1887

Microbiology Society logo

Volume 149, Issue 12

PepN is the major aminopeptidase in : insights on substrate specificity and role during sodium-salicylate-induced stress Free

Abstract

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in , the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two mutants, 9218 and DH5Δ. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both mutants. This decreased ability to cleave these substrates is -specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in mutants lacking other peptidases, , or . PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in . Further studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in , demonstrating a physiological role for this aminoendopeptidase under some stress conditions.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): AMC, 7-amino-4-methylcoumarin , Boc, butoxycarbonyl , Cbz, benzoyloxycarbonyl , NaSal, sodium salicylate , pNA, p-nitroanilide and βNA, β-naphthylamide
SGM
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26518-0
2003-12-01
2024-04-19
Loading full text...

Full text loading...

/deliver/fulltext/micro/149/12/mic1493437.html?itemId=/content/journal/micro/10.1099/mic.0.26518-0&mimeType=html&fmt=ahah

References

  1. Bally M., Murgier M., Lazdunski A. 1983; Molecular cloning and amplification of the gene for aminopeptidase N of Escherichia coli . FEMS Microbiol Lett 19:261–265
     [Google Scholar]
  2. Bally M., Murgier M., Lazdunski A. 1984; Cloning and orientation of the gene encoding aminopeptidase N in Escherichia coli . Mol Gen Genet 195:507–510
     [Google Scholar]
  3. Basten D. E., Visser J., Schaap P. J. 2001; Lysine aminopeptidase of Aspergillus niger . Microbiology 147:2045–2050
     [Google Scholar]
  4. Beninga J., Rock K. L., Goldberg A. L. 1998; Interferon- γ can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J Biol Chem 273:18734–18742
     [Google Scholar]
  5. Bradshaw R. A., Brickey W. W., Walker K. W. 1998; N-terminal processing: the methionine aminopeptidase and N alpha-acetyl transferase families. Trends Biochem Sci 23:263–267
     [Google Scholar]
  6. Brandstetter H., Kim J. S., Groll M., Huber R. 2001; Crystal structure of the tricorn protease reveals a protein disassembly line. Nature 414:466–470
     [Google Scholar]
  7. Braun V., Gunthner K., Hantke K., Zimmermann L. 1983; Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium . J Bacteriol 156:308–315
     [Google Scholar]
  8. Caprioglio D. R., Padilla C., Werner-Washburne M. 1993; Isolation and characterization of AAP1 . A gene encoding an alanine/arginine aminopeptidase in yeast. J Biol Chem 268:14310–14315
     [Google Scholar]
  9. Chandu D., Nandi D. 2002; From proteins to peptides to amino acids: comparative genomics of enzymes involved in downstream processing events during cytosolic protein degradation. Appl Genom Proteom 1:235–252
     [Google Scholar]
  10. Chandu D., Kumar A., Nandi D. 2003; PepN, the major Suc-LLVY-AMC-hydrolyzing enzyme in Escherichia coli , displays functional similarity with downstream processing enzymes in archaea and eukarya. Implications in cytosolic protein degradation. J Biol Chem 278:5548–5556
     [Google Scholar]
  11. Chang S. Y., McGary E. C., Chang S. 1989; Methionine aminopeptidase gene of Escherichia coli is essential for cell growth. J Bacteriol 171:4071–4072
     [Google Scholar]
  12. Chavagnat F., Casey M. G., Meyer J. 1999; Purification, characterization, gene cloning, sequencing, and overexpression of aminopeptidase N from Streptococcus thermophilus A. Appl Environ Microbiol 65:3001–3007
     [Google Scholar]
  13. Cohen S. P., Levy S. B., Foulds J., Rosner J. L. 1993; Salicylate induction of antibiotic resistance in Escherichia coli : activation of the mar operon and a mar -independent pathway. J Bacteriol 175:7856–7862
     [Google Scholar]
  14. Conlin C. A., Miller C. G. 1995; Dipeptidyl carboxypeptidase and oligopeptidase A from Escherichia coli and Salmonella typhimurium . Methods Enzymol 248:567–579
     [Google Scholar]
  15. Coux O., Tanaka K., Goldberg A. L. 1996; Structure and functions of the 20S and 26S proteasomes. Annu Rev Biochem 65:801–847
     [Google Scholar]
  16. Eisenthal R., Cornish-Bowden A. 1974; The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J 139:715–720
     [Google Scholar]
  17. Franzetti B., Schoehn G., Hernandez J. F., Jaquinod M., Ruigrok R. W., Zaccai G. 2002; Tetrahedral aminopeptidase: a novel large protease complex from archaea. EMBO J 21:2132–2138
     [Google Scholar]
  18. Fukasawa K., Fukasawa K. M., Kana M., Fujii S., Hirose J., Harada M. 1998; Dipeptidyl peptidase III is a zinc metallo-exopeptidase. Molecular cloning and expression. Biochem J 329:275–282
     [Google Scholar]
  19. Geier E., Pfeifer G., Wilm M., Luchhiari-Hartz M., Baumeister W., Eichmann K., Niedermann G. 1999; A giant protease with potential to substitute for some functions of the proteasome. Science 283:978–981
     [Google Scholar]
  20. Gharbi S., Belaich A., Murgier M., Lazdunski A. 1985; Multiple controls exerted on in vivo expression of the pepN gene in Escherichia coli : studies with pepN – lacZ operon and protein fusion strains. J Bacteriol 163:1191–1195
     [Google Scholar]
  21. Goettig P., Groll M., Kim J. S., Huber R., Brandstetter H. 2002; Structures of the tricorn-interacting aminopeptidase F1 with different ligands explain its catalytic mechanism. EMBO J 21:5343–5352
     [Google Scholar]
  22. Gonzales T., Robert-Baudouy J. 1996; Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18:319–344
     [Google Scholar]
  23. Gottesman S. 1996; Proteases and their targets in Escherichia coli . Annu Rev Genet 30:465–506
     [Google Scholar]
  24. Ishino Y., Shinagawa H., Makino K., Amemura M., Nakata A. 1987; Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli , and identification of the gene product. J Bacteriol 169:5429–5433
     [Google Scholar]
  25. Kisselev A. F., Akopian T. N., Woo K. M., Goldberg A. L. 1999; The sizes of peptides generated from protein by mammalian 26S and 20S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274:3363–3371
     [Google Scholar]
  26. Knipfer N., Shrader T. E. 1997; Inactivation of the 20S proteasome in Mycobacterium smegmatis . Mol Microbiol 25:375–383
     [Google Scholar]
  27. Koldamova R. P., Lefterova I. M., Gadjeva V. G., Lazo J. S. 1998; Essential binding and functional domains of human bleomycin hydrolase. Biochemistry 37:2282–2290
     [Google Scholar]
  28. Kuroda A., Nomura K., Ohtomo R., Kata J., Ikeda T., Takiguchi N., Ohtake H., Kornberg A. 2001; Role of inorganic phosphate in promoting ribosomal protein degradation by the Lon protease in E. coli . Science 293:705–708
     [Google Scholar]
  29. Larsen R. A., Knox T. M., Miller C. G. 2001; Aspartic peptide hydrolases in Salmonella enterica serovar typhimurium. J Bacteriol 183:3089–3097
     [Google Scholar]
  30. Latil M., Murgier M., Lazdunski A., Lazdunski C. 1976; Isolation and genetic mapping of Escherichia coli aminopeptidase mutants. Mol Gen Genet 148:43–47
     [Google Scholar]
  31. Lazdunski A., Murgier M., Lazdunski C. 1975a; Evidence for an aminoendopeptidase localized near the cell surface of Escherichia coli . Regulation of synthesis by inorganic phosphate. Eur J Biochem 60:349–355
     [Google Scholar]
  32. Lazdunski C., Busuttil J., Lazdunski A. 1975b; Purification and properties of a periplasmic aminoendopeptidase from Escherichia coli . Eur J Biochem 60:363–369
     [Google Scholar]
  33. Lowe J., Stock D., Jap B., Zwickl P., Baumeister W., Huber R. 1995; Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3. 4 Å resolution. Science 268:533–539
     [Google Scholar]
  34. Maurizi M. R. 1987; Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli . J Biol Chem 262:2696–2703
     [Google Scholar]
  35. McCaman M. T., Villarejo M. R. 1982; Structural and catalytic properties of peptidase N from Escherichia coli K-12. Arch Biochem Biophys 213:384–394
     [Google Scholar]
  36. McCaman M. T., McPartland A., Villarejo M. R. 1982; Genetics and regulation of peptidase N in Escherichia coli K-12. J Bacteriol 152:848–854
     [Google Scholar]
  37. Mierau I., Kunji E. R., Leenhouts K. J., Hellendoorn M. A., Haandrikman A. J., Poolman B., Konings W. N., Venema G., Kok J. 1996; Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk. J Bacteriol 178:2794–2803
     [Google Scholar]
  38. Miller C. G., Schwartz G. 1978; Peptidase-deficient mutants of Escherichia coli . J Bacteriol 135:603–611
     [Google Scholar]
  39. Nishimura T., Okitani A., Katakai R., Kato H. 1983; Mode of action towards oligopeptides and proteins of hydrolase H, a high-molecular-weight aminoendopeptidase from rabbit skeletal muscle. Eur J Biochem 137:23–27
     [Google Scholar]
  40. Okitani A., Nishimura T., Kato H. 1981; Characterization of hydrolase H, a new muscle protease possessing aminoendopeptidase activity. Eur J Biochem 115:269–274
     [Google Scholar]
  41. Osada T., Watanabe G., Kondo S., Toyoda M., Sakaki Y., Takeuchi T. 2001a; Male reproductive defects caused by puromycin-sensitive aminopeptidase deficiency in mice. Mol Endocrinol 15:960–971
     [Google Scholar]
  42. Osada T., Watanabe G., Sakaki Y., Takeuchi T. 2001b; Puromycin-sensitive aminopeptidase is essential for the maternal recognition of pregnancy in mice. Mol Endocrinol 15:882–893
     [Google Scholar]
  43. Osmulski P. A., Gaczynska M. 1998; A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast. Curr Biol 8:1023–1026
     [Google Scholar]
  44. Pomposiello P. J., Bennik M. H., Demple B. 2001; Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate. J Bacteriol 183:3890–3902
     [Google Scholar]
  45. Price C. T. D., Lee I. R., Gustafson J. E. 2000; The effects of salicylate on bacteria. Int J Biochem Cell Biol 32:1029–1043
     [Google Scholar]
  46. Ramani N., Boakye K. 2001; Salicylate inhibits the translation and transcription of ompF in Escherichia coli . Can J Microbiol 47:1053–1057
     [Google Scholar]
  47. Richmond C. S., Glasner J. D., Mau R., Jin H., Blattner F. R. 1999; Genome-wide expression profiling in Escherichia coli K-12. Nucleic Acids Res 27:3821–3835
     [Google Scholar]
  48. Ruepp A., Eckerskorn C., Bogyo M., Baumeister W. 1998; Proteasome function is dispensable under normal but not heat shock conditions in Thermoplasma acidophilum . FEBS Lett 425:87–90
     [Google Scholar]
  49. Saric T., Beninga J., Graef C. I., Akopian T. N., Rock K. L., Goldberg A. L. 2001; Major histocompatibility complex class I-presented antigenic peptides are degraded in cytosolic extracts primarily by thimet oligopeptidase. J Biol Chem 276:36474–36481
     [Google Scholar]
  50. Singh H., Kalnitsky G. 1980; α - N -Benzoylarginine- β -naphthylamide hydrolase, an aminoendopeptidase from rabbit lung. J Biol Chem 255:369–374
     [Google Scholar]
  51. Slonim D. K. 2002; From patterns to pathways: gene expression data analysis comes of age. Nat Genet 32 Suppl:502–508
     [Google Scholar]
  52. Stoltze L., Schirle M., Schwarz G. & 7 other authors; 2000; Two new proteases in the MHC class I processing pathway. Nat Immunol 1:413–418
     [Google Scholar]
  53. Suzuki H., Kamatani S., Kim E. S., Kumagai H. 2001; Aminopeptidases A, B, and N and dipeptidase D are the four cysteinylglycinases of Escherichia coli K-12. J Bacteriol 183:1489–1490
     [Google Scholar]
  54. Tamura N., Lottspeich F., Baumeister W., Tamura T. 1998; The role of tricorn protease and its aminopeptidase-interacting factors in cellular protein degradation. Cell 95:637–648
     [Google Scholar]
  55. Tamura T., Tamura N., Cejka Z., Hegerl R., Lottspeich F., Baumeister W. 1996; Tricorn protease – the core of a modular proteolytic system. Science 274:1385–1388
     [Google Scholar]
  56. Turk V., Turk B., Turk D. 2001; Lysosomal cysteine proteases: facts and opportunities. EMBO J 20:4629–4633
     [Google Scholar]
  57. van Allen-Boerrigter I. J., Baankreis R., de Vos W. M. 1991; Characterization and overexpression of the Lactococcus lactis pepN gene and localization of its product, aminopeptidase N. Appl Environ Microbiol 57:2555–2561
     [Google Scholar]
  58. Vanhoof G., Goossens F., De Meester I., Hendriks D., Scharpe S. 1995; Proline motifs in peptides and their biological processing. FASEB J 9:736–744
     [Google Scholar]
  59. Wang E. W., Kessler B. M., Borodovsky A., Cravatt B. F., Bogyo M., Ploegh H. L., Glas R. 2000; Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity. Proc Natl Acad Sci U S A 97:9990–9995
     [Google Scholar]
  60. Yen C., Green L., Miller C. G. 1980; Degradation of intracellular protein in Salmonella typhimurium peptidase mutants. J Mol Biol 143:21–33
     [Google Scholar]
  61. York I. A., Mo A. X., Lemerise K., Zeng W., Shen Y., Abraham C. R., Saric T., Goldberg A. L., Rock K. L. 2003; The cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of MHC class I antigen presentation. Immunity 18:429–440
     [Google Scholar]
  62. Zwickl P., Baumeister W., Steven A. 2000; Dis-assembly lines: the proteasome and related ATPase-assisted proteases. Curr Opin Struct Biol 10:242–250
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26518-0
Loading
PepN is the major aminopeptidase in Escherichia coli: insights on substrate specificity and role during sodium-salicylate-induced stress
Microbiology 149, 3437 (2003); https://doi.org/10.1099/mic.0.26518-0
/content/journal/micro/10.1099/mic.0.26518-0
/content/journal/micro/10.1099/mic.0.26518-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error