Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells

Denise A. Berti, Cain Morano, Lilian C. Russo, Leandro M. Castro, Fernanda M. Cunha, Xin Zhang, Juan Sironi, Clécio F. Klitzke, Emer S. Ferro, Lloyd D. Fricker

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Abstract

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

Original languageEnglish (US)
Pages (from-to)14105-14116
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number21
DOIs
StatePublished - May 22 2009

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thimet oligopeptidase
Kidney
Peptides
Substrates
Antigen Presentation
Antigens
Mass spectrometry
Mass Spectrometry
High pressure liquid chromatography
Amino Acids
Signal transduction

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells. / Berti, Denise A.; Morano, Cain; Russo, Lilian C.; Castro, Leandro M.; Cunha, Fernanda M.; Zhang, Xin; Sironi, Juan; Klitzke, Clécio F.; Ferro, Emer S.; Fricker, Lloyd D.

In: Journal of Biological Chemistry, Vol. 284, No. 21, 22.05.2009, p. 14105-14116.

Research output: Contribution to journalArticle

Berti, DA, Morano, C, Russo, LC, Castro, LM, Cunha, FM, Zhang, X, Sironi, J, Klitzke, CF, Ferro, ES & Fricker, LD 2009, 'Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells', Journal of Biological Chemistry, vol. 284, no. 21, pp. 14105-14116. https://doi.org/10.1074/jbc.M807916200
Berti, Denise A. ; Morano, Cain ; Russo, Lilian C. ; Castro, Leandro M. ; Cunha, Fernanda M. ; Zhang, Xin ; Sironi, Juan ; Klitzke, Clécio F. ; Ferro, Emer S. ; Fricker, Lloyd D. / Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 21. pp. 14105-14116.
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