A Giant Protease with Potential to Substitute for Some Functions of the Proteasome
10.1126/science.283.5404.978
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin–hydrolyzing protease particle copurifying with 26 S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26 S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor–adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.

Bibliography

Geier, E., Pfeifer, G., Wilm, M., Lucchiari-Hartz, M., Baumeister, W., Eichmann, K., & Niedermann, G. (1999). A Giant Protease with Potential to Substitute for Some Functions of the Proteasome. Science, 283(5404), 978–981.

Authors 7
  1. Elke Geier (first)
  2. Günter Pfeifer (additional)
  3. Matthias Wilm (additional)
  4. Maria Lucchiari-Hartz (additional)
  5. Wolfgang Baumeister (additional)
  6. Klaus Eichmann (additional)
  7. Gabriele Niedermann (additional)
References 32 Referenced 303
  1. 10.1146/annurev.bi.65.070196.004101
  2. Ciechanover A., Schwartz A., Proc. Natl. Acad. Sci. U.S.A. 95, 2727 (1998); (10.1073/pnas.95.6.2727) / Proc. Natl. Acad. Sci. U.S.A. by Ciechanover A. (1998)
  3. Rock K. L., et al., Cell 78, 761 (1994); (10.1016/S0092-8674(94)90462-6) / Cell by Rock K. L. (1994)
  4. ; V. J. Palombella O. J. Rando A. L. Goldberg T. Maniatis ibid. p. 773; R. W. King
  5. Deshaies R. J., Peters J.-M., Kirschner M. W., Science 274, 1652 (1996) . (10.1126/science.274.5293.1652) / Science by Deshaies R. J. (1996)
  6. Glas R., Bogyo M., McMaster J. S., Gaczynska M., Ploegh H. L., Nature 392, 618 (1998). (10.1038/33443) / Nature by Glas R. (1998)
  7. Murine EL4 thymoma cells (8 × 10 9 to 1 × 10 10 cells) were harvested and resuspended at a density of 3 × 10 7 cells per milliliter in imidazole buffer [20 mM imidazole-HCl (pH 6.8) 100 mM KCl 20 mM EGTA 2 mM MgCl 2 10% sucrose 1 mM adenosine triphosphate (ATP)] and lysed by freeze-thawing. The lysate was differentially centrifuged (15 min at 1500 g 15 min at 15 000 g 60 min at 100 000 g 3 hours at 100 000 g ) and the resulting pellet of HMW proteins was resuspended in 50 mM tris-HCl (pH 7.4) 20% glycerol 5 mM MgCl 2 0.5 mM β-mercaptoethanol 1 mM ATP) and stored at −80°C. Samples of the resuspended material were separated on a Mono Q (HR16/10) anion-exchange column [buffer A: 20 mM Hepes (pH 7.2) 15% glycerol 1 mM ATP; buffer B: 20 mM Hepes (pH 7.2) 15% glycerol 1 M NaCl 1 mM ATP] followed by gel filtration on a Superose 6-column [2 cm by 50 cm; elution buffer: 20 mM Hepes (pH 7.2) 100 mM NaCl 15% glycerol 1 mM ATP]. All purification steps were carried out at 4°C. Samples of collected fractions were used to measure hydrolysis of Suc-LLVY-AMC and AAF-AMC (4).
  8. Peptidase assays were performed as described (14).
  9. The protein was digested with trypsin in a 50 mM NH 4 HCO 3 5 mM CaCl 2 buffer containing 33% isotopically labeled H 2 18 O. Peptides are isotopically labeled to 33% at their COOH-terminus thereby allowing COOH-terminal fragments (y-ions) to be distinguished from other fragment ions by their specific 16 O/ 18 O isotopic distribution. The peptides were sequenced on a API III triple quadrupole instrument (PE-Sciex Toronto Canada) equipped with a nanoelectrospray source by differential scanning (M. Wilm G. Neubauer L. Taylor A. Shevchenko A. Bachi in preparation).
  10. Tomkinson B., Biochem. J. 304, 517 (1994). (10.1042/bj3040517) / Biochem. J. by Tomkinson B. (1994)
  11. Rose C., et al., Nature 380, 403 (1996). (10.1038/380403a0) / Nature by Rose C. (1996)
  12. Balöw R. M., Ragnasson U., Zetterqvist Ö., J. Biol. Chem. 258, 11622 (1983); (10.1016/S0021-9258(17)44273-6) / J. Biol. Chem. by Balöw R. M. (1983)
  13. Balöw R. M., Tomkinson B., Ragnarsson U., Zetterqvist Ö., ibid. 261, 2409 (1986); / ibid. by Balöw R. M. (1986)
  14. Macpherson E., Tomkinson B., Balöw R. M., Höglund S., Zetterqvist Ö., Biochem. J. 248, 259 (1987). (10.1042/bj2480259) / Biochem. J. by Macpherson E. (1987)
  15. 10.1006/jsbi.1998.3958
  16. 10.1016/S0092-8674(00)80942-3
  17. Tamura T., et al., Science 274, 1385 (1996). (10.1126/science.274.5291.1385) / Science by Tamura T. (1996)
  18. Fenteany G., et al., ibid. 268, 726 (1995). / ibid. by Fenteany G. (1995)
  19. E. Geier K. Eichmann G. Niedermann data not shown.
  20. Niedermann G., et al., Proc. Natl. Acad. Sci. U.S.A. 93, 8572 (1996). (10.1073/pnas.93.16.8572) / Proc. Natl. Acad. Sci. U.S.A. by Niedermann G. (1996)
  21. Kirschke H., et al., Acta Biol. Med. Ger. 36, 185 (1977); / Acta Biol. Med. Ger. by Kirschke H. (1977)
  22. Nishimura T., Okitani A., Katakai R., Kato H., Eur. J. Biochem. 137, 23 (1983); (10.1111/j.1432-1033.1983.tb07790.x) / Eur. J. Biochem. by Nishimura T. (1983)
  23. Bermpohl F., Löster K., Reutter W., Baum O., FEBS Lett. 428, 152 (1998). (10.1016/S0014-5793(98)00515-8) / FEBS Lett. by Bermpohl F. (1998)
  24. Imajoh-Ohmi S., et al., Biochem. Biophys. Res. Commun. 217, 1070 (1995); (10.1006/bbrc.1995.2878) / Biochem. Biophys. Res. Commun. by Imajoh-Ohmi S. (1995)
  25. Wang X., Luo H., Chen H., Duguid W., Wu J., J. Immunol. 160, 788 (1998). (10.4049/jimmunol.160.2.788) / J. Immunol. by Wang X. (1998)
  26. Zhou M., Wu X., Ginsberg H. N., J. Biol. Chem. 271, 24769 (1996); (10.1074/jbc.271.40.24769) / J. Biol. Chem. by Zhou M. (1996)
  27. Bush K. T., Goldberg A. L., Nigam S. K., ibid. 272, 9086 (1997); / ibid. by Bush K. T. (1997)
  28. ; A. B. Meriin V. L. Gabai J. Yaglom V. I. Shifrin M. Y. Sherman ibid. 273 6373 (1998). (10.1074/jbc.273.11.6373)
  29. Tamura N., Lottspeich F., Baumeister W., Tamura T., Cell 95, 637 (1998). (10.1016/S0092-8674(00)81634-7) / Cell by Tamura N. (1998)
  30. Benham A. H., Gromme M., Neefjes J., J. Immunol. 161, 63 (1998). (10.4049/jimmunol.161.1.83) / J. Immunol. by Benham A. H. (1998)
  31. For each time point 6 ng of TPPII was incubated with 3 μg of synthetic peptide corresponding to the ovalbumin amino acids 37 to 77 at 37°C in a total volume of 100 μl of assay buffer [20 mM Hepes (pH 7.8) 1 mM EGTA 0.5 mM EDTA 5 mM MgCl 2 0.5 mM 2-mercaptoethanol 5% glycerol]. The analysis of peptide products was performed as described (14).
  32. We thank S. Gaedicke for technical assistance E. J. Corey for supplying lactacystin K. Peters and A. Würch for helpful discussions and I. Haidl for critical reading of the manuscript. Supported by a grant of the Deutsche Forschungsgemeinschaft (NI 368/2-1) to G.N.
Dates
Type When
Created 23 years ago (July 27, 2002, 5:40 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 10:06 p.m.)
Indexed 3 days, 12 hours ago (Aug. 21, 2025, 1:26 p.m.)
Issued 26 years, 6 months ago (Feb. 12, 1999)
Published 26 years, 6 months ago (Feb. 12, 1999)
Published Print 26 years, 6 months ago (Feb. 12, 1999)
Funders 0

None

@article{Geier_1999, title={A Giant Protease with Potential to Substitute for Some Functions of the Proteasome}, volume={283}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.283.5404.978}, DOI={10.1126/science.283.5404.978}, number={5404}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Geier, Elke and Pfeifer, Günter and Wilm, Matthias and Lucchiari-Hartz, Maria and Baumeister, Wolfgang and Eichmann, Klaus and Niedermann, Gabriele}, year={1999}, month=feb, pages={978–981} }