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bioRxiv · 10.1101/266882

SETD3 protein is the actin-specific histidine N-methyltransferase

Kwiatkowski, S.·Seliga, A. K.·Veiga-da-Cunha, M.·Vertommen, D.·Terreri, M.·Ishikawa, T.·Grabowska, I.·Jagielski, A. K.·Drozak, J.
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Abstract

Protein histidine methylation is rarely studied posttranslational modification of unknown biochemical importance. In vertebrates, only a few methylhistidne-containing proteins have been reported so far, including {beta}-actin as an essential example. The evolutionary conserved methylation of {beta}-actin H73 residue is catalyzed by a specific histidine N-methyltransferase that has never been identified molecularly. In the present investigation, we have purified actin-specific histidine N-methyltransferase from rat muscles about 1200-fold. Its activity was studied by the radiochemical assay employing either homogeneous recombinant human {beta}-actin produced in E. coli or its mutated form exhibiting substitution of H73 by Ala residue (H73A) as substrates. Three polypeptides of {approx}65, 75 and 90 kDa coeluting with the enzyme activity were identified in the preparation. Mass spectrometry analysis of these polypeptides resulted in the identification of SETD3 methyltransferase as the only plausible candidate. Rat SETD3 and its human ortholog were expressed in COS-7 cells, purified to homogeneity and shown to catalyze methylation of {beta}-actin at H73 residue as confirmed by mass spectrometry analysis. The SETD3 enzyme was active towards a synthetic peptide corresponding to residues 69-77 of {beta}-actin, but not to its mutated form exhibiting His-to-Ala substitution. Finally, Setd3-deficient HAP1 cells were devoid of methylated H73 in {beta}-actin and exhibited phenotypic changes, including a decrease in F-actin content and an increased glycolytic activity. We conclude that SETD3 is the actin-specific histidine N-methyltransferase. The data show for the first time the molecular identity of protein histidine N-methyltransferase in vertebrates and throw new light on the substrate specificity of SET-domain-containing enzymes.

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Kwiatkowski, S., Seliga, A. K., Veiga-da-Cunha, M., Vertommen, D., Terreri, M., Ishikawa, T., Grabowska, I., Jagielski, A. K., Drozak, J.. 2018-02-20. SETD3 protein is the actin-specific histidine N-methyltransferase. https://doi.org/10.1101/266882

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