1887

Abstract

A low-molecular-mass aspartic protease inhibitor was isolated from a novel sp. The inhibitor was purified to homogeneity, as shown by reversed-phase HPLC and SDS-PAGE. The of the inhibitor was 1585 and the amino acid composition showed the presence of D, D, D, E, A, K, L, Y, H, I and W residues. The steady-state kinetic interactions of aspartic protease with the inhibitor revealed the reversible, competitive, time-dependent tight-binding nature of the inhibitor, with IC and values of 1.8 and 0.85 µM, respectively. Fluorescence spectroscopy and circular dichroism analysis showed that inactivation of the enzyme was due to binding of the inhibitor to the active site. The inhibitor was found to inhibit mycelial growth and spore germination of and with MIC values of 1.65 and 0.30 µg ml, respectively. This study will potentially open the way towards the development of a tight-binding peptidic inhibitor against fungal aspartic proteases to combat human fungal infections.

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2012-07-01
2024-04-19
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References

  1. Amsterdam D. ( 1991). Susceptibility testing of antimicrobials in liquid media. Antibiotics in Laboratory Medicine72–78 Lorian V. Baltimore, MD: Williams & Wilkins;
    [Google Scholar]
  2. Andrade M. A., Chacón P., Merelo J. J., Morán F. ( 1993). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6:383–390[PubMed]
    [Google Scholar]
  3. Arnon R. ( 1970). Proteolytic enzymes. Methods in Enzymology226–244 Perlmann G. E., Lorand L. New York: Academic Press;
    [Google Scholar]
  4. Bradford M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
    [Google Scholar]
  5. Broekaert W. F., Terras F. R. G., Cammue B. P. A., Vanderleyden J. ( 1990). An automated quantitative assay for fungal growth inhibition. FEMS Microbiol Lett 69:55–59 [View Article]
    [Google Scholar]
  6. Cammue B. P. A., De Bolle M. F. C., Terras F. R. G., Proost P., Van Damme J., Rees S. B., Vanderleyden J., Broekaert W. F. ( 1992). Isolation and characterization of a novel class of plant antimicrobial peptides from Mirabilis jalapa L. seeds. J Biol Chem 267:2228–2233[PubMed]
    [Google Scholar]
  7. Cavallini D., Graziani M. T., Dupré S. ( 1966). Determination of disulphide groups in proteins. Nature 212:294–295 [View Article][PubMed]
    [Google Scholar]
  8. Cha S., Agarwal R. P., Parks R. E. Jr ( 1975). Tight-binding inhibitors–II. Non-steady state nature of inhibition of milk xanthine oxidase by allopurinol and alloxanthine and of human erythrocytic adenosine deaminase by coformycin. Biochem Pharmacol 24:2187–2197 [View Article][PubMed]
    [Google Scholar]
  9. Cheung H. C. ( 1991). Principles. Topics in Fluorescence Spectroscopy127–176 Lakowicz J. R. New York: Plenum Press;
    [Google Scholar]
  10. Dagenais T. R. T., Keller N. P. ( 2009). Pathogenesis of Aspergillus fumigatus in invasive aspergillosis. Clin Microbiol Rev 22:447–465[PubMed]
    [Google Scholar]
  11. Dash C., Phadtare S., Deshpande V., Rao M. ( 2001a). Structural and mechanistic insight into the inhibition of aspartic proteases by a slow-tight binding inhibitor from an extremophilic Bacillus sp.: correlation of the kinetic parameters with the inhibitor induced conformational changes. Biochemistry 40:11525–11532 [View Article][PubMed]
    [Google Scholar]
  12. Dash C., Ahmad A., Nath D., Rao M. ( 2001b). Novel bifunctional inhibitor of xylanase and aspartic protease: implications for inhibition of fungal growth. Antimicrob Agents Chemother 45:2008–2017 [View Article][PubMed]
    [Google Scholar]
  13. Dash C., Kulkarni A., Dunn B., Rao M. ( 2003). Aspartic protease inhibitors: implication in drug development. Crit Rev Biochem Mol 38:89–119
    [Google Scholar]
  14. Denning D. W., Stevens D. A. ( 1990). Antifungal and surgical treatment of invasive aspergillosis: review of 2,121 published cases. Rev Infect Dis 12:1147–1201 [View Article][PubMed]
    [Google Scholar]
  15. Dixon M. ( 1953). The determination of enzyme inhibitor constants. Biochem J 55:170–171[PubMed]
    [Google Scholar]
  16. Dos Santos A. L. ( 2010). HIV aspartyl protease inhibitors as promising compounds against Candida albicans . World J Biol Chem 1:21–30 [View Article][PubMed]
    [Google Scholar]
  17. Dos Santos A. L. ( 2011). Protease expression by microorganisms and its relevance to crucial physiological/pathological events. World J Biol Chem 2:48–58 [View Article][PubMed]
    [Google Scholar]
  18. Grant G. A., Eisen A. Z., Bradshaw R. A. ( 1970). Proteolytic enzymes. Methods in Enzymology722–734 Perlmann G. E., Lorand L. New York: Academic Press;
    [Google Scholar]
  19. Hartmann T., Cairns T. C., Olbermann P., Morschhäuser J., Bignell E. M., Krappmann S. ( 2011). Oligopeptide transport and regulation of extracellular proteolysis are required for growth of Aspergillus fumigatus on complex substrates but not for virulence. Mol Microbiol 82:917–935[PubMed]
    [Google Scholar]
  20. Hogan L. H., Klein B. S., Levitz S. M. ( 1996). Virulence factors of medically important fungi. Clin Microbiol Rev 9:469–488[PubMed]
    [Google Scholar]
  21. Kothary M. H., Chase T. Jr, Macmillan J. D. ( 1984). Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice. Infect Immun 43:320–325[PubMed]
    [Google Scholar]
  22. Kulkarni A., Rao M. ( 2007). Biochemical characterization of an aspartic protease from Vigna radiata: kinetic interactions with the classical inhibitor pepstatin implicating a tight binding mechanism. Biochim Biophys Acta 1774:619–627[PubMed] [CrossRef]
    [Google Scholar]
  23. Kumar S., Tamura K., Jakobsen I. B., Nei M. ( 2001). mega2: molecular evolutionary genetics analysis software. Bioinformatics 17:1244–1245 [View Article][PubMed]
    [Google Scholar]
  24. Lakowicz J. R. ( 1983). Principles of Fluorescence Spectroscopy New York: Plenum Press; [CrossRef]
    [Google Scholar]
  25. Lee J. D., Kolattukudy P. E. ( 1995). Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung. Infect Immun 63:3796–3803[PubMed]
    [Google Scholar]
  26. Makimura K., Murayama S. Y., Yamaguchi H. ( 1994). Detection of a wide range of medically important fungi by the polymerase chain reaction. J Med Microbiol 40:358–364 [View Article][PubMed]
    [Google Scholar]
  27. Merelo J. J., Andrade M. A., Prieto A., Moran F. ( 1994). Proteinotopic feature maps. Neurocomputing 6:443–454 [View Article]
    [Google Scholar]
  28. Monod M., Capoccia S., Léchenne B., Zaugg C., Holdom M., Jousson O. ( 2002). Secreted proteases from pathogenic fungi. Int J Med Microbiol 292:405–419 [View Article][PubMed]
    [Google Scholar]
  29. Mueller J. H., Hinton J. ( 1941). A protein-free medium for primary isolation of the Gonococcus and Meningococcus . Exp Biol Med 48:330–333 [CrossRef]
    [Google Scholar]
  30. Nguyen J.-T., Hamada Y., Kimura T., Kiso Y. ( 2008). Design of potent aspartic protease inhibitors to treat various diseases. Arch Pharm (Weinheim) 341:523–535 [View Article][PubMed]
    [Google Scholar]
  31. Pawagi A. B., Deber C. M. ( 1990). Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein. Biochemistry 29:950–955 [View Article][PubMed]
    [Google Scholar]
  32. Rao M. B., Tanksale A. M., Ghatge M. S., Deshpande V. V. ( 1998). Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597–635[PubMed]
    [Google Scholar]
  33. Reich J. G. ( 1992). Curve Fitting and Modelling for Scientists and Engineers New York: McGraw-Hill;
    [Google Scholar]
  34. Rhodes J. C., Bode R. B., McCuan-Kirsch C. M. ( 1988). Elastase production in clinical isolates of Aspergillus . Diagn Microbiol Infect Dis 10:165–170[PubMed]
    [Google Scholar]
  35. Richards A. D., Roberts R., Dunn B. M., Graves M. C., Kay J. ( 1989). Effective blocking of HIV-1 proteinase activity by characteristic inhibitors of aspartic proteinases. FEBS Lett 247:113–117 [View Article][PubMed]
    [Google Scholar]
  36. Saitou N., Nei M. ( 1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425[PubMed]
    [Google Scholar]
  37. Schägger H. ( 2006). Tricine-SDS-PAGE. Nat Protoc 1:16–22 [View Article][PubMed]
    [Google Scholar]
  38. Segal B. H., Walsh T. J. ( 2006). Current approaches to diagnosis and treatment of invasive aspergillosis. Am J Respir Crit Care Med 173:707–717 [View Article][PubMed]
    [Google Scholar]
  39. Sharon H., Hagag S., Osherov N. ( 2009). Transcription factor PrtT controls expression of multiple secreted proteases in the human pathogenic mold Aspergillus fumigatus . Infect Immun 77:4051–4060 [View Article][PubMed]
    [Google Scholar]
  40. Spande T. F., Witkop B. ( 1967). Determination of tryptophan content of proteins with N-bromosuccimide. Methods in Enzymology498–506 Hirs C. H. W. New York: Academic Press; [View Article]
    [Google Scholar]
  41. Sriranganadane D., Reichard U., Salamin K., Fratti M., Jousson O., Waridel P., Quadroni M., Neuhaus J. M., Monod M. ( 2011). Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium. Microbiology 157:1541–1550 [View Article][PubMed]
    [Google Scholar]
  42. Szedlacsek S. E., Duggleby R. G., Vlad M. O. ( 1991). Enzyme catalysis as a chain reaction. Biochem J 279:855–861[PubMed]
    [Google Scholar]
  43. Umezawa H. ( 1976). Structures and activities of protease inhibitors of microbial origin. Methods Enzymol 45:678–695 [View Article][PubMed]
    [Google Scholar]
  44. Walsh K. A. ( 1970). Proteolytic enzymes. Methods in Enzymology41–63 Perlmann G. E., Lorand L. New York: Academic Press;
    [Google Scholar]
  45. Walsh T. J., Raad I., Patterson T. F., Chandrasekar P., Donowitz G. R., Graybill R., Greene R. E., Hachem R., Hadley S. & other authors ( 2007). Treatment of invasive aspergillosis with posaconazole in patients who are refractory to or intolerant of conventional therapy: an externally controlled trial. Clin Infect Dis 44:2–12 [View Article][PubMed]
    [Google Scholar]
  46. Williams J. W., Morrison J. F. ( 1979). The kinetics of reversible tight-binding inhibition. Methods Enzymol 63:437–467 [View Article][PubMed]
    [Google Scholar]
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