Analysis of intracellular substrates and products of thimet oligopeptidase in human embryonic kidney 293 cells.
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.
Berti
Denise A
DA
Department of Cell Biology and Development , Biomedical Science Institute, University of São Paulo, São Paulo SP 05508-900, Brazil.
Morano
Cain
C
Russo
Lilian C
LC
Castro
Leandro M
LM
Cunha
Fernanda M
FM
Zhang
Xin
X
Sironi
Juan
J
Klitzke
Clécio F
CF
Ferro
Emer S
ES
Fricker
Lloyd D
LD
D000595:Amino Acid Sequence; D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000201:enzymology*; D007553:Isotope Labeling; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D020539:Sequence Analysis, Protein; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity / Q000187:drug effects
D002457:Cell Extracts; D010455:Peptides; D000644:Quaternary Ammonium Compounds; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase