14‐3‐3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Carreño, Flávia Regina; Goñi, Camila N.; Castro, Leandro M.; Ferro, Emer S.

doi:10.1111/j.1471-4159.2004.02967.x

Cited by 33 publications

(66 citation statements)

References 83 publications

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“…Whereas most secreted proteins and neuropeptides precursors are known to contain a signal peptide sequence that drives their entry into the secretory pathway, the unconventional secretion of cytoplasmic proteins and bioactive peptides without involvement of the secretory pathway is also well known (53)(54)(55). ATP-binding cassette (ABC) transporters have been shown to carry antigenic peptides from the cytosol into the endoplasmic reticulum as well as to function in shuttling peptides across the plasma membrane (56)(57)(58).…”

Section: Discussionmentioning

confidence: 99%

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

Castro

Berti²,

Russo³

et al. 2010

AAPS J

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Abstract. 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.

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Section: Discussionmentioning

confidence: 99%

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

Castro

Berti²,

Russo³

et al. 2010

AAPS J

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“…Rat testis EP24.15 full-length cDNA was subcloned into the a modified pShooter vector (Invitrogen) inframe with the c-Myc epitope for C-terminal fusion to the recombinant proteins, as described previously (19). DNA sequencing confirmed the correct frame orientation for all constructs (data not shown).…”

Section: Methodsmentioning

confidence: 99%

“…This enzyme was first described as a neuropeptide-degrading enzyme present in the soluble fraction of brain homogenates (18). Whereas EP24.15 can be secreted (19,20), its predominant location in the cytosol and nucleus suggests that the primary function of this enzyme is not the extracellular degradation of neuropeptides and hormones (21,22). EP24.15 was shown in vivo to participate in antigen presentation through MHC-I (23)(24)(25) and in vitro to bind (26) or degrade (27) some MHC-I associated peptides.…”

mentioning

confidence: 99%

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

Berti

Morano

Russo

et al. 2009

Journal of Biological Chemistry

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106

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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...

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“…Analysis of Peptide Entry and Stability within Cells-The entry of peptides into CHO-S cells was analyzed by HPLC coupled to a fluorescence detector. CHO-S cells grown under standard conditions (43) were incubated with the studied peptides (20 M) for 30 min at 37°C. Cells were then scraped from 6-well plates, suspended in 6 ml of phosphate-buffered saline, and centrifuged at 800 ϫ g for 10 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Cells-In order to investigate the effect of EP24.15 overexpression on the signal transduction pathways of AT1 and ␤-adrenergic receptors, CHO-S and HEK293 cells were transfected with the set of luciferase vectors described above plus 1 g of either the vector coding for wild type EP24.15 (WT-EP24.15) or the "mock" control vector (43). After 48 h, cells were stimulated with ang II or isoproterenol, and luciferase activity was subsequently measured after 5 h, as detailed above.…”

Section: Ep2415 Overexpression In Cho-s and Hek293mentioning

confidence: 99%

Intracellular Peptides as Natural Regulators of Cell Signaling

Cunha

Berti

Ferreira

et al. 2008

Journal of Biological Chemistry

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138

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Protein degradation by the ubiquitin proteasome system releases large amounts of oligopeptides within cells. To investigate possible functions for these intracellularly generated oligopeptides, we fused them to a cationic transactivator peptide sequence using reversible disulfide bonds, introduced them into cells, and analyzed their effect on G protein-coupled receptor (GPCR) signal transduction. A mixture containing four of these peptides (20 -80 M) significantly inhibited the increase in the extracellular acidification response triggered by angiotensin II (ang II) in CHO-S cells transfected with the ang II type 1 receptor (AT1R-CHO-S). Subsequently, either alone or in a mixture, these peptides increased luciferase gene transcription in AT1R CHO-S cells stimulated with ang II and in HEK293 cells treated with isoproterenol. These peptides without transactivator failed to affect GPCR cellular responses. All four functional peptides were shown in vitro to competitively inhibit the degradation of a synthetic substrate by thimet oligopeptidase. Overexpression of thimet oligopeptidase in both CHO-S and HEK293 cells was sufficient to reduce luciferase activation triggered by a specific GPCR agonist. Moreover, using individual peptides as baits in affinity columns, several proteins involved in GPCR signaling were identified, including ␣-adaptin A and dynamin 1. These results suggest that before their complete degradation, intracellular peptides similar to those generated by proteasomes can actively affect cell signaling, probably representing additional bioactive molecules within cells.

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14‐3‐3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Cited by 33 publications

References 83 publications

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

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

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

Intracellular Peptides as Natural Regulators of Cell Signaling

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