Valosin-containing protein (VCP, also called p97) is an essential and highly conserved adenosine triphosphate-dependent chaperone implicated in a wide range of cellular processes in eukaryotes, and mild VCP mutations can cause severe neurodegenerative disease. Here we show that mammalian VCP is trimethylated on Lys315 in a variety of cell lines and tissues, and that the previously uncharacterized protein mETTL21D (denoted here as VCP lysine methyltransferase, VCP-KmT) is the responsible enzyme. VCP methylation was abolished in three human VCPKmT knockout cell lines generated with zinc-finger nucleases. Interestingly, VCP-KmT was recently reported to promote tumour metastasis, and indeed, VCP-KmT-deficient cells displayed reduced growth rate, migration and invasive potential. Finally, we present data indicating that VCP-KmT, calmodulin-lysine methyltransferase and eight uncharacterized proteins together constitute a novel human protein methyltransferase family. The present work provides new insights on protein methylation and its links to human disease.

Background: The function of many proteins is regulated through post-translational methylation. Results: METTL21A was identified as a human protein methyltransferase targeting Hsp70 proteins, thereby altering their ability to interact with client proteins. Conclusion: METTL21A is a specific methyltransferase modulating the function of Hsp70 proteins. Significance: The activity of a human protein-modifying enzyme is unraveled, and the modification is demonstrated to have functional consequences.

Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where “x” is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.

Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many non-histone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1A methylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B-mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosomal profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.

Background: Many proteins are modified by lysine methylation. Results: It is shown that the previously uncharacterized enzyme METTL20 methylates electron transfer flavoprotein ␤ (ETF␤), thereby inhibiting its ability to mediate electron transfer from acyl-CoA dehydrogenases. Conclusion: METTL20-mediated methylation modulates the function of ETF␤. Significance: The first mitochondrial lysine-specific protein methyltransferase in animals is reported, and the resulting methylation is shown to have functional consequences.