that of the and that the of METTL9 occurs of its and to the functions of the Thus, we first the and in The that of the of METTL9 not to its binding to S100A9 the that the of METTL9 is not involved in the of S100A9, we the the S100A9 methylation in We METTL9 in and with either or the of mouse with expression LC–MS/MS analysis demonstrated that, the Nπ-methylhistidine of S100A9 was by METTL9 whereas of the with the and Nπ-methylhistidine to than observed in S100A9 from In that the of METTL9 does not to its and methylation of S100A9 in we to the functional significance of METTL9-mediated S100A9 methylation. S100A9 is known to a with the a by two A. M. A protein and 2018; PubMed Scopus Google Scholar), we first the that METTL9-mediated methylation may affect the formation of heterodimer in this with or siRNA and for using mouse We that siRNA of METTL9 did not affect the S100A9 and S100A8, a in the methylation of S100A9 and this we recombinant S100A9 by of by an in methylation with in the or the of and the binding affinity to In with the using we found that the was not by methylation we that Nπ-methylation of S100A9 at His-107 did not the of In addition to the involved in heterodimer and S100A9 are by a that of the motif A. M. A protein and 2018; PubMed Scopus Google Scholar, L. T. C. A. G. E. L. the of murine S100A9 protein by its Struct. Biol. 2021; PubMed Scopus Google Scholar). S100A9 two to the the second overlaps with the histidine residue that Nπ-methylation this to METTL9-mediated methylation of S100A9 its zinc-binding this we an the second motif as as a methylated of this at and zinc-binding using on acid with zinc. only one histidine Nπ-methylation of the S100A9 in a in zinc that METTL9-mediated methylation the zinc-binding of S100A9 In this study, we discovered a new histidine using siRNA screening by analysis of method is on the of protein and methyl amino including and are acid N at for LC–MS/MS analysis of the methylhistidine isomers to histidine methylation and histidine methylation of cellular there are no that specifically protein histidine methylation, our be a for the methylation of METTL9 as as screening novel protein histidine In of this et E. T. H. Liu T. A. J. L. T. M. et methyltransferase METTL9 modification in mammalian Commun. 2021; PubMed Scopus Google have first that METTL9 is a that introduces at is a in et M. D. Zhang L. C. Li S. Zhang P. L. X. Li C. A. Z. Zhang K. W. METTL9 methylation of zinc is required for Cell. 2021; Scopus Google also evidence for METTL9-mediated formation and that METTL9 an motif in the However, on METTL9 as one of the mammalian our to identify the enzymes responsible for histidine of S100A9 by using an unbiased siRNA screen this allows a novel MTase that catalyzes histidine methylation of such as and myosin chain (14Meyer H.E. Mayr G.W. N pi-methylhistidine in myosin-light-chain kinase.Biol. Chem. Hoppe Seyler. 1987; 368: 1607-1611Crossref PubMed Scopus (12) Google Scholar). we have successfully in in it be to an Nπ-methyltransferase from a MTase siRNA We observed that was localized in the in both and and it that of METTL9 was by a signal conserved of the did not its more than that of the and the and S100A9 in cells, it remains unclear the plays a functional role in we noted the of in the of an its of whereas no was observed the was that the is an in which occurs I. Protein in the endoplasmic Biol. PubMed Scopus Google Scholar), the may of The to be required for this this METTL9 the motif can be amino acid but I. Protein in the endoplasmic Biol. PubMed Scopus Google Scholar). is to the significance of the in the METTL9 in demonstrated that Nπ-methylation at His-107 of S100A9 in the of zinc binding that METTL9-mediated methylation the zinc-binding of S100A9. The also as is known to be in the of and plays a role in the two regulatory mechanisms zinc binding In antimicrobial of the human protein PubMed Scopus Google Scholar). The first is an of which exerts an antimicrobial activity, by chelation of essential for the growth of bacteria and A. A. of release of human Biol. Google Scholar, D. S100A9 of human by PubMed Scopus Google Scholar, D. of or in S100A9 and in the of role for Med. PubMed Scopus Google Scholar). The second is an of which have been to and of T. K. K. J. of and S100A9 in the and of PubMed Scopus Google Scholar). Thus, METTL9-mediated S100A9 methylation to be involved in the innate immune response to bacterial infection. In of this the from the that the expression of METTL9 as as S100A9 is in T. V. O. K. D. T. E. C. D. A. C. and in the of the human and mouse immune Sci. S. A. PubMed Scopus Google Scholar). the to no E. T. H. Liu T. A. J. L. T. M. et methyltransferase METTL9 modification in mammalian Commun. 2021; PubMed Scopus Google Scholar), more on the innate immune response light on the biological of We found that METTL9 catalyzes Nπ-methylhistidine formation in S100A9, whereas no Nτ-methylhistidine was with of S100A9, and SAM in In known histidine yeast and mouse SETD3 have been to catalyze only Nτ-methylhistidine formation in Rpl3 and respectively (13Webb K.J. Zurita-Lopez C.I. Al-Hadid Q. Laganowsky A. Young B.D. Lipson R.S. Souda P. Faull K.F. Whitelegge J.P. Clarke S.G. A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase.J. Biol. Chem. 2010; 285: 37598-37606Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, A.W. Diep J. Dai S. Liu S. Ooi Y.S. Song D. Li T.M. Horton J.R. Zhang X. Liu C. Trivedi D.V. Ruppel K.M. Vilches-Moure J.G. Casey K.M. Mak J. et al.SETD3 is an actin histidine methyltransferase that prevents primary dystocia.Nature. 2019; 565: 372-376Crossref PubMed Scopus (49) Google Scholar, 20Kwiatkowski S. Seliga A.K. Vertommen D. Terreri M. Ishikawa T. Grabowska I. Tiebe M. Teleman A.A. Jagielski A.K. Veiga-da-Cunha M. Drozak J. SETD3 protein is the actin-specific histidine N-methyltransferase.Elife. 2018; 7e37921Crossref PubMed Scopus (38) Google Scholar). to be to arginine methylation by the protein arginine methyltransferase type enzymes catalyze the of arginine, whereas type enzymes catalyze the of arginine K. Kim A. of biological methylation on 2019; PubMed Scopus Google Scholar). Given the it is that the in which the position of the nitrogen is methylated in residues has for the of protein of the mechanisms the and methylation of S100A9 by METTL9 are required to this of the METTL9 with that of SETD3 the protein histidine methylation phenomenon Q. S. S. W. H. G. X. J. Drozak J. C. histidine methylation on 2019; PubMed Scopus Google Scholar). response light on the biological of The biological of METTL9 has to be however, our a METTL9 and innate immune response to bacterial histidine methylation of S100A9. an of human and METTL9 with one that is by a Thus, understanding the of METTL9-mediated histidine methylation of S100A9 in to immune response the of new and for bacterial with from mouse or and with and mouse with or S100A9, and mouse and by using by and from from S100A9 either or Nπ-methylhistidine at His-107 The in this and affinity by using and in with and of and siRNA was using and to the The siRNA in the siRNA screening from and in and in and by an affinity In the the E. The bacteria in with the at and protein expression was with at for 3 After in with 1 on for the bacteria by in 1 and and proteins using was to in 1 and 1 and the with at for or in and with by with for 3 at After with the for three proteins with in for at and the at for 1 The to which a of and for and for at The by LC–MS/MS as using a the of histidine and as an the protein by or with of with N at for and the was in was a with a at A and as the of acid and was at using A and as to to from to to to from to and from to at of was by an in using After Nπ-methylhistidine and Nτ-methylhistidine with the first in the with and are of the of the methyl Nπ-methylhistidine and Nτ-methylhistidine with the The on the of histidine, and as and at in the with for and other are as of of of of of 3 of of of of and of and and with the for A of and with on the of using the for histidine and and in the of to The observed and of the in from the and at His-107 of was using the LC–MS/MS The from with and was an with an at A and acid and acid in was at using A and as to to from to to from to to to from to and from to at In the of and methylated peptides of predominantly observed in both of with and and the of and and The and the as the on the from the for and in the of to The observed and of the in from the In methylation with 3 or with 3 to proteins in the of 1 in After at for 2 by and with by in for The at for 1 and to at for 3 and and with using for with for at After with for three with in for at by with with and for 1 was with in with and for 2 at with with and for 1 at with in After with with with A as an and 1 as a for at The with an and with with METTL9 or METTL9 in and with and proteins with Protein with mouse was as After with the by by analysis using and and in the E. and using and S100A9 with of or METTL9 and for The with and in the with by with the or proteins on for 3 at After with the proteins by to a with and with the for was from using After the was to using and to analysis using on a The mRNA expression was on the The for analysis was as was as K. A. H. T. H. H. H. E. A. A for of by Commun. PubMed Scopus (12) Google Scholar). the with zinc at a of After the S100A9 peptides either or Nπ-methylhistidine at His-107 on the at and binding was to for 1 at by the After the was with The of both was by LC–MS/MS The three are this and in this are upon The that have no of with the of this We for the in We Kim at the of of for the with We the of for the We for H. K. and A. H. D. and K. K. M. T. and A. H. K. T. T. H. and T. M. T. T. H. and S. H. D. K. K. and A. and A. was by the for the of for to H. for to H. D. and to K. and for to A. from the of and and a from to A.

Topics & Concepts