Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of a Highly Active Fluorescence-Based Detector for Yeast G Protein-Coupled Receptor Ste2p

  • Hong, Jin Woo (School of Biotechnology, Yeungnam University) ;
  • Ahn, Hee Jun (School of Biotechnology, Yeungnam University) ;
  • Baek, Jee Su (School of Biotechnology, Yeungnam University) ;
  • Hong, Eun young (School of Biotechnology, Yeungnam University) ;
  • Jin, Dong Hoon (Asan Institute for Life Science, Department of Convergence Medicine, College of Medicine, University of Ulsan) ;
  • Khang, Yong Ho (School of Biotechnology, Yeungnam University) ;
  • Hong, Nam Joo (School of Biotechnology, Yeungnam University)
  • Received : 2018.05.02
  • Accepted : 2018.09.17
  • Published : 2018.10.28
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Abstract

Twenty analogs of $[Orn^6,D-Ala^9]{\alpha}-factor$ were synthesized and assayed for their biological activities: seven analogs of $[Orn^6,X^9]{\alpha}-factor$, seven analogs of $[X^6,D-Ala^9]{\alpha}-factor$, five analogs of $[X^5,X^6,D-Ala^9]{\alpha}-factor$, and native ${\alpha}-factor$ (X = amino acids). Their biological activities (halo, gene induction, and affinity) were measured using S. cerevisiae Y7925 and LM102 and compared with those of native ${\alpha}-factor$ (100%). G protein-coupled receptor was expressed in strain LM102 containing pESC-LEU-STE2 vector. $[Dap^6,D-Ala^9]{\alpha}-factor$ with weak halo activity (10%) showed the highest receptor affinity (> 230%) and the highest gene induction activity (167%). $[Arg^6,D-Ala^9]{\alpha}-factor$ showed the highest halo activity (2,000%). The number of active binding sites per cell (about 20,000 for strain LM102) was determined using a newly-designed fluorescence-based detector, $[Arg^6,D-Ala^9]{\alpha}-factor-Edan$, with high sensitivity (12,500-fold higher than the absorption-based detector $[Orn^6]{\alpha}-factor-[Cys]_3$).

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References

  1. Dohlamn HG. 2002. G proteins and pheromone signaling. Annu. Rev. Physiol. 64: 129-152. https://doi.org/10.1146/annurev.physiol.64.081701.133448
  2. Schoneberg T, Schulz A, Biebermann H, Hermsdorf T, Rompler H, Sangkuhl K. 2004. Mutant G-protein-coupled receptors as a cause of human diseases. Pharmac. Ther. 104: 173-206. https://doi.org/10.1016/j.pharmthera.2004.08.008
  3. Klabunde T, Hessler G. 2002. Drug design strategies for targeting G-protein-coupled receptors. Chembiochem 3: 928 -944. https://doi.org/10.1002/1439-7633(20021004)3:10<928::AID-CBIC928>3.0.CO;2-5
  4. Naider F, Becker JM. 2004. The alpha-factor mating pheromone of Saccharomyces cerevisiae: a model for studying the interaction of peptide hormones and G protein-coupled receptors. Peptides 25: 1441-1463. https://doi.org/10.1016/j.peptides.2003.11.028
  5. Stotzler D, Duntze W. 1976. Isolation and characterization of four related peptides exhibiting alpha factor activity from Saccharomyces cerevisiae. Eur. J. Biochem. 65: 257-262. https://doi.org/10.1111/j.1432-1033.1976.tb10412.x
  6. Naider F, Becker JM. 1986. Structure-activity relationships of the yeast alpha-factor. CRC Crit. Rev. Biochem. 21: 225-248. https://doi.org/10.3109/10409238609113612
  7. Garbow JR, Breslav M, Antohi O, Naider F. 1994. Conformational analysis of the Saccharomyces cerevisiae tridecapeptide mating pheromone by 13C, 15N rotational-echo double resonance nuclear magnetic resonance spectroscopy. Biochemistry 33: 10094-10099. https://doi.org/10.1021/bi00199a037
  8. Henry LK, Khare S, Son C, Babu VV, Naider F, Becker JM. 2002. Identification of a contact region between the tridecapeptide alpha-factor mating pheromone of Saccharomyces cerevisiae and its G protein-coupled receptor by photoaffinity labeling. Biochemistry 41: 6128-6139. https://doi.org/10.1021/bi015863z
  9. Ding FX, Lee BK, Hauser M, Davenport L, Becker JM, Naider F. 2001. Probing the binding domain of the Saccharomyces cerevisiae alpha-mating factor receptor with rluorescent ligands. Biochemistry 40: 1102-1108. https://doi.org/10.1021/bi0021535
  10. Abel MG, Zhang YL, Lu HF, Naider F, Becker JM. 1998. Structure-function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine-scanned analogs. J. Pept. Res. 52: 95-106.
  11. Gounarides JS, Broido MS, Becker JM, Naider F. 1993. Conformational analysis of [D-Ala9]alpha-factor and [L-Ala9]alpha-factor in solution and in the presence of lipid. Biochemistry 32: 908-917. https://doi.org/10.1021/bi00054a023
  12. Ahn HJ, Hong EY, Jin DH, Hong NJ. 2014. Highly active analogs of $\alpha$-factor and their activities against Saccharomyces cerevisiae. Bull. Korean Chem. Soc. 35: 1365-1374. https://doi.org/10.5012/bkcs.2014.35.5.1365
  13. Ahn HJ, Kim HJ, Jin DH, Hong NJ. 2015. Spectrophotometric determination of affinities of $\alpha$-factors for their G protein-coupled receptors in Saccharomyces cerevisiae. Bull. Korean Chem. Soc. 36: 1885-1896. https://doi.org/10.1002/bkcs.10367
  14. Kim KM, Lee YH, Naider F, Uddin MS, Akal-Strader A, Hauser M, et al. 2012. Multiple regulatory roles of the carboxy terminus of Ste2p a yeast GPCR. Pharmacol. Res. 65: 31-40. https://doi.org/10.1016/j.phrs.2011.11.002
  15. Hanahan D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580. https://doi.org/10.1016/S0022-2836(83)80284-8
  16. Gietz RD, Schiestl RH. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2: 31-34. https://doi.org/10.1038/nprot.2007.13
  17. Hong NJ. 2010. Structure-activity relationships of 13- and 14-membered cyclic partial retro-inverso pentapeptides related to enkephalin. Bull. Korean Chem. Soc. 31: 874-880. https://doi.org/10.5012/bkcs.2010.31.04.874
  18. Kim DH, Hong NJ. 2012. Activity profiles of linear, cyclic monomer and cyclic dimer of enkephalin. Bull. Korean Chem. Soc. 33: 261-269. https://doi.org/10.5012/bkcs.2012.33.1.261
  19. Li J, Wang S, VanDusen WJ, Schultz LD, George HA, Herber WK, et al. 2000. Green fluorescent protein in Saccharomyces cerevisiae: real-time studies of the GAL1 promoter. Biotechnol. Bioeng. 70: 187-196. https://doi.org/10.1002/1097-0290(20001020)70:2<187::AID-BIT8>3.0.CO;2-H
  20. Taylor RG, Walker DC, McInnes RR. 1993. E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing. Nucleic Acids Res. 21: 1677-1678. https://doi.org/10.1093/nar/21.7.1677
  21. Bimboim HC, Doly J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7: 1513-1523. https://doi.org/10.1093/nar/7.6.1513
  22. Chrambach A, Rodbard D. 1971. Polyacrylamide gel electrophoresis. Science 172: 440-451. https://doi.org/10.1126/science.172.3982.440
  23. Thibodeau SA, Fang R, Joung JK. 2004. High-throughput $\beta$-galactosidase assay for bacterial cell-based reporter systems. Biotechniques 36: 410-415. https://doi.org/10.2144/04363BM07
  24. Vidal-Aroca, Giannattasio M, Brunelli E, Vezzoli A, Plevani P, Muzi-Falconi M, et al. 2006. One-step high-throughput assay for quantitative detection of beta-galactosidase activity in intact gram-negative bacteria, yeast, and mammalian cells. Biotechniques 40: 433-434. https://doi.org/10.2144/000112145
  25. Plovins A, Alvarez AM, Ibanez M, Molina M, Nombela C. 1994. Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells. Appl. Environ. Microbiol. 60: 4638-4641.
  26. Raths SK, Naider F, Becker JM. 1988 . Peptide Analogues Compete with the Binding of $\alpha$-factor to Its Receptor in Saccharomyces cerevisiae. J. Biol. Chem. 263: 17333-17341.
  27. Kippert F. 1995. A rapid permeabilization procedure for accurate quantitative determination of $\beta$-galactosidase activity in yeast cells. FEMS Microbiol Lett. 128: 201-206.
  28. Jenness DD, Sprtrick P. 1986. Down regulation of the $\alpha$-factor pheromone receptor in S. cerevisiae. Cell 46: 345-353. https://doi.org/10.1016/0092-8674(86)90655-0
  29. Mathew E, Bajaj A, Connelly SM, Sargsyan H, Ding FX, Hajduczok AJ, et al. 2011. Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p. J. Mol. Biol. 409: 513-528. https://doi.org/10.1016/j.jmb.2011.03.059
  30. Son CD, Sargsyan H, Naider F, Becker JM. 2004. Identification of ligand binding regions of the Saccharomyces cerevisiae $\alpha$-factor pheromone receptor by photoaffinity cross-linking. Biochemistry 43: 13193-13203. https://doi.org/10.1021/bi0496889
  31. Xue CB, Mckinney A, Lu HF, Jiang Y, Becker JM, Naider F. 1996. Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs. Int. J. Pept. Protein. Res. 47: 131-141.
  32. Banerjee R, Chattopadhyay S, Basu G. 2009. Conformational preferences of a short Aib/Ala-based water-soluble peptide as a function of temperature. Proteins 76: 184-200. https://doi.org/10.1002/prot.22337
  33. Wakamatsu K, Okada A, Miyazawa T, Masui Y, Sakakibara S, Higashijima T. 1987. Conformations of yeast $\alpha$-mating factor and analog peptides as bound to phospholipid bilayer. Eur. J. Biochem. 163: 331-338. https://doi.org/10.1111/j.1432-1033.1987.tb10804.x
  34. Higashijima T, Masui Y, Chino N, Sakakibara S, Kita H, Miyazawa T. 1984. Conformations of tridecapeptide $\alpha$-mating factor from yeast Saccharomyces cerevisiae and analog peptides in aqueous solution. Eur. J. Biochem. 140: 163-171. https://doi.org/10.1111/j.1432-1033.1984.tb08081.x
  35. Marsh L. 1992. Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist. Mol. Cell. Biol. 12: 3959-3966. https://doi.org/10.1128/MCB.12.9.3959
  36. Liu S, Henry LK, Lee BK, Becker JM, Naider F. 2000. Position 13 analogs of the tridecapeptide mating pheromone from Saccharomyces cerevisiae: design of an iodinatable ligand for receptor binding. J. Pept. Res. 56: 24-34. https://doi.org/10.1034/j.1399-3011.2000.00730.x
  37. Widmann C, Gibson S, Jarpe MB, Johnson GL. 1999. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79: 143-180. https://doi.org/10.1152/physrev.1999.79.1.143
  38. Oehlen LJ, Mckinney JD, Cross FR. 1996. Ste12 and Mcm1 regulate cell cycle-dependent transcription of FAR1. Mol. Cell. Biol. 16: 2830-2837. https://doi.org/10.1128/MCB.16.6.2830
  39. Bardwell L, Cook JG, Chang EC, Cairns BR, Thorner J. 1996. Signaling in the yeast pheromone response pathway: specific and high-affinity interaction of the mitogen-activated protein (MAP) kinases Kss1 and FUS3 with the upstream MAP kinase kinase Ste7. Mol. Cell. Biol. 16: 3637-3650. https://doi.org/10.1128/MCB.16.7.3637