The Legionella pneumophila Methyltransferase RomA Methylates Also Non-histone Proteins during Infection

J Mol Biol. 2018 Jun 22;430(13):1912-1925. doi: 10.1016/j.jmb.2018.04.032. Epub 2018 May 4.

Abstract

RomA is a SET-domain containing protein lysine methyltransferase encoded by the Gram-negative bacterium Legionella pneumophila. It is exported into human host cells during infection and has been previously shown to methylate histone H3 at lysine 14 [Rolando et al. (2013), Cell Host Microbe, 13, 395-405]. Here, we investigated the substrate specificity of RomA on peptide arrays showing that it mainly recognizes a G-K-X-(PA) sequence embedded in a basic amino acid sequence context. Based on the specificity profile, we searched for possible additional RomA substrates in the human proteome and identified 34 novel peptide substrates. For nine of these, the corresponding full-length protein or protein domains could be cloned and purified. Using radioactive and antibody-based methylation assays, we showed that seven of them are methylated by RomA, four of them strongly, one moderately, and two weakly. Mutagenesis confirmed for the seven methylated proteins that methylation occurs at target lysine residues fitting to the specificity profile. Methylation of one novel substrate (AROS) was investigated in HEK293 cells overexpressing RomA and during infection with L. pneumophila. Methylation could be detected in both conditions, confirming that RomA methylates non-histone proteins in human cells. Our data show that the bacterial methyltransferase RomA methylates also human non-histone proteins suggesting a multifaceted role in the infection process.

Keywords: Legionella pneumophila; enzyme specificity; non-histone substrate; protein lysine methyltransferase; protein methylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism
  • Cloning, Molecular
  • HEK293 Cells
  • HeLa Cells
  • Histone-Lysine N-Methyltransferase / metabolism*
  • Humans
  • Legionella pneumophila / enzymology
  • Legionella pneumophila / pathogenicity*
  • Legionnaires' Disease / metabolism*
  • Lysine / metabolism
  • Methylation
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Peptides / analysis*
  • Peptides / chemistry
  • Peptides / genetics
  • Protein Processing, Post-Translational
  • Substrate Specificity
  • Transcription Factors / chemistry
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*

Substances

  • Bacterial Proteins
  • Nuclear Proteins
  • Peptides
  • RPS19BP1 protein, human
  • Transcription Factors
  • Histone-Lysine N-Methyltransferase
  • Lysine