Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

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.Intracellular protein turnover is a crucial step for cell functioning, and if this process is impaired, the elevated levels of aged proteins usually lead to the formation of intracellular insoluble aggregates that can cause severe pathologies (1). In mammalian cells, most proteins destined for degradation are initially tagged with a polyubiquitin chain in an energy-dependent process and then digested to small peptides by the 26 S proteasome, a large proteolytic complex involved in the regulation of cell division, gene expression, and other key processes (2, 3). In eukaryotes, 30 -90% of newly synthesized proteins may be degraded by proteasomes within minutes of synthesis (3, 4). In addition to proteasomes, other extralysosomal proteolytic systems have been reported (5, 6). The proteasome cleaves proteins into peptides that are typically 2-20 amino acids in length (7). In most cases, these peptides are thought to be rapidly hydrolyzed into amino acids by aminopeptidases (8 -10). However, some intracellular peptides escape complete degradation and are imported into the endoplasmic reticulum where they associate with major histocompatibility complex class I (MHC-I) 3 molecules and traffic to the cell surface for presentation to the immune system (10 -12). Additionally, based on the fact that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions such as gene reg...

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

“…However, destruction of defined epitopes by TOP as studied by CTL recognition of target cells with suppressed expression of TOP, has thus far only been shown for the mouse H2-K b -presented SIINFEKL epitope from ovalbumin 5 . The role of TOP in the production of the PRA [190][191][192][193][194][195][196][197][198] and MART-1 epitopes is in accordance with a recent study that demonstrated that TOP in the cytosol both destroys and generates peptides of the length of class I ligands 40 . Thus the role of TOP in antigen processing appears to be twofold.…”

A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

“…Although the intracellular peptides have not yet been shown to directly modulate protein-protein interactions in vivo, the in vitro use of surface plasmon resonance demonstrates that at concentrations of 1-50 M, several intracellular peptides can modulate the interactions of calmodulin and 14-3-3⑀ with proteins from the mouse brain cytoplasm or with recombinant EP24.15. One of these peptides (VFDVELL; VFD-7), shown to be a proteasome product (24), increases the free cytosolic Ca 2ϩ concentration in a dose-dependent manner but only if introduced into HEK293 cells (27).…”

“…These peptides were used for affinity chromatography and were suggested to bind to a specific set of proteins, many involved in protein and vesicular traffic (23). In addition to the proteasome, thimet oligopeptidase (EC 3.4.24.15; EP24.15), which is an intracellular peptidase that only degrades small peptides (ϳ5-17 amino acids), was also shown to participate in intracellular peptide metabolism (24). By manipulating intracellular EP24.15 activity either by overexpressing the enzyme or inhibiting its activity by means of siRNA, it was possible to modulate G-protein-coupled receptor signal transduction in HEK293 and CHO-S cells (23,25).…”

Limitations of Mass Spectrometry-Based Peptidomic Approaches

“…These three databases were composed of published studies [20,22,24,25,28,30,[33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48] and a few additional studies that have not yet been published; these unpublished studies used identical methods, differing only in the treatment groups (which is not relevant to the present meta-analysis). The mouse database contains all peptidomic results from studies using mouse tissues-mainly brain regions, but also whole brain as well as heart, spleen, and testis.…”