Magnus E. Jakobsson

Lysine methylation of VCP by a member of a novel human protein methyltransferase family.

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 VCP-KMT 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.

Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation

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. Hsp70 proteins constitute an evolutionarily conserved protein family of ATP-dependent molecular chaperones involved in a wide range of biological processes. Mammalian Hsp70 proteins are subject to various post-translational modifications, including methylation, but for most of these, a functional role has not been attributed. In this study, we identified the methyltransferase METTL21A as the enzyme responsible for trimethylation of a conserved lysine residue found in several human Hsp70 (HSPA) proteins. This enzyme, denoted by us as HSPA lysine (K) methyltransferase (HSPA-KMT), was found to catalyze trimethylation of various Hsp70 family members both in vitro and in vivo, and the reaction was stimulated by ATP. Furthermore, we show that HSPA-KMT exclusively methylates 70-kDa proteins in mammalian protein extracts, demonstrating that it is a highly specific enzyme. Finally, we show that trimethylation of HSPA8 (Hsc70) has functional consequences, as it alters the affinity of the chaperone for both the monomeric and fibrillar forms of the Parkinson disease-associated protein α-synuclein.

Identification and characterization of a novel evolutionarily conserved lysine-specific methyltransferase targeting eukaryotic translation elongation factor 2 (eEF2).

Background: The function of many proteins is regulated through post-translational methylation. Results: The previously uncharacterized human methyltransferase FAM86A and its yeast homologue Yjr129c methylate eukaryotic translation elongation factor 2 (eEF2), altering translational frameshifting. Conclusion: Evolutionarily conserved FAM86A methyltransferase modulates the function of eEF2. Significance: The activity of a novel protein-modifying enzyme is discovered and is shown to have functional consequences. The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.

Saccharomyces cerevisiae Eukaryotic Elongation Factor 1A (eEF1A) Is Methylated at Lys-390 by a METTL21-Like Methyltransferase

The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024cΔ strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).

The novel lysine specific methyltransferase METTL21B affects mRNA translation through inducible and dynamic methylation of Lys-165 in human eukaryotic elongation factor 1 alpha (eEF1A)

Abstract 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.

Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

Abstract Many cellular proteins are methylated on lysine residues and this has been most intensively studied for histone proteins. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. Several recently discovered KMTs belong to the so-called seven-β-strand (7BS) class of MTases and we have here investigated an uncharacterized human 7BS MTase currently annotated as part of the endothelin converting enzyme 2, but which should be considered a separate enzyme. Combining in vitro enzymology and analyzes of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we show that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site and we demonstrate the importance of this motif for catalytic activity.

Hsp70 (HSPA1) Lysine Methylation Status as a Potential Prognostic Factor in Metastatic High-Grade Serous Carcinoma

Cellular proteins are subject to frequent methylation on lysine residues, introduced by specific methyltransferases, and each lysine residue can receive up to three methyl groups. Histone methylations, which are key determinants of chromatin state and transcriptional status, have been subject to particularly intense studies, but methylations on non-histone protein substrates are also abundant and biologically significant. Numerous studies have addressed lysine methylation in the realm of cancer biology. A recent study used an antibody-based approach to investigate the methylation of Lys-561 of the stress-inducible Hsp70 protein HSPA1, focusing exclusively on dimethylated HSPA1, concluding that it was elevated in cancer [Cho et al. (2012), Nat. Commun.,3, 1072]. In the present study, we have performed a more extensive analysis of HSPA1 methylation status in cancer samples, using protein mass spectrometry. We found that the four methylation states of Lys561 on HSPA1 (un-, mono-, di- and trimethylated) could be measured accurately and reproducibly in samples from carcinomas. We investigated HSPA1 methylation in 70 effusions, representing 53 high-grade serous ovarian carcinomas and 17 breast carcinomas. Notably, we found the trimethylated form of HSPA1 to be predominant in the cancer samples. HSPA1 methylation was studied for association with clinicopathologic parameters, including chemotherapy response and survival. The trimethylated form was more prevalent in breast carcinoma effusions (p = 0.014), whereas the dimethylated (p = 0.025), monomethylated (p = 0.004) and unmethylated (p = 0.021) forms were overrepresented in the ovarian carcinomas. For the ovarian carcinomas, the monomethylated (p = 0.028) and unmethylated (p = 0.007) forms were significantly related to the presence of higher residual disease volume, while the unmethylated form was significantly associated with poor overall (p = 0.015) and progression-free (p = 0.012) survival. In conclusion, lysine methylation of HSPA1 differs between metastatic breast and ovarian carcinoma, and unmethylated HSPA1 shows potential as a prognostic marker in high-grade serous carcinoma.

Correspondence: On the enzymology and significance of HSPA1 lysine methylation

Cho et al.1 recently reported that the methyltransferase SETD1A catalyses dimethylation of the stress-inducible Hsp70 protein HSPA1 at lysine 561 (HSPA1-K561me2), while we2 and others3 have later reported that a different enzyme, METTL21A, methylates HSPA1-K561 in vitro and in vivo. Moreover, Cho et al.1 reported that HSPA1-K561me2 specifically localizes to the cell nucleus, where it activates Aurora kinase B, and that this modification is increased in various cancers. Here, we present data that conflict with the conclusions drawn by Cho et al.1, indicating that SETD1A does not play a direct role in HSPA1-K561 methylation and that HSPA1-K561me2 is not specifically localized to the nucleus. To further investigate the roles of human METTL21A and SETD1A in HSPA1-K561 methylation in vivo, the HSPA1-K561 methylation status in a METTL21A knockout cell line was assessed by mass spectrometry. HSPA1-K561 was found to be mostly trimethylated (HSPA1-K561me3) in wild-type KBM-7 cells (Fig. 1a), a methylation pattern similar to what we previously observed in human cell lines and cancer samples2,4. In contrast, HSPA-K561 was exclusively unmethylated in the corresponding METT21A knockout cells, which expressed SETD1A (Fig. 1a). This shows that METTL21A is required for HSPA1-K561 methylation, and suggests no involvement of SETD1A. However, Cho et al.1 suggested that SETD1A specifically mediates the formation of the dimethylated species, HSPA1-K561me2, and previous in vitro experiments, using high enzyme concentrations, have only demonstrated METTL21A-mediated formation of HSPA1-K561me3. Thus, the possibility existed that METTL21A is a processive enzyme responsible only for the formation of HSPA1-K561me3, whereas SETD1A is involved in introducing lower methylation states, such as HSPA1-K561me2. To further explore this, we incubated recombinant HSPA1 with varying amounts of METTL21A in vitro, and, subsequently determined the HSPA1-K561 methylation status by protein mass spectrometry. Clearly, HSPA1 incubated with intermediate amounts of METTL21A displayed a mixture of the three methylated forms (me1, me2 and me3), demonstrating that METTL21A is a non-processive enzyme capable of generating all methylation states on HSPA1-K561 (Fig. 1b). Cho et al.1 observed alterations in HSPA1 methylation upon modulating SETD1A levels by knockdown and overexpression. However, as SETD1A is already established as a histone methyltransferase that mono-, di- and trimethylates lysine 4 in histone H3 (ref. 5), and thereby regulates gene expression6, the likely possibility exists that the observed effects were indirect and not reflecting SETD1A-mediated HSPA1 methylation. Moreover, Cho et al.1 presented no biochemical evidence that SETD1A can catalyse HSPA1-K561 methylation in vitro. Therefore, the above data, taken together with the published literature2,3 on METTL21A-mediated HSPA1 methylation, strongly indicates that METTL21A is the sole enzyme responsible for methylation of HSPA1-K561. Figure 1 METTL21A-mediated methylation of HSPA1-K561. Cho et al.1 reported that HSPA1-K561me2 localizes predominantly to the nucleus of cancer cells. Using subcellular fractionation in combination with protein mass spectrometry, we have here further investigated the reported methylation-dependent nuclear localization of HSPA1. We observed, in agreement with Cho et al.1 and the published literature7, HSPA1 in both the cytosolic and nuclear fractions (Fig. 2). Notably, we found, using two different cell lines, HeLa and HEK-293, the HSPA1-K561 methylation pattern to be indistinguishable between the nuclear and cytosolic fractions, with HSPA1-K561me3 as the predominant form (Fig. 2). The nuclear accumulation of HSPA1 reported by Cho et al.1 was entirely based on nuclear immunostaining observed with a HSPA1-K561me2-specific antibody, and the above results suggest to us that this antibody, despite apparently recognizing HSPA1-K561me2 in western blotting, recognizes a nuclear protein other than HSPA1 when used for immunofluorescence and imaging. Cho et al.1 further reported that HSPA1-K561me2, compared to unmethylated HSPA1, was a stronger activator of Aurora kinase B, thereby linking HSPA1-K561me2 to cell proliferation. However, we question these conclusions, since they largely rely upon the assumptions that HSPA1-K561 is dimethylated in the nucleus and unmethylated in the cytoplasm, and that differences between wild-type HSPA1 and a K561R mutant with respect to Aurora kinase B binding and activation reflect HSPA1-K561me2-dependent effects. In our opinion, these reported effects are likely caused by the mutation of a highly conserved residue (the residue corresponding to K561 is conserved in most eukaryotic and prokaryotic Hsp70 homologues), and not related to HSPA1 methylation. Figure 2 Methylation status of HSPA1-K561 in subcellular fractions. In summary, we have here presented data and arguments that put into question the role of SETD1A in mediating the methylation of HSPA1 and question whether HSPA1-K561me2 is specifically localized to the nucleus.

Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

Lysine methylation is an important and much-studied posttranslational modification of nuclear and cytosolic proteins but is present also in mitochondria. However, the responsible mitochondrial lysine-specific methyltransferases (KMTs) remain largely elusive. Here, we investigated METTL12, a mitochondrial human S-adenosylmethionine (AdoMet)-dependent methyltransferase and found it to methylate a single protein in mitochondrial extracts, identified as citrate synthase (CS). Using several in vitro and in vivo approaches, we demonstrated that METTL12 methylates CS on Lys-395, which is localized in the CS active site. Interestingly, the METTL12-mediated methylation inhibited CS activity and was blocked by the CS substrate oxaloacetate. Moreover, METTL12 was strongly inhibited by the reaction product S-adenosylhomocysteine (AdoHcy). In summary, we have uncovered a novel human mitochondrial KMT that introduces a methyl modification into a metabolic enzyme and whose activity can be modulated by metabolic cues. Based on the established naming nomenclature for similar enzymes, we suggest that METTL12 be renamed CS-KMT (gene name CSKMT).

Regulation of eukaryotic elongation factor 1 alpha (eEF1A) by dynamic lysine methylation

ABSTRACT Lysine methylation is a frequent post-translational protein modification, which has been intensively studied in the case of histone proteins. Lysine methylations are also found on many non-histone proteins, and one prominent example is eukaryotic elongation factor 1 alpha (eEF1A). Besides its essential role in the protein synthesis machinery, a number of non-canonical functions have also been described for eEF1A, such as regulation of the actin cytoskeleton and the promotion of viral replication. The functional significance of the extensive lysine methylations on eEF1A, as well as the identity of the responsible lysine methyltransferases (KMTs), have until recently remained largely elusive. However, recent discoveries and characterizations of human eEF1A-specific KMTs indicate that lysine methylation of eEF1A can be dynamic and inducible, and modulates mRNA translation in a codon-specific fashion. Here, we give a general overview of eEF1A lysine methylation and discuss its possible functional and regulatory significance, with particular emphasis on newly discovered human KMTs.