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• W2085450186 abstract "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.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. 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. Many cellular proteins are post-translationally methylated. These modifications are introduced by protein methyltransferases (MTases), 3The abbreviations used are: MTasemethyltransferase7BSseven-beta strandSAMS-adenosylmethionineVCPvalosin-containing proteinNBDnucleotide-binding domainα-Synα-synucleinCBPcalmodulin-binding peptideSBPstreptavidin-binding peptideTAPtandem affinity purificationBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. usually on amine groups on the side chains of lysine and arginine residues (1.Clarke S.G. Protein methylation at the surface and buried deep: thinking outside the histone box.Trends Biochem. Sci. 2013; 38: 243-252Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The functional role of protein methylation has been most thoroughly studied in the case of histones, where methylation is described as a key element in the “histone code,” which is a strong determinant of chromatin state and gene transcription status (2.Greer E.L. Shi Y. Histone methylation: a dynamic mark in health, disease and inheritance.Nat. Rev. Genet. 2012; 13: 343-357Crossref PubMed Scopus (1425) Google Scholar). Lysine can accept up to three methyl groups, and many of the so-called “readers” of the histone code represent protein domains that specifically interact with a lysine of a certain methylation state, leading to the recruitment of transcriptional modulators and/or chromatin-modifying enzymes (3.Margueron R. Trojer P. Reinberg D. The key to development: interpreting the histone code?.Curr. Opin. Genet. Dev. 2005; 15: 163-176Crossref PubMed Scopus (603) Google Scholar, 4.Taverna S.D. Li H. Ruthenburg A.J. Allis C.D. Patel D.J. How chromatin-binding modules interpret histone modifications: lessons from professional pocket pickers.Nat. Struct. Mol. Biol. 2007; 14: 1025-1040Crossref PubMed Scopus (1169) Google Scholar). In addition to the frequent lysine methylations on histone proteins, many non-histone proteins are also subject to such methylation (1.Clarke S.G. Protein methylation at the surface and buried deep: thinking outside the histone box.Trends Biochem. Sci. 2013; 38: 243-252Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). methyltransferase seven-beta strand S-adenosylmethionine valosin-containing protein nucleotide-binding domain α-synuclein calmodulin-binding peptide streptavidin-binding peptide tandem affinity purification 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. Besides methylating proteins, MTases are responsible for methylation of a wide variety of cellular macromolecules and metabolites (5.Schubert H.L. Blumenthal R.M. Cheng X. Many paths to methyltransfer: a chronicle of convergence.Trends Biochem. Sci. 2003; 28: 329-335Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 6.Bestor T.H. The DNA methyltransferases of mammals.Hum. Mol. Genet. 2000; 9: 2395-2402Crossref PubMed Scopus (1597) Google Scholar). Bioinformatics analysis has predicted that MTases constitute ∼1% of all human protein-coding genes, and most of the >200 corresponding enzymes remain uncharacterized (7.Petrossian T.C. Clarke S.G. Uncovering the human methyltransferasome.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M110.000976)Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). MTases are subdivided into five different classes based on their three-dimensional structure. Of these, the “seven-beta strand” (7BS) MTases are the most abundant, comprising 126 enzymes in humans (7.Petrossian T.C. Clarke S.G. Uncovering the human methyltransferasome.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M110.000976)Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). The 7BS-MTases adopt a characteristic Rossmann fold-like twisted β-sheet and contain conserved sequence motifs involved in the binding of the methyl donor S-adenosylmethionine (SAM), facilitating their identification by sequence homology searches (8.Katz J.E. Dlakić M. Clarke S. Automated identification of putative methyltransferases from genomic open reading frames.Mol. Cell. Proteomics. 2003; 2: 525-540Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The second largest class of MTases is constituted by the SET domain proteins, which are lysine-specific protein MTases responsible for methylations on the histone tails, as well as several methylations on non-histone substrates (9.Del Rizzo P.A. Trievel R.C. Substrate and product specificities of SET domain methyltransferases.Epigenetics. 2011; 6: 1059-1067Crossref PubMed Scopus (98) Google Scholar). However, some 7BS-MTases are also lysine-specific, and so far, three such human enzymes have been characterized. These are the histone-specific MTase DOT1L, which methylates Lys-79 in the globular part of histone H3; CaM-KMT, which trimethylates Lys-115 in calmodulin; and VCP-KMT, which trimethylates Lys-315 in the molecular chaperone valosin-containing protein (VCP) (see below) (10.Feng Q. Wang H. Ng H.H. Erdjument-Bromage H. Tempst P. Struhl K. Zhang Y. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain.Curr. Biol. 2002; 12: 1052-1058Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 11.Magnani R. Dirk L.M. Trievel R.C. Houtz R.L. Calmodulin methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin.Nat. Commun. 2010; 1: 43Crossref PubMed Scopus (60) Google Scholar, 12.Kernstock S. Davydova E. Jakobsson M. Moen A. Pettersen S. Mælandsmo G.M. Egge-Jacobsen W. Falnes P.Ø. Lysine methylation of VCP by a member of a novel human protein methyltransferase family.Nat. Commun. 2012; 3: 1038Crossref PubMed Scopus (91) Google Scholar). Hsp70 (heat shock protein of ∼70 kDa) proteins represent ATP-dependent molecular chaperones present in all three domains of life and constitute one of the most evolutionarily conserved protein families (13.Hageman J. van Waarde M.A. Zylicz A. Walerych D. Kampinga H.H. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.Biochem. J. 2011; 435: 127-142Crossref PubMed Scopus (125) Google Scholar). Humans have 13 genes encoding Hsp70s (HSPA proteins), and whereas some of these proteins are induced by heat shock and other types of stress, others are constitutively expressed (14.Kampinga H.H. Hageman J. Vos M.J. Kubota H. Tanguay R.M. Bruford E.A. Cheetham M.E. Chen B. Hightower L.E. Guidelines for the nomenclature of the human heat shock proteins.Cell Stress Chaperones. 2009; 14: 105-111Crossref PubMed Scopus (878) Google Scholar). Although Hsp70s have been extensively studied, several remain largely uncharacterized. Some human Hsp70s are associated with specific subcellular compartments, such as the endoplasmic reticulum resident HSPA5 (BiP/Grp78), mitochondrial HSPA9 (mortalin), cytosolic constitutively expressed HSPA8 (Hsc70), and cytosolic/nuclear classical stress-inducible HSPA1 (Hsp70). Other members are testis-specific HSPA2 and cytosolic HSPA6, which has been reported to chaperone p53 (13.Hageman J. van Waarde M.A. Zylicz A. Walerych D. Kampinga H.H. The diverse members of the mammalian HSP70 machine show distinct chaperone-like activities.Biochem. J. 2011; 435: 127-142Crossref PubMed Scopus (125) Google Scholar). Hsp70s share a domain architecture consisting of an N-terminal nucleotide-binding domain (NBD), followed by a substrate-binding domain, which can be further subdivided into a peptide-binding domain, which binds stretches of hydrophobic residues, and a C-terminal “lid” domain, which can fold back onto the peptide-binding domain and lock client proteins to Hsp70 (15.Hartl F.U. Bracher A. Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis.Nature. 2011; 475: 324-332Crossref PubMed Scopus (2190) Google Scholar). This process, and thus the affinity for unfolded substrates and the binding/release cycles of the chaperone, is allosterically regulated by ATP hydrolysis catalyzed by the NBD, which in turn is stimulated by co-chaperones from the Hsp40/DNAJ family and by nucleotide exchange factors. Together, these proteins are often referred to as the “Hsp70 chaperone machinery” (16.Kampinga H.H. Craig E.A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity.Nat. Rev. Mol. Cell Biol. 2010; 11: 579-592Crossref PubMed Scopus (1146) Google Scholar). In a recent study (12.Kernstock S. Davydova E. Jakobsson M. Moen A. Pettersen S. Mælandsmo G.M. Egge-Jacobsen W. Falnes P.Ø. Lysine methylation of VCP by a member of a novel human protein methyltransferase family.Nat. Commun. 2012; 3: 1038Crossref PubMed Scopus (91) Google Scholar), we reported that the previously uncharacterized human putative MTase METTL21D was responsible for trimethylating Lys-315 in the abundant and essential molecular chaperone VCP, and we denoted this enzyme VCP-KMT. Moreover, our results indicated that VCP-KMT and its close relatives METLL21A, METTL21B, and METTL21C, together with calmodulin (CaM-KMT) and five uncharacterized MTases, constitute a family of human protein MTases (12.Kernstock S. Davydova E. Jakobsson M. Moen A. Pettersen S. Mælandsmo G.M. Egge-Jacobsen W. Falnes P.Ø. Lysine methylation of VCP by a member of a novel human protein methyltransferase family.Nat. Commun. 2012; 3: 1038Crossref PubMed Scopus (91) Google Scholar). In this work, we report the function of METTL21A and show by a combination of enzymatic and cellular studies that it catalyzes the trimethylation of a conserved Lys residue found in the lid domain of several human Hsp70s. Moreover, we show that trimethylation of HSPA8 modulates its chaperone activity, manifested as a reduced ability to sequester the Parkinson disease-associated protein α-synuclein (α-Syn) in a soluble state and to bind to preformed α-Syn fibrils. NCBI BLAST was used for identification of proteins homologous to human Hsp70s and METTL21A (17.McGinnis S. Madden T.L. BLAST: at the core of a powerful and diverse set of sequence analysis tools.Nucleic Acids Res. 2004; 32: W20-W25Crossref PubMed Scopus (1217) Google Scholar). Multiple protein sequence alignments were performed using the Muscle algorithm embedded in Jalview v2.8 (18.Waterhouse A.M. Procter J.B. Martin D.M. Clamp M. Barton G.J. Jalview Version 2–a multiple sequence alignment editor and analysis workbench.Bioinformatics. 2009; 25: 1189-1191Crossref PubMed Scopus (5690) Google Scholar). The secondary structure of proteins was predicted from the primary sequence using the PSIPRED algorithm (19.McGuffin L.J. Bryson K. Jones D.T. The PSIPRED protein structure prediction server.Bioinformatics. 2000; 16: 404-405Crossref PubMed Scopus (2726) Google Scholar). All plasmids used in this study and the detailed cloning strategy used to generate them are listed in supplemental Table S1. Briefly, ORFs were amplified with Phusion DNA polymerase (Thermo Scientific) using primers that generated PCR products with restriction sites flanking the ORF. The PCR product was then digested with the indicated restriction enzymes (New England Biolabs) and cloned into pNTAPa (Agilent Technologies), pGEX-6P (GE Healthcare), or pET28a (Novagen). To generate the pcDNA5/FRT/TO-METL21A-CBP-SBP construct, METTL21A was first cloned into pNTAPa and then subcloned into pcDNA5/FRT/TO (Invitrogen) using ApaI and NotI. Mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The identity of all constructs was verified by DNA sequencing. A cell line for inducible overexpression of calmodulin-binding peptide (CBP)- and streptavidin-binding peptide (SBP)-tagged METTL21A was generated using the Flp-InTM T-RExTM HEK-293 system (Invitrogen) according to the manufacturer's instructions. Briefly, Flp-In T-REx HEK-293 cells were cotransfected with pcDNA5/FRT/TO-METTL21A-CBP-SBP and pOG44 (Invitrogen) using Lipofectamine® 2000 (Invitrogen). Successful incorporation of the METTL21A construct was selected for with 200 μg/ml hygromycin B (Invitrogen). Cells were thereafter maintained in high-glucose DMEM (Lonza) supplemented with tetracycline-reduced 10% (v/v) FBS (AH diagnostics), l-glutamine (Lonza), 5 μg/ml blasticidin S (Invitrogen), 10 μg/ml streptomycin (Invitrogen), and 10 units/ml penicillin (Invitrogen). Tandem affinity purification (TAP) was performed using the InterPlay mammalian TAP system (Agilent) according to the manufacturer's instructions with a few exceptions. Briefly, the Flp-In T-REx HEK-293 METTL21A-CBP-SBP cell line was expanded onto 20 T175 flasks. Overexpression of the bait was induced in 10 flasks with 1 μg/ml doxycycline (Sigma-Aldrich D9891) for 24 h, whereas the remaining flasks were untreated to serve as a negative control. As our prior experience indicated that S-adenosyl-l-homocysteine may stabilize 7BS-MTase·substrate complexes, all buffers were supplemented with 100 μm S-adenosyl-l-homocysteine, and 1 mm PMSF (Sigma-Aldrich) and protease inhibitor mixture (Sigma-Aldrich) were added to promote protein stability throughout the purification. Proteins were eluted by boiling in 2× NuPAGE gel loading buffer (Invitrogen) and stored at −20 °C until analyzed. pET28a- and pGEX-6P-derived plasmids harboring recombinant His6- and GST-tagged proteins, respectively, were transfected into the Escherichia coli BL21-CodonPlus (DE3)-RIPL expression strain (Agilent). Cells were cultured with appropriate antibiotics at 37 °C and shaking at 250 rpm until the absorbance at 600 nm reached 0.5. The temperature was then reduced to 18 °C, and the expression of His6- or GST-tagged proteins was induced by 100 or 500 μm isopropyl β-d-thiogalactopyranoside, respectively, and allowed to proceed for 16 h. Cells were thereafter harvested by centrifugation. For purification of His6-tagged proteins, cell pellets were resuspended in lysis buffer (50 mm Tris (pH 8.0), 500 mm NaCl, 10% (w/v) glycerol, 0.5% (w/v) Nonidet P-40, 30 mm imidazole, 3 mm 2-mercaptoethanol, Complete protease inhibitor mixture (Roche Applied Science), 0.5 mg/ml lysozyme (Sigma-Aldrich), and 25 units/ml Benzonase (Sigma-Aldrich)). Imidazole was omitted for GST-tagged constructs. Cell lysates were cleared by centrifugation, and recombinant proteins were allowed to bind to nickel-nitrilotriacetic acid-agarose (His6-tagged proteins; Qiagen) or glutathione-Sepharose 4B (GST-tagged proteins; GE Healthcare) at 4 °C for 16 h. Glutathione-Sepharose 4B affinity resins were extensively washed with wash buffer (50 mm Tris (pH 7.5), 500 mm NaCl, and 10% (w/v) glycerol), whereas nickel-nitrilotriacetic acid-agarose resins were washed with wash buffer supplemented with 30 mm imidazole. Recombinant proteins were eluted in elution buffer (50 mm Tris-HCl (pH 8.0) and 500 mm NaCl) supplemented with either 300 mm imidazole (His6-tagged proteins) or 15 mm reduced glutathione (GST-tagged proteins). Eluates were then buffer-exchanged to storage buffer (100 mm NaCl, 20 mm Tris (pH 6.8), and 1 mm DTT) by sequential concentration and dilution using Vivaspin 20 ultrafiltration columns with a molecular mass cutoff of 10 or 50 kDa (Sartorius AG). Proteins were aliquoted and stored at −80 °C, and concentrations were determined using the BCA protein assay kit (Pierce). For tag removal, GST-fused HSPA proteins were incubated with PreScission protease (GE Healthcare) at 4 °C for 16 h to allow proteolytic cleavage. Free GST, GST-fused HSPA proteins, and protease were removed with glutathione-Sepharose according to the manufacturer's instructions, and untagged HSPA proteins were buffer-exchanged to storage buffer as described above. Methyltransferase reactions were performed in 50-μl volumes for 1 h at 37 °C in methyltransferase reaction buffer (50 mm Tris (pH 7.8), 50 mm KCl, 5 mm MgCl2, 1 mm ATP, 13 μm [3H]SAM (2 μCi; PerkinElmer Life Sciences), 4 μm substrate, and 2 μm METTL21A) unless stated otherwise. For mass spectrometric analysis, radiolabeled SAM was replaced with 1.2 mm nonradioactive SAM (New England Biolabs). Reactions were stopped by precipitating proteins with TCA for liquid scintillation counting of radioactivity or by denaturation in NuPAGE sample buffer for SDS-PAGE analysis (followed by MS or fluorographic analysis). For fluorography, reactions were separated on NuPAGE 4–12% gradient Bis-Tris gels (Invitrogen), and proteins were transferred to PVDF membranes using the XCell SureLockTM system (Invitrogen). Membranes were then stained with Ponceau S, treated with the scintillation enhancer EN3HANCE (PerkinElmer Life Sciences), and exposed to Carestream Kodak BioMax MS film (Sigma-Aldrich) for 24–72 h at −80 °C. For liquid scintillation counting, proteins were precipitated in 10% (w/v) TCA at 4 °C for 1 h. Acid-insoluble material was retained on glass fiber filters (Whatman GF/C) by vacuum filtration. Filters were washed with 10% (w/v) TCA and EtOH and placed in scintillation fluid (Ultima GoldTM XR, PerkinElmer Life Sciences), and incorporated radioactivity was measured by scintillation counting. LC-MS/MS of proteolytic peptides was performed using a Hypercarb 5-μm particle size column (G&T Septech AS) for sample concentration and a C18 column (GlycproSIL C18–80Å, Glycpromass) for analytical separation of peptides. Samples were washed with a mobile phase (0.1% (v/v) formic acid and 2.5% (v/v) acetonitrile) and eluted with a binary gradient of increasing acetonitrile up to 60% (v/v). The LC setup was coupled to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific) via nanoelectrospray. In-gel proteolytic digestion was performed with Asp-N (Roche Applied Science) or trypsin (Sigma-Aldrich). Peptide samples were analyzed with a collision-induced dissociation fragmentation method, acquiring one Orbitrap survey scan in the mass range of m/z 200–2000, followed by MS/MS of the most intense ions in the Orbitrap. Mass spectrometric data were analyzed with searches using Proteome Discoverer SEQUESTTM (Thermo Scientific) against in-house maintained databases of either HSPA proteins or the human proteome. The mass tolerances for fragment ions and parent ions were set to 0.5 Da and 10 ppm, respectively. Methionine oxidation and lysine mono-, di-, and trimethylation and acetylation were selected as variable modifications. MS/MS spectra of relevant peptides were manually extracted using Qual Browser (v2.0.7). Chromatograms representing various methylation states of peptides were generated by gating for m/z ratios corresponding to un-, mono-, di-, and trimethylated species of the predominant charge state using the BOXCAR algorithm and a sensitivity of 10 ppm in Qual Browser (v2.0.7). To achieve comparable chromatograms, the y axis was normalized with respect to signal intensity for the different methylation states. Relative quantification of peptides was performed by integrating area under curves using Qual Browser (v2.0.7). The following primary antibodies were used (dilution in Western blotting is indicated): anti-HSPA1 (Abcam ab79852; 1:10,000), anti-HSPA8 (Abcam EP1531Y/ab51052; 1:1000), anti-GAPDH (Ambion AM4300; 1:4000), and anti-CBP (Millipore 07-482; 1:4000). For detection of HSPA1-K561me3 (HSPA1(me3)), a custom antibody was used at a dilution of 1:10,000 in Western blots. The antibody was obtained by affinity purification of serum from rabbits immunized with a synthetic peptide (acetyl-CDEGLKGK(me3)ISEA-amide; New England Peptide). Transfer of samples to PVDF membrane was performed as described above. Membranes were blocked with 5% (w/v) BSA in TBS containing 0.05% (w/v) Tween 20 and incubated with the appropriate HRP-conjugated secondary antibodies. Blots were thereafter incubated with SuperSignal West Dura (Thermo Scientific), and chemiluminescence signals were detected with a Image Station 4000R Pro system (Kodak). For knockdown assays, Flp-In T-REx HEK-293 cells were transfected with either a pool of siRNAs versus the METTL21A transcript (Sigma-Aldrich SASI_Hs01_00047371, SASI_Hs01_00047373, and SASI_Hs01_00047374) or a scrambled sequence as a negative control (Sigma-Aldrich SIC001), or RNA was omitted. Lipofectamine RNAiMAX (Invitrogen) was used as the transfection agent according to the manufacturer's instructions, and knockdown was assayed by Western blotting and MS after 72 h. Reactions were performed in 50-μl volumes in a 96-well format in assay buffer (25 mm HEPES (pH 7.4), 1 mm DTT, 5 mm MgCl2, and 50 mm KCl) supplemented with HSPA1 (1 μm), HSPA8 (1 μm), DNAJB1 (1 μm), HSPA-KMT (0.5 μm), SAM (1 mm), and ATP (100 μm) as indicated. After incubation of the plate for 60 min at 37 °C in a humidity chamber, 100 μl of BIOMOL Green reagent (Enzo Life Sciences) was added. After additional incubation of the plate at room temperature for 30 min, the absorbance at 620 nm was measured. The concentration of liberated free phosphate was determined from a standard curve. Recombinant α-Syn was purified as described previously (20.Ghee M. Melki R. Michot N. Mallet J. PA700, the regulatory complex of the 26S proteasome, interferes with α-synuclein assembly.FEBS J. 2005; 272: 4023-4033Crossref PubMed Scopus (66) Google Scholar). The α-Syn concentration was determined spectrophotometrically using an extinction coefficient of 5960 m−1 cm−1 at 280 nm. Pure α-Syn (0.5 mm) in 50 mm Tris-HCl (pH 7.5) and 150 mm KCl was passed through sterile 0.22-μm filters and stored at −80 °C. Assembly of α-Syn fibrils was performed as described previously (21.Pemberton S. Madiona K. Pieri L. Kabani M. Bousset L. Melki R. Hsc70 protein interaction with soluble and fibrillar α-synuclein.J. Biol. Chem. 2011; 286: 34690-34699Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Briefly, soluble α-Syn was incubated with continuous shaking in an Eppendorf Thermomixer set at 600 rotations/min at 37 °C. At regular time intervals, aliquots were withdrawn, and the assembly process was monitored using thioflavin T binding by averaging the emission signal at 480 nm for an excitation wavelength of 440 nm over 30 s using a Cary Eclipse spectrofluorometer (Varian Inc., Palo Alto, CA). The morphology and structure of α-Syn assemblies were assessed using a Jeol 1400 transmission electron microscope following adsorption of the samples onto carbon-coated 200-mesh grids and negative staining with 1% (w/v) uranyl acetate. The images were recorded with a Gatan ORIUS CCD camera. Human METTL21A belongs, together with METTL21B, METTL21C, and VCP-KMT (METTL21D), to a subgroup of four closely related enzymes within MTase family 16 (pfam PF10294) (12.Kernstock S. Davydova E. Jakobsson M. Moen A. Pettersen S. Mælandsmo G.M. Egge-Jacobsen W. Falnes P.Ø. Lysine methylation of VCP by a member of a novel human protein methyltransferase family.Nat. Commun. 2012; 3: 1038Crossref PubMed Scopus (91) Google Scholar). To identify putative METTL21A orthologs in other organisms, BLAST searches were performed to identify proteins with substantial sequence similarity to METTL21A (expect value < 10−15). Next, these candidate proteins were used as queries in reciprocal BLAST searches versus human sequences, and those that yielded METTL21A, rather than its paralogs, as the best human hit were categorized as putative orthologs. According to this analysis, METTL21A orthologs are found in a wide range of eukaryotes, including all vertebrates and several invertebrates, but appear to be absent in bacteria (Table 1). However, METTL21A shows a rather scattered distribution among eukaryotes; for example, METTL21A orthologs are absent in the common model organisms Arabidopsis thaliana, Caenorhabditis elegans, and Schizosaccharomyces pombe but are present in other members of the respective organism groups, i.e. plants, nematodes, and fungi (Table 1).TABLE 1Presence of METTL21A-like sequences in various organismsClosest METTL21A homologReciprocal BLAST versus human (expect value)OrganismExpect valueAccession no.METTL21A (NP_660323.3)METTL21B (NP_056248.2)METTL21C (NP_001010977.1)METTL21D (NP_078834.2)VertebratesH. sapiens (human)NP_660323.31E-161aBoldface indicates best human hit in a reciprocal search.1E-486E-235E-28Mus musculus (mouse)3E-133NP_080240.15E-1453E-472E-243E-25D. rerio (zebrafish)1E-80NP_001013584.12E-924E-495E-263E-24Gallus gallus (chicken)2E-94XP_421949.13E-1064E-432E-285E-26InsectsAcyrthosiphon pisum (pea aphid)bBest hit within a group (insect, nematode, etc.).5E-20XP_001945214.15E-225E-254E-152E-38Drosophila melanogaster (fruit fly)4E-07NP_573368.29E-95E-110.000020.024NematodesA. suum (pig roundworm)bBest hit within a group (insect, nematode, etc.).5E-31ADY46645.15E-331E-223E-111E-10C. elegans6E-10NP_001122759.17E-129E-217E-135E-29PlantsZ. mays (maize)bBest hit within a group (insect, nematode, etc.).1E-25NP_001142219.12E-272E-203E-189E-25A. thaliana (thale cress)1E-21AAU94386.12E-232E-243E-184E-24FungiSerpula lacrymans (dry rot fungus)bBest hit within a group (insect, nematode, etc.).7E-29EGO015761E-303E-182E-144E-28S. cerevisiae (budding yeast)1E-17NP_014374.11E-195E-132E-112E-16S. pombe (fission yeast)9E-11NP_593178.12E-126E-114E-81E-14OtherscBest hits among invertebrates.Crassostrea gigas (Pacific oyster)7E-69EKC35706.19E-713E-444E-224E-24N. vectensis (starlet sea anemone)7E-64XP_001623378.18E-662E-341E-224E-30Strongylocentrotus purpuratus (sea urchin)4E-51XP_781765.34E-587E-311E-221E-17a Boldface indicates best human hit in a reciprocal search.b Best hit within a group (insect, nematode, etc.).c Best hits among invertebrates. Open table in a new tab METTL21A was predicted to be a 7BS-MTase in a recent bioinformatics study aimed at uncovering the human methyltransferasome (7.Petrossian T.C. Clarke S.G. Uncovering the human methyltransferasome.Mol. Cell. Proteomics. 2011; 10 (10.1074/mcp.M110.000976)Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Accordingly, a sequence alignment of putative METTL21A orthologs from various organisms revealed the presence of conserved sequence motifs corresponding to motifs typical of the 7BS-MTases, denoted Motif 1, Post 1, Motif 2, and Motif 3, as well as a hallmark (D/E)XX(Y/F) motif found in members of MTase family 16 (Fig. 1) (8.Katz J.E. Dlakić M. Clarke S. Automated identification of putative methyltransferases from genomic open reading frames.Mol. Cell. Proteomics. 2003; 2: 525-540Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 12.Kernstock S. Davydova E. Jakobsson M. Moen A. Pettersen S. Mælandsmo G.M. Egge-Jacobsen W. Falnes P.Ø. Lysine methylation of VCP by a member of a novel human protein methyltransferase family.Nat. Commun. 2012; 3: 1038Crossref PubMed Scopus (91) Google Scholar). Furthermore, second" @default.
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• W2085450186 title "Identification and Characterization of a Novel Human Methyltransferase Modulating Hsp70 Protein Function through Lysine Methylation" @default.
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