A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity

Abstract

Methylation is a post-translational modification that can affect numerous features of proteins, notably cellular localization, turnover, activity, and molecular interactions. Recent genome-wide analyses have considerably extended the list of human genes encoding putative methyltransferases. Studies on protein methyltransferases have revealed that the regulatory function of methylation is not limited to epigenetics, with many non-histone substrates now being discovered. We present here our findings on a novel family of distantly related putative methyltransferases. Affinity purification coupled to mass spectrometry shows a marked preference for these proteins to associate with various chaperones. Based on the spectral data, we were able to identify methylation sites in substrates, notably trimethylation of K135 of KIN/Kin17, K561 of HSPA8/Hsc70 as well as corresponding lysine residues in other Hsp70 isoforms, and K315 of VCP/p97. All modification sites were subsequently confirmed in vitro. In the case of VCP, methylation by METTL21D was stimulated by the addition of the UBX cofactor ASPSCR1, which we show directly interacts with the methyltransferase. This stimulatory effect was lost when we used VCP mutants (R155H, R159G, and R191Q) known to cause Inclusion Body Myopathy with Paget's disease of bone and Fronto-temporal Dementia (IBMPFD) and/or familial Amyotrophic Lateral Sclerosis (ALS). Lysine 315 falls in proximity to the Walker B motif of VCP's first ATPase/D1 domain. Our results indicate that methylation of this site negatively impacts its ATPase activity. Overall, this report uncovers a new role for protein methylation as a regulatory pathway for molecular chaperones and defines a novel regulatory mechanism for the chaperone VCP, whose deregulation is causative of degenerative neuromuscular diseases.

Figure 1. Computational analysis defines a novel family of putative protein methyltransferases.

(A) Unrooted phylogenetic tree of a family of human putative methyltransferases distantly related to PRMTs. FAM86 represents a number of genetic variants (FAM86A, B1, B2, C, and D) whose duplication is observed only in primates. Branch lengths are not proportional to the actual evolutionary distances between the sequences. (B) Secondary structure organization of the Rossmann fold domain of PRMTs responsible for the methyltransferase activity. Arrows represent β strands and rectangles correspond to α helices, including typically ill-defined or inexistent α helix C. (C) Multiple sequence alignment of the Rossmann fold of all members within this family as generated by ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Red residues are small, hydrophobic, aromatic; blue are acidic; magenta are basic; and all other residues are green. Primary sequence alignment corresponds nicely with secondary structure prediction by Jpred3 (http://www.compbio.dundee.ac.uk/www-jpred/). Overhead β strands and α helices are shown as in (B). Conserved motif I, the site of S-adenosylmethionine binding, is also marked.

Figure 2. Tandem-affinity purification coupled to mass spectrometry reveals protein interaction network of putative methyltransferases.

Purification of 10 TAP-tagged putative methyltransferases from ponasterone-inducible strains of HEK 293 cells. Eluted proteins were separated by SDS-PAGE. Gels were silver stained and cut in slices that were then trypsin digested before protein identification by LC-MS/MS. Tagged baits and major interactors are marked.

Figure 3. KIN, VCP, and a number of hsp70 isoforms are each trimethylated on lysine residues by specific methyltransferases within this family.

(A) Linear representation of all identified substrates with domain architecture. Residues delineating each domain are marked below. ZnF, Zinc Finger; WH, Winged Helix; SH3, Src Homology 3; SBD, substrate binding domain. Position of the methylated lysines is shown above. (B) Multiple sequence alignment of the region surrounding trimethylated lysines (boxed) in human VCP, KIN, and Hsp70 isoforms compared to paralogous genes in various organisms. Hs, Homo sapiens; Sc, Saccharomyces cerevisiae; Dm, Drosophila melanogaster; At, Arabidopsis thaliana; Pf, Plasmodium falciparum. Color code is as in Figure 1. Strong and weak residue similarity are represented by a colon (:) and period (.), respectively, and asterisk (*) denotes identity. (C–E) In vitro methylation assays with tritium-labeled S-adenosylmethionine of KIN-His with GST-METTL22 (C), VCP-His with GST-METTL21D (D), and three His-tagged hsp70 isoforms (HSPA1, HSPA5, and HSPA8) with GST-METTL21A (E). In each case, substitution of the methylated lysine by an arginine leads to loss of methylation signal as detected by autoradiography. Coomassie staining of the gel shows total proteins loaded onto the gel and serves as control.

Figure 4. Intracellular distribution of putative methyltransferases and colocalization with identified substrates as shown by immunofluorescence.

(A) HeLa cells were transiently transfected with vectors encoding C-terminally FLAG-tagged putative methyltransferases. Localization of the recombinant proteins is revealed by immunofluorescence with a Cy3-labelled antibody. DNA is stained with To-Pro-3 to determine the position of the nucleus, and overall morphology of the cell is shown by Differential Interference Contrast microscopy (DIC). (B) Comparative localization of methyltransferase with associated proteins is shown by transient cotransfection of vectors for the expression of FLAG-tagged methyltransferase and C-terminally GFP-tagged interactors.

Figure 5. ASPSCR1 promotes methylation of VCP by METTL21D.

(A) In vitro methylation assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Various combinations of the UBX proteins were added to reactions containing VCP. Coomassie staining of the gel is shown. (B) Linear representation of ASPSCR1 showing domain architecture of the protein (UBL, UBiquitin-Like domain; SHP, SHP box; UBX, UBiquitin regulatory X domain; CC, Coiled-Coil domain) and localization of residue 280 which marks the boundary between the N- and C- terminal fragments used in these experiments. (C) In vitro GST pull-down assays of VCP-His, UBXN6-His, ASPSCR1-His and fragments of ASPSCR1 with GST-METTL21D. Combinations of full-length ASPSCR1 and its fragments were once again employed with VCP. (D) Linear representation of VCP showing domain architecture of the protein (including double Ψ barrel superfold and 4-stranded β barrel of the N-terminal domain, Walker A and B motifs, as well as 4 α helices bundle of ATPase domains D1 and D2 as well as linker regions L1 and L2 and C-terminal domain) and localization of the mutants used in this study as well as the site of methylation. (E) In vitro methylation assays of wild-type VCP-His and IBMPFD and ALS-causing mutations R155H, R159G and R191Q in presence or absence of ASPSCR1-His. (F) In vitro GST pull-down assays of the same combination of proteins as in (E).

Figure 6. Methylation of VCP decreases the activity of its N-terminal ATPase domain.

(A) Linear representation of a fragment of VCP encompassing its N-terminal and first ATPase domain employed in the ATPase assay. Proximity of the methylated lysine to the Walker B motif is highlighted above. (B) In vitro methylation assays of 1–481_VCP-His fragment by GST-METTL21D as compared to full length VCP. (C, D) Colorimetric assays to measure released phosphate (C) and relative ATPase activity (D) by the 1–481 fragment of VCP. The experiment was done in triplicate. Data from the last 3 time points (9 measurements in total for each condition) was compiled to generate the graph shown in (D). (E) In vitro GST pull-down assay of VCP-His with GST-METTL21D. In all experiments the effect of un methylatable VCP mutant K135R, catalytically inactive METTL21D mutant E73Q, and methylation inhibitor S-adenosylhomocysteine is shown.