IDENTIFICATION OF THE AMYLOIDOGENIC DOMAIN IN THE INTEGRASE OF YEAST RETROTRANSPOSON TY1



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Abstract

Background: Retrotransposons are mobile genetic elements that replicate via reverse transcription and constitute substantial fractions of eukaryotic genomes; they are also considered evolutionary precursors of retroviruses, and can affect host fitness. Intriguingly, some retrotransposon proteins share motifs with amyloids – fibrous protein aggregates with cross-β architecture that readily self-assemble into polymeric structures and can, in some cases, self-propagate in an infectious manner (prions).

Aim: To identify and characterize potential amyloid-forming regions within the integrase of Saccharomyces cerevisiae Ty1 retrotransposon, which mediates integration of transposon copies into the host genome.

Methods: Computational analysis of the Ty1 integrase sequence was performed with the ArchCandy algorithm to identify putative amyloidogenic motifs. To evaluate the amyloidogenic potential of candidate regions, we employed a yeast-based nucleation assay. Aggregation was visualized by expressing Ty1Int(AD)-GFP fusion constructs. Colocalization of Ty1 amyloidogenic domain with full length in yeast cells was evaluated by confocal microscopy.

Results: We identified and experimentally validated an amyloidogenic region within the Ty1 integrase, designated Ty1Int(AD). ArchCandy predicted the region with amyloidogenic potential, and these predictions were confirmed in a yeast prion‑nucleation assay and by expression of the Ty1Int(AD)–GFP fragment, which formed detergent‑resistant aggregates. Confocal microscopy showed co‑localization of these aggregates with native Ty1 integrase fused with YFP, indicating recruitment of the full‑length protein into inclusions.

Conclusion: Together, these results identify a previously unrecognized amyloidogenic region within Ty1 integrase, possessing amyloid-like properties, and suggest that aggregation of this domain may regulate retrotransposon activity by altering integrase availability and/or function. This is therefore important for the design, optimization, and biosafety assessment of retrotransposon‑based vectors and other GMO constructs.

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About the authors

Andrew A. Zelinsky

Saint Petersburg State University

Email: andrew_zelinsky@mail.ru
ORCID iD: 0000-0003-2068-3024
SPIN-code: 5832-1192

M. Sci. (Biol.), Researcher, Laboratory of Amyloid Biology

Russian Federation, 7/9 Universitetskaya amb., Saint Petersburg, 199034, Russia

Marina V. Ryabinina

Saint Petersburg State University

Email: marina.ryabinina.v@gmail.com
ORCID iD: 0000-0002-5504-7362
SPIN-code: 7113-6941

PhD student, Junior reasercher , Laboratory of Amyloid Biology

Russian Federation, 7/9 Universitetskaya amb., Saint Petersburg, 199034, Russia

Andrey V. Kajava

University of Montpellier

Email: andrey.kajava@crbm.cnrs.fr
ORCID iD: 0000-0002-2342-6886
SPIN-code: 5631-3525

PhD, Professor

France, Place Eugène Bataillon, 34293 Montpellier Cedex 5 Montpellier, France;

Yury O. Chernoff

Georgia Institute of Technology

Email: yury.chernoff@biology.gatech.edu
ORCID iD: 0000-0002-8934-9051
SPIN-code: 6201-0359
ResearcherId: J-2833-2014

School of Biological Sciences, Cand. Sci. (Biol.), Professor

United States, Atlanta, GA, USA

Aleksandr A. Rubel

St. Petersburg State University

Author for correspondence.
Email: arubel@mail.ru
ORCID iD: 0000-0001-6203-2006
SPIN-code: 3961-4690
Scopus Author ID: 23981106300
ResearcherId: D-2903-2013

PhD, Head of the Laboratory of Amyloid biology

Russian Federation, 7/9 Universitetskaya amb., Saint Petersburg, 199034, Russi

References

  1. Kim JM, Vanguri S, Boeke JD, Gabriel A, Voytas DF. Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence. Genome Res. 1998;8(5):464–478. doi: 10.1101/gr.8.5.464.
  2. Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10(10):691–703. doi: 10.1038/nrg2640.
  3. Boeke JD, Garfinkel DJ, Styles CA, Fink GR. Ty elements transpose through an RNA intermediate. Cell. 1985;40(3):491–500. doi: 10.1016/0092-8674(85)90197-7.
  4. Garfinkel DJ, Boeke JD, Fink GR. Ty element transposition: reverse transcriptase and virus-like particles. Cell. 1985;42(2):507–517. doi: 10.1016/0092-8674(85)90108-4.
  5. Finnegan DJ. Eukaryotic transposable elements and genome evolution. Trends Genet. 1989;5(4):103–107. doi: 10.1016/0168-9525(89)90039-5.
  6. Kazazian HH Jr, Wong C, Youssoufian H, Scott AF, Phillips DG, Antonarakis SE. Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man. Nature. 1988;332(6160):164–166. doi: 10.1038/332164a0.
  7. van de Lagemaat LN, Gagnier L, Medstrand P, Mager DL. Genomic deletions and precise removal of transposable elements mediated by recombination between direct repeats. Genome Res. 2005;15(1):95–103. doi: 10.1101/gr.3910705.
  8. Scheifele LZ, Cost GJ, Zupancic ML, Caputo EM, Boeke JD. Retrotransposon overdose and genome integrity. Proc Natl Acad Sci USA. 2009;106(33):13927–13932. doi: 10.1073/pnas.0906552106.
  9. Mount SM, Rubin GM. Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins. Mol Cell Biol. 1985;5(7):1630–1638. doi: 10.1128/mcb.5.7.1630-1638.1985.
  10. Cottee MA, Letham SC, Young GR, Harding M, et al. Structure of a Ty1 restriction factor reveals the molecular basis for retrotransposition restriction. Nat Commun. 2021;12:5471. doi: 10.1038/s41467-021-25849-0.
  11. Kim A, Terzian C, Santamaria P, Pélisson A, Prud'homme N, Bucheton A. Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster. Proc Natl Acad Sci USA. 1994;91(4):1285–1289. doi: 10.1073/pnas.91.4.1285.
  12. Wang J, Han GZ. A missing link between retrotransposons and retroviruses. mBio. 2022;13(2):e00187-22. doi: 10.1128/mbio.00187-22.
  13. UNAIDS. Global AIDS Update 2024. Geneva: UNAIDS; 2024. Available from: https://www.unaids.org/en/resources/fact-sheet
  14. Wensing AM, Calvez V, Ceccherini-Silberstein F, Charpentier C, Günthard HF, Paredes R, et al. 2022 update of the drug resistance mutations in HIV‑1. Top Antivir Med. 2022;30(4):559–574.
  15. Carr M, Bensasson D, Bergman CM. Evolutionary genomics of transposable elements in Saccharomyces cerevisiae. PLoS One. 2012;7(11):e50978. doi: 10.1371/journal.pone.0050978.
  16. Mellor J, Fulton SM, Dobson MJ, Wilson W, Kingsman SM, Kingsman AJ. A retrovirus-like strategy for expression of a fusion protein encoded by yeast transposon Ty1. Nature. 1985;313(5999):243–246. doi: 10.1038/313243a0.
  17. Adams SE, Mellor J, Gull K, Sim RB, Tuite MF, Kingsman SM, Kingsman AJ. The functions and relationships of Ty-VLP proteins in yeast. Cell. 1987;49(1):111–119. doi: 10.1016/0092-8674(87)90761-6.
  18. Garfinkel DJ, Hedge AM, Youngren SD, Copeland TD. Proteolytic processing of pol-TYB proteins from the yeast retrotransposon Ty1. J Virol. 1991;65(9):4573–4581. doi: 10.1128/JVI.65.9.4573-4581.1991.
  19. Devine SE, Boeke JD. Integration of the yeast retrotransposon Ty1 is targeted to regions upstream of genes transcribed by RNA polymerase III. Genes Dev. 1996;10(5):620–633. doi: 10.1101/gad.10.5.620.
  20. Katz RA, Skalka AM. The retroviral enzymes. Annu Rev Biochem. 1994;63:133–173. doi: 10.1146/annurev.bi.63.070194.001025.
  21. Bridier-Nahmias A, Tchalikian-Cosson A, Baller JA, Menouni R, Fayol H, Flores A, et al. An RNA polymerase III subunit determines sites of retrotransposon integration. Science. 2015;348(6234):585–588. doi: 10.1126/science.1259114.
  22. Asif-Laidin A, Conesa C, Coulon A, Menouni R, Fayol H, Lefrançois P, et al. A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes. EMBO J. 2020;39(17):e104337. doi: 10.15252/embj.2019104337.
  23. Nguyen PQ, Conesa C, Rabut E, Bragagnolo G, Gouzerh C, Fernández-Tornero C, Lesage P, Reguera J, Acker J. Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro. J Biol Chem. 2021;297(4):101093. doi: 10.1016/j.jbc.2021.101093.
  24. Barkova A, Adhya I, Conesa C, Asif-Laidin A, Bonnet A, Rabut E, Chagneau C, Lesage P, Acker J. A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition. Mob DNA. 2022. 13(1):26. doi: 10.1186/s13100-022-00284-0.
  25. Uversky VN, Fink AL. Conformational constraints for amyloid fibrillation: the importance of being unfolded. Biochim Biophys Acta. 2004;1698(2):131–153. doi: 10.1016/j.bbapap.2003.12.008.
  26. Murray KA, Hughes MP, Hu CJ, Sawaya MR, Salwinski L, Pan H, et al. Identifying amyloid-related diseases by mapping mutations in low-complexity protein domains to pathologies. Nat Struct Mol Biol. 2022;29(6):529–536. doi: 10.1038/s41594-022-00774-y.
  27. Tzotzos S, Doig AJ. Amyloidogenic sequences in native protein structures. Protein Sci. 2010;19(2):327-348. doi: 10.1002/pro.314.
  28. Benson MD, Buxbaum JN, Eisenberg DS, Merlini G, Saraiva MJM, Sekijima Y, Sipe JD, Westermark P. Amyloid nomenclature 2020: update and recommendations by the International Society of Amyloidosis (ISA) nomenclature committee. Amyloid. 2020. 27(4):217-222. doi: 10.1080/13506129.2020.
  29. Rubel MS, Fedotov SA, Grizel AV, Sopova JV, Malikova OA, Chernoff YO, Rubel AA. Functional mammalian amyloids and amyloid-like proteins. Life (Basel). 2020;10(9):156. doi: 10.3390/life10090156.
  30. Kulichikhin KY, Malikova OA, Zobnina AE, Zalutskaya NM, Rubel AA. Interaction of Proteins Involved in Neuronal Proteinopathies. Life (Basel). 2023. 13(10):1954. doi: 10.3390/life13101954.
  31. McKinley MP, Bolton DC, Prusiner SB. A protease-resistant protein is a structural component of the scrapie prion. Cell. 1983;35:57–62. doi: 10.1016/0092-8674(83)90207-6.
  32. Takeda A, Hashimoto M, Mallory M, et al. Abnormal distribution of the non-Abeta component of Alzheimer’s disease amyloid precursor/alpha-synuclein in Lewy body disease as revealed by proteinase K and formic acid pretreatment. Lab Invest. 1998;78:1169–1177.
  33. Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, Kushnirov VV. Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J Biol Chem. 2003;278(49):49636–49643. doi: 10.1074/jbc.M307996200.
  34. Collins SR, Douglass A, Vale RD, Weissman JS. Mechanism of prion propagation: amyloid growth occurs by monomer addition. PLoS Biol. 2004;2(10):e321. doi: 10.1371/journal.pbio.0020321.
  35. Castello F, Paredes JM, Ruedas-Rama MJ, Martin M, Orte A, Crovetto L. Two-step amyloid aggregation: sequential lag phase intermediates. Sci Rep. 2017;7:40065. doi: 10.1038/srep40065.
  36. Beckwith SL, Nomberg EJ, Newman AC, Taylor JV, Guerrero-Ferreira RC, Garfinkel DJ. An interchangeable prion-like domain is required for Ty1 retrotransposition. Proc Natl Acad Sci U S A. 2023. 120(30):e2303358120. doi: 10.1073/pnas.2303358120.
  37. Chandramowlishwaran P, Sun M, Casey KL, Romanyuk AV, Grizel AV, Sopova JV, et al. Mammalian amyloidogenic proteins promote prion nucleation in yeast. J Biol Chem. 2018;293(9):3436–3450. doi: 10.1074/jbc.M117.809004.
  38. Ahmed AB, Znassi N, Château M-T, Kajava AV. A structure-based approach to predict predisposition to amyloidosis. Alzheimers Dement. 2015; doi: 10.1016/j.jalz.2014.06.007.
  39. Redl I, Fisicaro C, Dutton O, Hoffmann F, Henderson L, Owens BMJ, Heberling M, Paci E, Tamiola K. ADOPT: intrinsic protein disorder prediction through deep bidirectional transformers. NAR Genom Bioinform. 2023. 5(2):lqad041. doi: 10.1093/nargab/lqad041.
  40. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1989.
  41. Allen KD, Chernova TA, Tennant EP, Wilkinson KD, Chernoff YO. Effects of ubiquitin system alterations on the formation and loss of a yeast prion. J Biol Chem. 2007;282(5):3004–3013. doi: 10.1074/jbc.M609597200.
  42. Chernoff YO, Newnam GP, Kumar J, Allen K, Zink AD. Role of the chaperone Ssb in formation, stability and toxicity of the [PSI+] prion. Mol Cell Biol. 1999;19:8103–8112. doi: 10.1128/MCB.19.12.8103.
  43. Gietz RD, Schiestl RH. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2(1):31–34. doi: 10.1038/nprot.2007.14.
  44. Curcio MJ, Garfinkel DJ. Single-step selection for Ty1 element retrotransposition. Proc Natl Acad Sci U S A. 1991.;88(3):936-40. doi: 10.1073/pnas.88.3.936.
  45. Rubel AA, Saĭfitdinova AF, Lada AG, Nizhnikov AA, Inge-Vechtomov SG, Galkin AP. [Yeast chaperone Hspl04 regulates gene expression on the posttranscriptional level]. Mol Biol (Mosk). 2008. 42(1):123-30. Russian. doi: 10.1134/s0026893308010184.
  46. Sikorski RS, Hieter P. A system of shuttle vectors andyeast host strains designed for efficient manipulationof DNA in Saccharomyces cerevisiae. Genetics. 1989. 122(1):19-27. doi: 10.1093/genetics/122.1.19.
  47. Chernoff YO, Grizel AV, Rubel AA, Zelinsky AA, Chandramowlishwaran P, Chernova TA. Application of yeast to studying amyloid and prion diseases. Adv Genet. 2020;105:293-380. doi: 10.1016/bs.adgen.2020.01.002.
  48. Kachkin D, Zelinsky AA, Romanova NV, Kulichikhin KY, Zykin PA, Khorolskaya JI, Deckner ZJ, Kajava AV, Rubel AA, Chernoff YO. Prion-like Properties of Short Isoforms of Human Chromatin Modifier PHC3. Int J Mol Sci. 2025 Feb 11;26(4):1512. doi: 10.3390/ijms26041512.
  49. Bagriantsev SN, Kushnirov VV, Liebman SW. Analysis of amyloid aggregates using agarose gel electrophoresis. Methods Enzymol. 2006; 412:33-48. doi: 10.1016/S0076-6879(06)12003-0.

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