Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Effects of Mutation at Two Conserved Aspartate Residues and a Serine Residue on Functions of Yeast TSA 1

Saccharomyces cerevisiae TSA1의 보존된 아스파트산 잔기 및 세린 잔기의 변이가 과산화효소 활성 및 샤페론 활성에 미치는 영향

  • Lee, Songmi (Department of Food and Nutrition, Dongshin University) ;
  • Cho, Eun Yi (College of Pharmacy, Ajou University) ;
  • Kim, Kanghwa (Department of Food and Nutrition, Chonnam National University)
  • Received : 2017.02.15
  • Accepted : 2017.03.09
  • Published : 2017.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

Alignment of 967 reference sequences of the typical 2-Cys peroxiredoxin family of proteins revealed that 10 amino acids were conserved, with over 99% identity. To investigate whether the conserved aspartic acid residues and serine residue affect the peroxidase and chaperone activity of the protein, we prepared yeast TSA1 mutant proteins in which aspartic acids at positions 75 and 103 were replaced by valine or asparagine, and serine at position 73 was replaced by alanine. By non-reducing SDS-PAGE, TSA1 and the S73A, D75V and D75N mutants were detected in dimeric form, whereas the D103V and D103N mutants were detected in various forms, ranging from high molecular-weight to monomeric. Compared with wild type TSA1, the D75N mutant exhibited 50% thioredoxin peroxidase activity, and the S73A and D75V mutants showed 25% activity. However, the D103V and D103N mutants showed no peroxidase activity. All proteins, except for the D103V and D103N mutants, exhibited chaperone activity at $43^{\circ}C$. Our results suggest that the two conserved aspartic acid residues and serine residue of TSA1 play important roles in its thioredoxin peroxidase activity, and D103 plays a critical role in its chaperone activity.

퍼옥시레독신은 티오레독신, 티오레독신 환원효소, NADPH로 이루어진 티오레독신 시스템의 환원력을 이용하여 과산화물을 제거하는 티오레독신 과산화효소 활성과 다른 단백질의 열변성에 의한 응집을 막아주는 샤페론 활성을 갖는 효소이다. 정형 2-Cys Prx군에 속하는 퍼옥시레독신 참고서열 1,024개 중 부분적인 서열 등을 제외한 967개 서열을 정렬하였을 때 75번과 103번 아스파트산 잔기는 99% 보존되었고, 73번 세린 잔기는 97% 보존되었음에도 불구하고 잘 보존된 아스파트산 잔기와 세린 잔기에 대해 알려지지 않았다. 이 잔기가 TSA1의 두가지 효소 활성에 미치는 영향을 알아보기 위해 재조합 단백질을 이용하여 활성도를 알아보았다. in vitro 실험을 통하여 잘 보존된 잔기인 103번 아스파트산은 75번 아스파트산보다 티오레독신 퍼옥시레독신 활성 및 분자 샤페론 활성에 더 영향을 미치고, 103번의 음전하는 분자 샤페론 활성에 중요한 역할을 하며 과산화효소활성에는 75번과 103번의 음전하가 관여함을 알 수 있었다. 또한 73의 세린 잔기 역시 과산화효소에 영향을 미치는 잔기임을 알 수 있었다. 최근 출아 효모 퍼옥시레독신인 TSA2의 79번과 109번의 세린 잔기를 시스테인 잔기로 변이시킨 경우 두 변이 단백질 모두 과산화효소 활성과 샤페론 활성이 증가되었는데 이는 ${\beta}$-sheet 구조의 증가와 관련되는 것으로 보고하였다[28]. 이들 두 세린 잔기는 TSA1 구조에 의하면 모두 ${\alpha}$-나선 구조에 위치하였다. 반면에 73번의 세린 잔기는 ${\beta}$-sheet의 C-말단에 위치하는 잔기로 과산화효소 활성에 대한 영향이 다르게 나타나는 것으로 추정된다. 추후 생체 내 실험을 통하여 아스파트산 잔기의 변이가 과산화물 저항성이 미치는 영향 및 열 저항성(thermal stress)에 미치는 역할을 살펴볼 필요가 있다. 또한 아스파트산 잔기와 과산화물과의 반응 및 분자 샤페론과의 반응에 장애가 되는 요인이 무엇인지에 대한 추가 연구가 필요할 것이다.

Keywords

References

  1. Nordberg J, Arner ESJ. 2001. Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31: 1287-1312. https://doi.org/10.1016/S0891-5849(01)00724-9
  2. Rhee SG, Kang SW, Jeong W, Chang T-S, Yang K-S, Woo HA. 2005. Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr. Opin. Cell Biol. 17: 183-189. https://doi.org/10.1016/j.ceb.2005.02.004
  3. Panfili E, Sandri G, Ernster L. 1991. Distribution of glutathione peroxidases and glutathione reductase in rat brain mitochondria. FEBS 290: 35-37. https://doi.org/10.1016/0014-5793(91)81219-X
  4. Rhee SG, Chae HZ, Kim K. 2005. Peroxiredoxin: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling. Free Radic. Biol. Med. 38: 1543-1552. https://doi.org/10.1016/j.freeradbiomed.2005.02.026
  5. Karplus PA. 2015. A primer on peroxiredoxin biochemistry. Free Radic. Biol. Med. 80: 183-190. https://doi.org/10.1016/j.freeradbiomed.2014.10.009
  6. Martin RE, Cao Z, Bulleid NJ. 2014. Regulating the level of intracellular hydrogen peroxide: the role of peroxiredoxin IV. Biochem. Soc. Trans. 42: 42-46. https://doi.org/10.1042/BST20130168
  7. Hoyle NP, O’Neill JS. 2014. Oxidation-reduction cycle of peroxiredoxin proteins and non transcriptional aspects of timekeeping. Biochemistry 54: 184-193.
  8. Kang SW, Rhee SG, Chang TS, Jeong WJ, Chai MH. 2005. 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends in Mol. Med. 11: 571-578. https://doi.org/10.1016/j.molmed.2005.10.006
  9. Park SG, Cha MK, Jeong W, Kim IH. 2000. Distinct physiological functions of thiol peroxidase isoenzymes in Saccharomyces cerevisiae. J. Biol. Chem. 275: 5723-5732. https://doi.org/10.1074/jbc.275.8.5723
  10. Seo MS, Kang SW, Kim K, Baines I, Lee TH, Rhee SG. 2000. Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J. Biol. Chem. 275: 20346-20354. https://doi.org/10.1074/jbc.M001943200
  11. Jang HH, Lee KO, Chi YH, Jung BG, Park SK, Park JH, et al. 2004. Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function. Cell 117: 625-635. https://doi.org/10.1016/j.cell.2004.05.002
  12. Gething MJ, Sambrook J. 1992. Protein folding in the cell. Nature 355: 33-45. https://doi.org/10.1038/355033a0
  13. Hendrick JP, Hartl FU. 1993. Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62: 349-384. https://doi.org/10.1146/annurev.bi.62.070193.002025
  14. Ellis RJ. 1993. The general concept of molecular chaperones. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 339: 257-261. https://doi.org/10.1098/rstb.1993.0023
  15. Nover L. 2000. Heat stress response; a complex gene with chaperones and transcription factors. Procedings of EMBO Lecture Course.
  16. Feder ME, Hofmann GE. 1999. Heat-shock proteins, molecular chaperones, and the stress trsponse; evolutionary and ecological physiology. Annu. Rev. Physiol. 61: 243-282. https://doi.org/10.1146/annurev.physiol.61.1.243
  17. Chae HZ, Oubrahim H, Park JW, Rhee SG, Chock PB. 2011. Protein glutathionylation in the regulation of peroxidase that function as antioxidants, molecular chaperone, and signal modulators. Antioxidants & Redox Signaling 16: 506-523.
  18. Woo HA, Yim SH, Shin DH, Kang D, Yu D-Y, Rhee SG. 2010. Inactivation of peroxiredoxin I by phosphorylation allows localized $H_2O_2$ accumulation for cell signaling. Cell 140: 517-528. https://doi.org/10.1016/j.cell.2010.01.009
  19. Rhee SG. 2016. Overview on peroxiredoxin. Mol. Cells. 39:1-5. https://doi.org/10.14348/molcells.2016.2368
  20. Kim JS, Bang MA, Lee S, Chae HZ, Kim K. 2010. Distinct functional roles of peroxiredoxin isozymes and glutathione peroxidase from fission yeast, Schizosaccharomyces pombe. BMB Rep. 43: 170-175. https://doi.org/10.5483/BMBRep.2010.43.3.170
  21. Chae HZ, Chung SJ, Rhee SG. 1994. Thioredoxin-dependent Peroxidase Reductase from yeast. J. Biol. Chem. 269: 27670-27078.
  22. Lee S, Kim JS, Yun CH, Chae HZ, Kim K. 2009. Aspartyl aminopeptidase of Schizosaccharomyces pombe has a molecular chaperone function. BMB Rep. 42: 812-816. https://doi.org/10.5483/BMBRep.2009.42.12.812
  23. Wood ZA, Schroder E, Harris JR, Poole LB. 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem. Sci. 28: 32-40. https://doi.org/10.1016/S0968-0004(02)00003-8
  24. Montemaritini M, Kalisz HM, Hecht HJ. 1999. Activaion of active-site cysteine residues in the peroxiredoxin-type tryparedoxin peroxidase of Crithidia fasciculate. Eur. J. Biochem. 264: 516-524. https://doi.org/10.1046/j.1432-1327.1999.00656.x
  25. Flohe L, Budde H, Bruns K, Castro H, Clos J, Hofmann B, et al. 2002. Tryparedoxin peroxidase of Leishmanis donovani : molecular cloning, heterogous expression, specificity and catalytic mechanism. Arch. Biochem. Biophys. 397: 324-335. https://doi.org/10.1006/abbi.2001.2688
  26. Schroder E, Littlechid JA, Lebedev AA, Errington N, Vagin AA, Isupov MN. 2000. Crystal structure of decameric 2-Cys peroxiredoxin from human erythrocytes at 1.7 A. Structure 8: 605-615. https://doi.org/10.1016/S0969-2126(00)00147-7
  27. Chae HZ, Uhm TB, Rhee SG. 1994. Dimerization of thiol-specific antioxidant and essential role of cysteine 47. 1994. Proc. Natl. Acad. Sci. USA 91:7022-7026. https://doi.org/10.1073/pnas.91.15.7022
  28. Hong SH, Lee SS, Chung JM, Jung HS, Singh S, Mondal S, et al. 2017. Protoplasma 254: 327-334. https://doi.org/10.1007/s00709-016-0948-0