Biochemical characterization of Ty1 retrotransposon protease


Autoři: Lívia Diána Gazda aff001;  Krisztina Joóné Matúz aff001;  Tibor Nagy aff002;  János András Mótyán aff001;  József Tőzsér aff001
Působiště autorů: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary aff001;  Department of Applied Chemistry, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary aff002
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
prolekare.web.journal.doi_sk: 10.1371/journal.pone.0227062

Souhrn

Ty1 is one of the many transposons in the budding yeast Saccharomyces cerevisiae. The life-cycle of Ty1 shows numerous similarities with that of retroviruses, e.g. the initially synthesized polyprotein precursor undergoes proteolytic processing by the protease. The retroviral proteases have become important targets of current antiretroviral therapies due to the critical role of the limited proteolysis of Gag-Pol polyprotein in the replication cycle and they therefore belong to the most well-studied enzymes. Comparative analyses of retroviral and retroviral-like proteases can help to explore the key similarities and differences which may help understanding how resistance is developed against protease inhibitors, but the available information about the structural and biochemical characteristics of retroviral-like, and especially retrotransposon, proteases is limited. To investigate the main characteristics of Ty1 retrotransposon protease of Saccharomyces cerevisiae, untagged and His6-tagged forms of Ty1 protease were expressed in E. coli. After purification of the recombinant proteins, activity measurements were performed using synthetic oligopeptide and fluorescent recombinant protein substrates, which represented the wild-type and the modified forms of naturally occurring cleavage sites of the protease. We investigated the dependence of enzyme activity on different reaction conditions (pH, temperature, ionic strength, and urea concentration), and determined enzyme kinetic parameters for the studied substrates. Inhibitory potentials of 10 different protease inhibitors were also tested. Ty1 protease was not inhibited by the inhibitors which have been designed against human immunodeficiency virus type 1 protease and are approved as antiretroviral therapeutics. A quaternary structure of homodimeric Ty1 protease was proposed based on homology modeling, and this structure was used to support interpretation of experimental results and to correlate some structural and biochemical characteristics with that of other retroviral proteases.

Klíčová slova:

Dimers – HIV-1 – Matrix-assisted laser desorption ionization time-of-flight mass spectrometry – Proteases – Recombinant proteins – Retrotransposons – Sequence motif analysis – Urea


Zdroje

1. Coffin JM, Hughes SH, Varmus HE. Retroviruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1997

2. Curcio MJ, Lutz S, Lesage P. The Ty1 LTR retrotransposon of budding yeast, Saccharomyces cerevisiae. Microbiol Spectr. 2015; 3(2): MDNA3-0053-2014

3. Belcourt MF, Farabaugh PJ. Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site. Cell. 1990 62(2): 339–352. doi: 10.1016/0092-8674(90)90371-k 2164889

4. Farabaugh PJ. Efficient translational frameshifting occurs within a conserved sequence of the overlap between the two genes of a yeast Ty1 transposon. Proc Natl Acad Sci USA. 1988; 85(18): 6816–6820. doi: 10.1073/pnas.85.18.6816 2842793

5. Adams SE, Mellor J, Gull K, Sim RB, Tuite MF, Kingsman SM, et al. The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins. Cell. 1987 49, 111–119. doi: 10.1016/0092-8674(87)90761-6 3030564

6. Müller F, Brühl KH, Freidel K, Kowallik KV, Ciriacy M. Processing of TY1 proteins and formation of Ty1 virus-like particles in Saccharomyces cerevisiae. Mol Gen Genet. 1987; 207(2–3): 421–429. doi: 10.1007/bf00331610 3039300

7. Youngren SD, Boeke JD, Sanders NJ, Garfinkel DJ. Functional organization of the retrotransposon Ty from Saccharomyces cerevisiae: Ty protease is required for transposition. Mol Cell Biol. 1988; 8(4): 1421–1431. doi: 10.1128/mcb.8.4.1421 2454391

8. Garfinkel DJ, Hedge AM, Youngren SD, Copeland TD. Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1. J Virol. 1991; 65(9): 4573–81. 1714514

9. Mellor J, Alexandra MF, Melanie JD, Nichola AR, Wilson W, Kingsman AJ, et al. The Ty transposon of Saccharomyces cerevisiae determines the synthesis of at least three proteins. Nucl Acids Res. 1985; 13(17): 6249–6263. doi: 10.1093/nar/13.17.6249 2995923

10. Brookman JL, Stott AJ, Cheeseman PJ, Adamson CS, Holmes D, Cole J, et al. Analysis of TYA protein regions necessary for formation of the Ty1 virus-like particle structure. Virology. 1995; 212(1): 69–76. doi: 10.1006/viro.1995.1454 7676650

11. Roth JF. The yeast Ty virus-like particles. Yeast. 2000; 16(9): 785–95. doi: 10.1002/1097-0061(20000630)16:9<785::AID-YEA550>3.0.CO;2-L 10861903

12. Curcio MJ, Garfinkel DJ. Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing. Mol Cell Biol. 1992; 12(6): 2813–2825. doi: 10.1128/mcb.12.6.2813 1317008

13. Curcio MJ, Derbyshire KM. The outs and ins of transposition:from mu to kangaroo. Nat Rev Mol Cell Biol. 2003; 4: 865–877. doi: 10.1038/nrm1241 14682279

14. Moore SP, Rinckel LA Garfinkel DJ. A Ty1 Integrase Nuclear Localization Signal Required for Retrotransposition. Mol Cell Biol. 1998; 18(2): 1105–1114. doi: 10.1128/mcb.18.2.1105 9448008

15. Moore SP and Garfinkel DJ. Correct Integration of Model Substrates by Ty1 Integrase. J Virol. 2000; 74(24): 11522–11530. doi: 10.1128/jvi.74.24.11522-11530.2000 11090149

16. Friedl AA, Kiechle M, Maxeiner HG, Schiestl RH, Eckardt-Schupp F. Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae. Mol Genet Genomics. 2010; 284(4): 231–242. doi: 10.1007/s00438-010-0561-4 20677012

17. Athauda SB, Yoshioka K, Shiba T, Takahashi K. Isolation and characterization of recombinant Drosophila Copia aspartic proteinase. Biochem J. 2006; 399(3):535–42. doi: 10.1042/BJ20060800 16813567

18. Rawlings ND, Barrett AJ, Thomas PD, Huang X, Bateman A, Finn RD. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 2018; 46, D624–D632. doi: 10.1093/nar/gkx1134 29145643

19. Malik HS, Eickbush TH. Phylogenetic analysis of ribonuclease H domains suggests a late, chimeric origin of LTR retrotransposable elements and retroviruses. Genome Res. 2001; 11(7):1187–97. doi: 10.1101/gr.185101 11435400

20. Merkulov GV, Swiderek KM, B. BC, Boeke JD. A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein. J Virol. 1996; 70: 5548–5556. 8764068

21. Merkulov GV, Lawler JF Jr, Eby Y, Boeke JD. Ty1 Proteolytic Cleavage Sites Are Required for Transposition: All Sites Are Not Created Equal. J Virol. 2001; 75(2): 638–644. doi: 10.1128/JVI.75.2.638-644.2001 11134277

22. Lawler JF Jr, Merkulov GV, Boeke JD. Frameshift signal transplantation and the unambiguous analysis of mutations in the yeast retrotransposon Ty1 Gag-Pol overlap region. J Virol. 2001; 75(15): 6769–6775. doi: 10.1128/JVI.75.15.6769-6775.2001 11435555

23. Lawler JF Jr, Haeusser DP, Dull A, Boeke JD, Keeney JB. Ty1 defect in proteolysis at high temperature. J Virol. 2002; 76(9):4233–40. doi: 10.1128/JVI.76.9.4233-4240.2002 11932388

24. Laco GS. HIV-1 protease substrate-groove: Role in substrate recognition and inhibitor resistance. Biochimie. 2015; 118: 90–103. doi: 10.1016/j.biochi.2015.08.009 26300060

25. Kirchner J, Sandmeyer S. Proteolytic processing of Ty3 proteins is required for transposition. J Virol. 1993; 67(1):19–28. 7677953

26. Eunenn KV, Bakker BM. The importance and challenges of in vivo-like enzyme kinetics. Perspectives in Science, 2014; 1: 126–130.

27. Tőzsér J, Bagossi P, Weber IT, Copeland TD, Oroszlan S. Comparative studies on the substrate specificity of avian myeloblastosis virus proteinase and lentiviral proteinases. J Biol Chem. 1996; 271: 6781–6788.

28. Mahdi M, Szojka Z, Mótyán JA, Tőzsér J. Inhibition Profiling of Retroviral Protease Inhibitors Using an HIV-2 Modular System. Viruses. 2015; 7(12): 6152–62. doi: 10.3390/v7122931 26633459

29. Matúz K, Mótyán J, Li M, Wlodawer A, Tőzsér J. Inhibition of XMRV and HIV-1 proteases by pepstatin A and acetyl-pepstatin. FEBS J. 2012; 279(17): 3276–3286. doi: 10.1111/j.1742-4658.2012.08714.x 22804908

30. Fehér A, Weber IT, Bagossi P, Boross P, Mahalingam B, Louis JM, et al. Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites. Eur J Biochem. 2002; 269(16): 4114–20. doi: 10.1046/j.1432-1033.2002.03105.x 12180988

31. Bozóki B, Gazda L, Tóth F, Miczi M, Mótyán JA, Tőzsér J. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening. Anal Biochem. 2018; 540–541: 52–63. doi: 10.1016/j.ab.2017.11.001 29122614

32. Mótyán J.A., Miczi M., Bozóki B., Tőzsér J. Data supporting Ni-NTA magnetic bead-based fluorescent protease assay using recombinant fusion protein substrates. Data in brief. 2018; 18: 203–208. doi: 10.1016/j.dib.2018.03.031 29896511

33. Bozóki B, Mótyán JA, Miczi M, Gazda LD, Tőzsér J. Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation. J Vis Exp. 2019; 143: e58824.

34. Yachdav G, Kloppmann E, Kajan L, Hecht M, Goldberg T, Hamp T, et al. PredictProtein—an open resource for online prediction of protein structural and functional features. Nucleic Acids Res. 2014; 42: W337–343. doi: 10.1093/nar/gku366 24799431

35. Dosztányi Z, Csizmok V, Tompa P, Simon I. IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content. Bioinformatics. 2005; 21: 3433–3434. doi: 10.1093/bioinformatics/bti541 15955779

36. Sirkis R, Gerst JE, Fass D. Ddi1, a eukaryotic protein with the retroviral protease fold. J Mol Biol. 2006; 364: 376–387. doi: 10.1016/j.jmb.2006.08.086 17010377

37. Sali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993; 234: 779–815. doi: 10.1006/jmbi.1993.1626 8254673

38. Kapust RB, Tözsér J, Fox JD, Anderson DE, Cherry S, Copeland TD, et al. Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 2001; 14: 993–1000. doi: 10.1093/protein/14.12.993 11809930

39. Louis JM, Oroszlan S, Tozser J. Stabilization from autoproteolysis and kinetic characterization of the human T-cell leukemia virus type 1 proteinase. J Biol Chem. 1999; 274: (10) pp. 6660–6666. doi: 10.1074/jbc.274.10.6660 10037763

40. Fenyöfalvi Gy, Bagossi P, Copeland TD, Oroszlan S, Boross P, Tőzsér J. Expression and characterization of human foamy virus proteinase. Febs Lett. 1999; 462: 397–401. doi: 10.1016/s0014-5793(99)01563-x 10622733

41. Boross P, Bagossi P, Copeland TD, Oroszlan S, Louis JM, Tözsér J. Effect of substrate residues on the P2' preference of retroviral proteinases. Eur J Biochem. 1999; 264(3): 921–929. doi: 10.1046/j.1432-1327.1999.00687.x 10491141

42. Nallamsetty S, Kapust RB, Tözsér J, Cherry S, Tropea JE, Copeland TD, et al. Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro. Protein Expr Purif. 2004; 38(1): 108–115. doi: 10.1016/j.pep.2004.08.016 15477088

43. Tozser J. Comparative Studies on Retroviral Proteases: Substrate Specificity. Viruses. 2010; 2:(1) pp. 147–165. doi: 10.3390/v2010147 21994605

44. Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357–69. doi: 10.1016/j.addr.2012.09.039 23026637

45. Li M, Gustchina A, Matúz K, Tözsér J, Namwong S, Goldfarb NE, et al. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease. FEBS J. 2011; 278(22):4413–24. doi: 10.1111/j.1742-4658.2011.08364.x 21951660

46. Menéndez-Arias L, Weber IT, Soss J, Harrison RW, Gotte D, Oroszlan S. Kinetic and modeling studies of subsites S4-S3' of Moloney murine leukemia virus protease. J Biol Chem. 1994; 269(24):16795–801. 8207003

47. Louis JM, Nashed NT, Parris KD, Kimmel AR, Jerina DM. Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein. Proc Natl Acad Sci U S A. 1994; 91(17): 7970–7974. doi: 10.1073/pnas.91.17.7970 8058744

48. Gustchina A, Kervinen J, Powell DJ, Zdanov A, Kay J, Wlodawer A. Structure of equine infectious anemia virus proteinase complexed with an inhibitor. Protein Sci. 1996; 5: 1453–1465. doi: 10.1002/pro.5560050802 8844837

49. Strisovsky K, Tessmer U, Langner J, Konvalinka J, Kräusslich HG. Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the “fireman’s grip” hypothesis. Protein Sci. 2000; 9(9): 1631–41. doi: 10.1110/ps.9.9.1631 11045610

50. Sperka T, Boross P, Eizert H, Tözsér J, Bagossi P. Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease. Protein Eng Des Sel. 2006; 19(8): 369–75. doi: 10.1093/protein/gzl021 16799151

51. Bagossi P, Sperka T, Fehér A, Kádas J, Zahuczky G, Miklóssy G, et al. Amino acid preferences for a critical substrate binding subsite of retroviral proteases in type 1 cleavage sites. J Virol. 2005; 79(7):4213–8. doi: 10.1128/JVI.79.7.4213-4218.2005 15767422

52. Eizert H, Bander P, Bagossi P, Sperka T, Miklóssy G, Boross P, et al. Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site. J Virol. 2008; 82(20):10111–7. doi: 10.1128/JVI.00418-08 18701588

53. Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982; 157(1):105–32. doi: 10.1016/0022-2836(82)90515-0 7108955


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