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.
Vyšlo v časopise: A Newly Uncovered Group of Distantly Related Lysine Methyltransferases Preferentially Interact with Molecular Chaperones to Regulate Their Activity. PLoS Genet 9(1): e32767. doi:10.1371/journal.pgen.1003210 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003210
1. MartinJL, McMillanFM (2002) SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold. Curr Opin Struct Biol 12 : 783–793.
2. PetrossianTC, ClarkeSG (2011) Uncovering the human methyltransferasome. Mol Cell Proteomics 10: M110 000976.
3. SchubertHL, BlumenthalRM, ChengX (2003) Many paths to methyltransfer: a chronicle of convergence. Trends Biochem Sci 28 : 329–335.
4. IshizawaT, NozakiY, UedaT, TakeuchiN (2008) The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC. Biochem Biophys Res Commun 373 : 99–103.
5. KlotzAV, LearyJA, GlazerAN (1986) Post-translational methylation of asparaginyl residues. Identification of beta-71 gamma-N-methylasparagine in allophycocyanin. J Biol Chem 261 : 15891–15894.
6. WebbKJ, Zurita-LopezCI, Al-HadidQ, LaganowskyA, YoungBD, et al. (2010) A novel 3-methylhistidine modification of yeast ribosomal protein Rpl3 is dependent upon the YIL110W methyltransferase. J Biol Chem 285 : 37598–37606.
7. PeggAE (1990) Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res 50 : 6119–6129.
8. SprungR, ChenY, ZhangK, ChengD, ZhangT, et al. (2008) Identification and validation of eukaryotic aspartate and glutamate methylation in proteins. J Proteome Res 7 : 1001–1006.
9. ClarkeS (1992) Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem 61 : 355–386.
10. TooleyCE, PetkowskiJJ, Muratore-SchroederTL, BalsbaughJL, ShabanowitzJ, et al. (2010) NRMT is an alpha-N-methyltransferase that methylates RCC1 and retinoblastoma protein. Nature 466 : 1125–1128.
11. WuJ, TolstykhT, LeeJ, BoydK, StockJB, et al. (2000) Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J 19 : 5672–5681.
12. FengQ, WangH, NgHH, Erdjument-BromageH, TempstP, et al. (2002) Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr Biol 12 : 1052–1058.
13. MartinC, ZhangY (2005) The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 6 : 838–849.
14. VolkelP, AngrandPO (2007) The control of histone lysine methylation in epigenetic regulation. Biochimie 89 : 1–20.
15. BarskiA, CuddapahS, CuiK, RohTY, SchonesDE, et al. (2007) High-resolution profiling of histone methylations in the human genome. Cell 129 : 823–837.
16. KochCM, AndrewsRM, FlicekP, DillonSC, KaraozU, et al. (2007) The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res 17 : 691–707.
17. RosenfeldJA, WangZ, SchonesDE, ZhaoK, DeSalleR, et al. (2009) Determination of enriched histone modifications in non-genic portions of the human genome. BMC Genomics 10 : 143.
18. ChuikovS, KurashJK, WilsonJR, XiaoB, JustinN, et al. (2004) Regulation of p53 activity through lysine methylation. Nature 432 : 353–360.
19. HoutzRL, StultsJT, MulliganRM, TolbertNE (1989) Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci U S A 86 : 1855–1859.
20. HuangJ, Perez-BurgosL, PlacekBJ, SenguptaR, RichterM, et al. (2006) Repression of p53 activity by Smyd2-mediated methylation. Nature 444 : 629–632.
21. KruiswijkT, KunstA, PlantaRJ, MagerWH (1978) Modification of yeast ribosomal proteins. Methylation. Biochem J 175 : 221–225.
22. LeeSW, BergerSJ, MartinovicS, Pasa-TolicL, AndersonGA, et al. (2002) Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR. Proc Natl Acad Sci U S A 99 : 5942–5947.
23. LevyD, KuoAJ, ChangY, SchaeferU, KitsonC, et al. (2011) Lysine methylation of the NF-kappaB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappaB signaling. Nat Immunol 12 : 29–36.
24. LhoestJ, LobetY, CostersE, ColsonC (1984) Methylated proteins and amino acids in the ribosomes of Saccharomyces cerevisiae. Eur J Biochem 141 : 585–590.
25. MorinoH, KawamotoT, MiyakeM, KakimotoY (1987) Purification and properties of calmodulin-lysine N-methyltransferase from rat brain cytosol. J Neurochem 48 : 1201–1208.
26. PolevodaB, MartzenMR, DasB, PhizickyEM, ShermanF (2000) Cytochrome c methyltransferase, Ctm1p, of yeast. J Biol Chem 275 : 20508–20513.
27. YangXD, HuangB, LiM, LambA, KelleherNL, et al. (2009) Negative regulation of NF-kappaB action by Set9-mediated lysine methylation of the RelA subunit. Embo J 28 : 1055–1066.
28. CloutierP, Al-KhouryR, Lavallee-AdamM, FaubertD, JiangH, et al. (2009) High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes. Methods 48 : 381–386.
29. CloutierP, CoulombeB (2010) New insights into the biogenesis of nuclear RNA polymerases? Biochem Cell Biol 88 : 211–221.
30. CojocaruM, BouchardA, CloutierP, CooperJJ, VarzavandK, et al. (2011) Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B. J Biol Chem 286 : 5012–5022.
31. CoulombeB, BlanchetteM, JeronimoC (2008) Steps towards a repertoire of comprehensive maps of human protein interaction networks: the Human Proteotheque Initiative (HuPI). Biochem Cell Biol 86 : 149–156.
32. ForgetD, LacombeAA, CloutierP, Al-KhouryR, BouchardA, et al. (2010) The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II. Mol Cell Proteomics 9 : 2827–2839.
33. JeronimoC, ForgetD, BouchardA, LiQ, ChuaG, et al. (2007) Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol Cell 27 : 262–274.
34. JeronimoC, LangelierMF, ZeghoufM, CojocaruM, BergeronD, et al. (2004) RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits. Mol Cell Biol 24 : 7043–7058.
35. KruegerBJ, JeronimoC, RoyBB, BouchardA, BarrandonC, et al. (2008) LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated. Nucleic Acids Res 36 : 2219–2229.
36. BaysNW, WilhovskySK, GoradiaA, Hodgkiss-HarlowK, HamptonRY (2001) HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins. Mol Biol Cell 12 : 4114–4128.
37. BraunS, MatuschewskiK, RapeM, ThomsS, JentschS (2002) Role of the ubiquitin-selective CDC48(UFD1/NPL4)chaperone (segregase) in ERAD of OLE1 and other substrates. Embo J 21 : 615–621.
38. JaroschE, TaxisC, VolkweinC, BordalloJ, FinleyD, et al. (2002) Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nat Cell Biol 4 : 134–139.
39. RabinovichE, KeremA, FrohlichKU, DiamantN, Bar-NunS (2002) AAA-ATPase p97/Cdc48p, a cytosolic chaperone required for endoplasmic reticulum-associated protein degradation. Mol Cell Biol 22 : 626–634.
40. YeY, MeyerHH, RapoportTA (2001) The AAA ATPase Cdc48/p97 and its partners transport proteins from the ER into the cytosol. Nature 414 : 652–656.
41. IoukTL, AitchisonJD, MaguireS, WozniakRW (2001) Rrb1p, a yeast nuclear WD-repeat protein involved in the regulation of ribosome biosynthesis. Mol Cell Biol 21 : 1260–1271.
42. MagnaniR, DirkLM, TrievelRC, HoutzRL (2010) Calmodulin methyltransferase is an evolutionarily conserved enzyme that trimethylates Lys-115 in calmodulin. Nat Commun 1 : 43.
43. ZhangK, YauPM, ChandrasekharB, NewR, KondratR, et al. (2004) Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3. Proteomics 4 : 1–10.
44. MeisterG, EggertC, BuhlerD, BrahmsH, KambachC, et al. (2001) Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln. Curr Biol 11 : 1990–1994.
45. AlexandruG, GraumannJ, SmithGT, KolawaNJ, FangR, et al. (2008) UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover. Cell 134 : 804–816.
46. OrmeCM, BoganJS (2012) The ubiquitin regulatory X (UBX) domain-containing protein TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi intermediate compartment. J Biol Chem 287 : 6679–6692.
47. JohnsonJO, MandrioliJ, BenatarM, AbramzonY, Van DeerlinVM, et al. (2010) Exome sequencing reveals VCP mutations as a cause of familial ALS. Neuron 68 : 857–864.
48. WattsGD, WymerJ, KovachMJ, MehtaSG, MummS, et al. (2004) Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein. Nat Genet 36 : 377–381.
49. HanzelmannP, SchindelinH (2011) The structural and functional basis of the p97/valosin-containing protein (VCP)-interacting motif (VIM): mutually exclusive binding of cofactors to the N-terminal domain of p97. J Biol Chem 286 : 38679–38690.
50. SchuberthC, BuchbergerA (2008) UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97. Cell Mol Life Sci 65 : 2360–2371.
51. YeungHO, KloppsteckP, NiwaH, IsaacsonRL, MatthewsS, et al. (2008) Insights into adaptor binding to the AAA protein p97. Biochem Soc Trans 36 : 62–67.
52. WangQ, SongC, YangX, LiCC (2003) D1 ring is stable and nucleotide-independent, whereas D2 ring undergoes major conformational changes during the ATPase cycle of p97-VCP. J Biol Chem 278 : 32784–32793.
53. DalalS, RosserMF, CyrDM, HansonPI (2004) Distinct roles for the AAA ATPases NSF and p97 in the secretory pathway. Mol Biol Cell 15 : 637–648.
54. WangC, LazaridesE (1984) Arsenite-induced changes in methylation of the 70,000 dalton heat shock proteins in chicken embryo fibroblasts. Biochem Biophys Res Commun 119 : 735–743.
55. WangC, LinJM, LazaridesE (1992) Methylations of 70,000-Da heat shock proteins in 3T3 cells: alterations by arsenite treatment, by different stages of growth and by virus transformation. Arch Biochem Biophys 297 : 169–175.
56. KleinJB, BaratiMT, WuR, GozalD, SachlebenLRJr, et al. (2005) Akt-mediated valosin-containing protein 97 phosphorylation regulates its association with ubiquitinated proteins. J Biol Chem 280 : 31870–31881.
57. KoikeM, FukushiJ, IchinoheY, HigashimaeN, FujishiroM, et al. (2010) Valosin-containing protein (VCP) in novel feedback machinery between abnormal protein accumulation and transcriptional suppression. J Biol Chem 285 : 21736–21749.
58. LiG, ZhaoG, SchindelinH, LennarzWJ (2008) Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD. Biochem Biophys Res Commun 375 : 247–251.
59. LivingstoneM, RuanH, WeinerJ, ClauserKR, StrackP, et al. (2005) Valosin-containing protein phosphorylation at Ser784 in response to DNA damage. Cancer Res 65 : 7533–7540.
60. Mori-KonyaC, KatoN, MaedaR, YasudaK, HigashimaeN, et al. (2009) p97/valosin-containing protein (VCP) is highly modulated by phosphorylation and acetylation. Genes Cells 14 : 483–497.
61. HalawaniD, LeBlancAC, RouillerI, MichnickSW, ServantMJ, et al. (2009) Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation. Mol Cell Biol 29 : 4484–4494.
62. MannoA, NoguchiM, FukushiJ, MotohashiY, KakizukaA (2010) Enhanced ATPase activities as a primary defect of mutant valosin-containing proteins that cause inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. Genes Cells 15 : 911–922.
63. AnguloJF, MoreauPL, MaunouryR, LaporteJ, HillAM, et al. (1989) KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein. Mutat Res 217 : 123–134.
64. BiardDS, MiccoliL, DesprasE, HarperF, PichardE, et al. (2003) Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes. Mol Cancer Res 1 : 519–531.
65. KannoucheP, MauffreyP, Pinon-LatailladeG, MatteiMG, SarasinA, et al. (2000) Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans. Carcinogenesis 21 : 1701–1710.
66. MiccoliL, FrouinI, NovacO, Di PaolaD, HarperF, et al. (2005) The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins. Mol Cell Biol 25 : 3814–3830.
67. MakarovEM, MakarovaOV, UrlaubH, GentzelM, WillCL, et al. (2002) Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome. Science 298 : 2205–2208.
68. RappsilberJ, RyderU, LamondAI, MannM (2002) Large-scale proteomic analysis of the human spliceosome. Genome Res 12 : 1231–1245.
69. DonlinLT, AndresenC, JustS, RudenskyE, PappasCT, et al. (2012) Smyd2 controls cytoplasmic lysine methylation of Hsp90 and myofilament organization. Genes Dev 26 : 114–119.
70. ZeghoufM, LiJ, ButlandG, BorkowskaA, CanadienV, et al. (2004) Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes. J Proteome Res 3 : 463–468.
71. PuigO, CasparyF, RigautG, RutzB, BouveretE, et al. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24 : 218–229.
72. RigautG, ShevchenkoA, RutzB, WilmM, MannM, et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17 : 1030–1032.
73. Lavallee-AdamM, CloutierP, CoulombeB, BlanchetteM (2010) Modeling contaminants in AP-MS/MS experiments. J Proteome Res 10 : 886–895.
74. PerkinsDN, PappinDJ, CreasyDM, CottrellJS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20 : 3551–3567.
75. InamitsuM, ItohS, HellmanU, Ten DijkeP, KatoM (2006) Methylation of Smad6 by protein arginine N-methyltransferase 1. FEBS Lett 580 : 6603–6611.
76. ZwijsenRM, BuckleRS, HijmansEM, LoomansCJ, BernardsR (1998) Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1. Genes Dev 12 : 3488–3498.
77. ApweilerR, Jesus MartinM, O'onovanC, MagraneM, Alam-FaruqueY, et al. (2012) Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 40: D71–75.
78. LarkinMA, BlackshieldsG, BrownNP, ChennaR, McGettiganPA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23 : 2947–2948.
79. WaterhouseAM, ProcterJB, MartinDM, ClampM, BartonGJ (2009) Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25 : 1189–1191.
80. SaitouN, NeiM (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4 : 406–425.
81. HenikoffS, HenikoffJG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A 89 : 10915–10919.
Zvýšte si kvalifikáciu online z pohodlia domova
Nemáte účet? Registrujte sa
Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.
