Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity

Retrovirology

Mark A Wainberg¹  Jean-Pierre Falgueyret²  Peter K Quashie¹  Arielle Sabbah³  Thibault Mesplède³  Diane N Singhroy³ 

[1] Division of Experimental Medicine, Faculty of Medicine, McGill University, Montreal, QC, Canada;Centre for Biological Application of Mass Spectrometry, Concordia University, Montreal, QC, Canada;McGill University AIDS Centre, Lady Davis for Medical Research, Jewish General Hospital, 3755 Cote Sainte Catherine, Montreal, QC, H3T 1E2, Canada

关键词: Antiretroviral activity;    Protein stability;    Automethylation;    HIV-1;    PRMT6;   

Others  :  1209102
DOI  :  10.1186/1742-4690-10-73

 received in 2012-07-12, accepted in 2013-07-09,  发布年份 2013

【 摘 要 】

Background

Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that methylates arginine residues on histones and transcription factors. In addition, PRMT6 inhibits HIV-1 replication in cell culture by directly methylating and interfering with the functions of several HIV-1 proteins, i.e. Tat, Rev and nucleocapsid (NC). PRMT6 also displays automethylation capacity but the role of this post-translational modification in its antiretroviral activity remains unknown.

Results

Here we report the identification by liquid chromatography-mass spectrometry of R35 within PRMT6 as the target residue for automethylation and have confirmed this by site-directed mutagenesis and in vitro and in vivo methylation assays. We further show that automethylation at position 35 greatly affects PRMT6 stability and is indispensable for its antiretroviral activity, as demonstrated in HIV-1 single-cycle TZM-bl infectivity assays.

Conclusion

These results show that PRMT6 automethylation plays a role in the stability of this protein and that this event is indispensible for its anti-HIV-1 activity.

【 授权许可】

   
2013 Singhroy et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Krause CD, Yang ZH, Kim YS, Lee JH, Cook JR, Pestka S: Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential. Pharmacol Ther 2007, 113:50-87.
• [2]Konig R, Zhou Y, Elleder D, Diamond TL, Bonamy GM, Irelan JT, Chiang CY, Tu BP, De Jesus PD, Lilley CE, et al.: Global analysis of host-pathogen interactions that regulate early-stage HIV-1 replication. Cell 2008, 135:49-60.
• [3]Brass AL, Dykxhoorn DM, Benita Y, Yan N, Engelman A, Xavier RJ, Lieberman J, Elledge SJ: Identification of host proteins required for HIV infection through a functional genomic screen. Science 2008, 319:921-926.
• [4]Le Clerc S, Limou S, Coulonges C, Carpentier W, Dina C, Taing L, Delaneau O, Labib T, Sladek R, Deveau C, et al.: Genomewide association study of a rapid progression cohort identifies new susceptibility alleles for AIDS (ANRS Genomewide Association Study 03). J Infect Dis 2009, 200:1194-1201.
• [5]Zhou H, Xu M, Huang Q, Gates AT, Zhang XD, Castle JC, Stec E, Ferrer M, Strulovici B, Hazuda DJ, Espeseth AS: Genome-scale RNAi screen for host factors required for HIV replication. Cell Host Microbe 2008, 4:495-504.
• [6]Liu L, Oliveira NM, Cheney KM, Pade C, Dreja H, Bergin AM, Borgdorff V, Beach DH, Bishop CL, Dittmar MT, McKnight A: A whole genome screen for HIV restriction factors. Retrovirology 2011, 8:94. BioMed Central Full Text
• [7]Kiernan RE, Vanhulle C, Schiltz L, Adam E, Xiao H, Maudoux F, Calomme C, Burny A, Nakatani Y, Jeang KT, et al.: HIV-1 tat transcriptional activity is regulated by acetylation. EMBO J 1999, 18:6106-6118.
• [8]Yang X, Goncalves J, Gabuzda D: Phosphorylation of Vif and its role in HIV-1 replication. J Biol Chem 1996, 271:10121-10129.
• [9]Gary JD, Clarke S: RNA and protein interactions modulated by protein arginine methylation. Prog Nucleic Acid Res Mol Biol 1998, 61:65-131.
• [10]Bedford MT, Clarke SG: Protein arginine methylation in mammals: who, what, and why. Mol Cell 2009, 33:1-13.
• [11]Miranda TB, Miranda M, Frankel A, Clarke S: PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity. J Biol Chem 2004, 279:22902-22907.
• [12]Bedford MT, Richard S: Arginine methylation an emerging regulator of protein function. Mol Cell 2005, 18:263-272.
• [13]Bedford MT: Arginine methylation at a glance. J Cell Sci 2007, 120:4243-4246.
• [14]Lakowski TM, Frankel A: A kinetic study of human protein arginine N-methyltransferase 6 reveals a distributive mechanism. J Biol Chem 2008, 283:10015-10025.
• [15]Lee YH, Stallcup MR: Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation. Mol Endocrinol 2009, 23:425-433.
• [16]Boisvert FM, Cote J, Boulanger MC, Richard S: A proteomic analysis of arginine-methylated protein complexes. Mol Cell Proteomics 2003, 2:1319-1330.
• [17]Frankel A, Yadav N, Lee J, Branscombe TL, Clarke S, Bedford MT: The novel human protein arginine N-methyltransferase PRMT6 is a nuclear enzyme displaying unique substrate specificity. J Biol Chem 2002, 277:3537-3543.
• [18]Michaud-Levesque J, Richard S: Thrombospondin-1 is a transcriptional repression target of PRMT6. J Biol Chem 2009, 284:21338-21346.
• [19]Hyllus D, Stein C, Schnabel K, Schiltz E, Imhof A, Dou Y, Hsieh J, Bauer UM: PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation. Genes Dev 2007, 21:3369-3380.
• [20]Waldmann T, Izzo A, Kamieniarz K, Richter F, Vogler C, Sarg B, Lindner H, Young NL, Mittler G, Garcia BA, Schneider R: Methylation of H2AR29 is a novel repressive PRMT6 target. Epigenetics Chromatin 2011, 4:11. BioMed Central Full Text
• [21]Xie B, Invernizzi CF, Richard S, Wainberg MA: Arginine methylation of the human immunodeficiency virus type 1 Tat protein by PRMT6 negatively affects Tat Interactions with both cyclin T1 and the Tat transactivation region. J Virol 2007, 81:4226-4234.
• [22]Boulanger MC, Liang C, Russell RS, Lin R, Bedford MT, Wainberg MA, Richard S: Methylation of Tat by PRMT6 regulates human immunodeficiency virus type 1 gene expression. J Virol 2005, 79:124-131.
• [23]Invernizzi CF, Xie B, Richard S, Wainberg MA: PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA. Retrovirology 2006, 3:93. BioMed Central Full Text
• [24]Invernizzi CF, Xie B, Frankel FA, Feldhammer M, Roy BB, Richard S, Wainberg MA: Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function. AIDS 2007, 21:795-805.
• [25]Suhasini M, Reddy TR: Cellular proteins and HIV-1 Rev function. Curr HIV Res 2009, 7:91-100.
• [26]Tange TO, Jensen TH, Kjems J: In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. J Biol Chem 1996, 271:10066-10072.
• [27]Thomas D, Lakowski TM, Pak ML, Kim JJ, Frankel A: Forster resonance energy transfer measurements of cofactor-dependent effects on protein arginine N-methyltransferase homodimerization. Protein Sci 2010, 19:2141-2151.
• [28]Kuhn P, Chumanov R, Wang Y, Ge Y, Burgess RR, Xu W: Automethylation of CARM1 allows coupling of transcription and mRNA splicing. Nucleic Acids Res 2011, 39:2717-2726.
• [29]Sivakumaran H, van der Horst A, Fulcher AJ, Apolloni A, Lin MH, Jans DA, Harrich D: Arginine methylation increases the stability of human immunodeficiency virus type 1 Tat. J Virol 2009, 83:11694-11703.
• [30]Cha B, Kim W, Kim YK, Hwang BN, Park SY, Yoon JW, Park WS, Cho JW, Bedford MT, Jho EH: Methylation by protein arginine methyltransferase 1 increases stability of Axin, a negative regulator of Wnt signaling. Oncogene 2011, 30:2379-2389.
• [31]Willemsen NM, Hitchen EM, Bodetti TJ, Apolloni A, Warrilow D, Piller SC, Harrich D: Protein methylation is required to maintain optimal HIV-1 infectivity. Retrovirology 2006, 3:92. BioMed Central Full Text
• [32]Shirakawa K, Takaori-Kondo A, Yokoyama M, Izumi T, Matsui M, Io K, Sato T, Sato H, Uchiyama T: Phosphorylation of APOBEC3G by protein kinase A regulates its interaction with HIV-1 Vif. Nat Struct Mol Biol 2008, 15:1184-1191.
• [33]Dussart S, Douaisi M, Courcoul M, Bessou G, Vigne R, Decroly E: APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles. J Mol Biol 2005, 345:547-558.
• [34]Douaisi M, Dussart S, Courcoul M, Bessou G, Lerner EC, Decroly E, Vigne R: The tyrosine kinases Fyn and Hck favor the recruitment of tyrosine-phosphorylated APOBEC3G into vif-defective HIV-1 particles. Biochem Biophys Res Commun 2005, 329:917-924.
• [35]Sayegh J, Webb K, Cheng D, Bedford MT, Clarke SG: Regulation of protein arginine methyltransferase 8 (PRMT8) activity by its N-terminal domain. J Biol Chem 2007, 282:36444-36453.
• [36]Lakowski TM, t Hart P, Ahern CA, Martin NI, Frankel A: Neta-substituted arginyl peptide inhibitors of protein arginine N-methyltransferases. ACS Chem Biol 2010, 5:1053-1063.
• [37]Choi S, Jung CR, Kim JY, Im DS: PRMT3 inhibits ubiquitination of ribosomal protein S2 and together forms an active enzyme complex. Biochim Biophys Acta 2008, 1780:1062-1069.
• [38]Jarmalavicius S, Trefzer U, Walden P: Differential arginine methylation of the G-protein pathway suppressor GPS-2 recognized by tumor-specific T cells in melanoma. FASEB J 2010, 24:937-946.
• [39]Yamamoto N, Tanaka C, Wu Y, Chang MO, Inagaki Y, Saito Y, Naito T, Ogasawara H, Sekigawa I, Hayashida Y: Analysis of human immunodeficiency virus type 1 integration by using a specific, sensitive and quantitative assay based on real-time polymerase chain reaction. Virus Genes 2006, 32:105-113.
• [40]Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA: Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 1986, 59:284-291.
• [41]Rasband WS: ImageJ. Bethesda, Maryland, USA: Health NIo ed; 1997-2011.
• [42]Donahue DA, Sloan RD, Kuhl BD, Bar-Magen T, Schader SM, Wainberg MA: Stage-dependent inhibition of HIV-1 replication by antiretroviral drugs in cell culture. Antimicrob Agents Chemother 2010, 54:1047-1054.
• [43]Schader SM, Oliveira M, Ibanescu RI, Moisi D, Colby-Germinario SP, Wainberg MA: In vitro resistance profile of the candidate HIV-1 microbicide drug dapivirine. Antimicrob Agents Chemother 2012, 56:751-756.