Lilian C. Russo

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.

Identification of intracellular peptides in rat adipose tissue: Insights into insulin resistance.

Intracellular peptides generated by the proteasome and oligopeptidases have been suggested to function in signal transduction and to improve insulin resistance in mice fed a high‐caloric diet. The aim of this study was to identify specific intracellular peptides in the adipose tissue of Wistar rats that could be associated with the physiological and therapeutic control of glucose uptake. Using semiquantitative mass spectrometry and LC/MS/MS analyses, we identified ten peptides in the epididymal adipose tissue of the Wistar rats; three of these peptides were present at increased levels in rats that were fed a high‐caloric Western diet (WD) compared with rats fed a control diet (CD). The results of affinity chromatography suggested that in the cytoplasm of epididymal adipose tissue from either WD or CD rats, distinctive proteins bind to these peptides. However, despite the observed increase in the WD animals, the evaluated peptides increased insulin‐stimulated glucose uptake in 3T3‐L1 adipocytes treated with palmitate. Thus, intracellular peptides from the adipose tissue of Wistar rats can bind to specific proteins and facilitate insulin‐induced glucose uptake in 3T3‐L1 adipocytes.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3ε and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+‐calmodulin (CaM) and 14‐3‐3ε, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1–50 μM, most of the peptides that are investigated in this study modulate the interactions of CaM and 14‐3‐3ε with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD‐7) increases the cytosolic Ca2+ concentration in a dose‐dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi‐quantitative intracellular peptidome analyses of siRNA‐transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1–10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.

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

Background: Intracellular peptides probably regulate several biological processes. Results: pep5 derived from G1/S cyclin D2 specifically increases during the S phase of the cell cycle and, reintroduced into the cell, induces apoptosis and necrosis. Conclusion: pep5 has potential therapeutic applications and could have biological functions. Significance: pep5 discovery advances our understanding of limited proteolysis. Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides.

Similar Intracellular Peptide Profile of TAP1/β2 Microglobulin Double-Knockout Mice and C57BL/6 Wild-Type Mice as Revealed by Peptidomic Analysis

Cells produce and use peptides in distinctive ways. In the present report, using isotope labeling plus semi-quantitative mass spectrometry, we evaluated the intracellular peptide profile of TAP1/β2m−/− (transporter associated with antigen-processing 1/ß2 microglobulin) double-knockout mice and compared it with that of C57BL/6 wild-type animals. Overall, 92 distinctive peptides were identified, and most were shown to have a similar concentration in both mouse strains. However, some peptides showed a modest increase or decrease (~2-fold), whereas a glycine-rich peptide derived from the C-terminal of neurogranin (KGPGPGGPGGAGGARGGAGGGPSGD) showed a substantial increase (6-fold) in TAP1/β2m−/− mice. Thus, TAP1 and β2microglobulin have a small influence on the peptide profile of neuronal tissue, suggesting that the presence of peptides derived from intracellular proteins in neuronal tissue is not associated with antigens of the class I major histocompatibility complex. Therefore, it is possible that these intracellular peptides play a physiological role.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide‐metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium‐dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca2+, with an estimated Kd value of 0.52 μm. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187‐stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187‐stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

AGH is a new hemoglobin alpha-chain fragment with antinociceptive activity

Limited proteolysis of certain proteins leads to the release of endogenous bioactive peptides. Hemoglobin-derived peptides such as hemorphins and hemopressins are examples of intracellular protein-derived peptides that have antinociceptive effects by modulating G-protein coupled receptors activities. In the present study, a previously characterized substrate capture assay that uses a catalytically inactive form of the thimet oligopeptidase was combined with isotopic labeling and mass spectrometry in order to identify new bioactive peptides. Indeed, we have identified the peptide AGHLDDLPGALSAL (AGH), a fragment of the hemoglobin alpha-chain, which specifically bind to the inactive thimet oligopeptidase in the substrate capture assay. Previous peptidomics studies have identified the AGH as well as many other natural peptides derived from hemoglobin alpha-chain containing this sequence, further suggesting that AGH is a natural endogenous peptide. Pharmacological assays suggest that AGH inhibits peripheral inflammatory hyperalgesic responses through indirect activation of mu opioid receptors, without having a central nervous system activity. Therefore, we have successfully used the substrate capture assay to identify a new endogenous bioactive peptide derived from hemoglobin alpha-chain.

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways

BACKGROUND Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown. METHODS Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed. RESULTS The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation. CONCLUSION The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair. GENERAL SIGNIFICANCE The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.

Interferon-gamma activity is potentiated by an intracellular peptide derived from the human 19S ATPase regulatory subunit 4 of the proteasome.

Hundreds of intracellular peptides that are neither antigens nor neuropeptides are present in mammalian cells and tissues. These peptides correspond to fragments of cytosolic, nuclear or mitochondrial proteins. Proteasome inhibition affects the levels of the intracellular peptides in human cell lines. Here, the effect of immuneproteasome expression on the intracellular peptide profile was evaluated, and its functional significance was investigated. The expression of the immuneproteasome in HeLa cells was induced by interferon gamma treatment, and the relative concentrations of the intracellular peptides were compared to those of the control cells using isotope labeling and electron spray mass spectrometry. One of the peptides identified, VGSELIQKY (EL28), corresponds to amino acids 251-259 of the human 19S ATPase regulatory subunit 4. This peptide was increased in the extracts of HeLa cells that had been treated with interferon gamma compared to those of control cells. In vitro, EL28 increased the chymotrypsin, trypsin and caspase-like proteasome activities. In vivo, when covalently linked to a cell-penetrating peptide, EL28 potentiated the ability of interferon gamma to stimulate the expression of the immuneproteasome β5i subunit and to increase the proliferation of CD8+ T-cells. The EL28/cell-penetrating peptide construct also improved and positively modulated the secondary IgG anti-bovine serum albumin immune responsiveness elicited in high antibody-responder mice. Together, these results suggest that EL28 is a functional intracellular peptide that can potentiate interferon gamma activity. BIOLOGICAL SIGNIFICANCE The functional identification of EL28 advances our understanding regarding the bioactive peptides generated by limited proteolysis within cells.