Inhibition of Thimet Oligopeptidase by Sirna Alters Specific

FEBS Letters 586 (2012) 3287–3292

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Inhibition of thimet oligopeptidase by siRNA alters speciﬁc intracellular peptides and potentiates isoproterenol signal transduction ⇑ Lilian C. Russo a, Leandro M. Castro a, Fabio C. Gozzo b, Emer S. Ferro a, a Department of Cell Biology and Development, Biomedical Sciences Institute, University of São Paulo, São Paulo 05508-000, Brazil b Chemistry Institute, Campinas State University, Campinas 13083-970, SP, Brazil article info abstract

Article history: Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal Received 4 May 2012 proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in Revised 14 June 2012 human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative Accepted 2 July 2012 intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific Available online 10 July 2012 intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased Edited by Gianni Cesareni expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1–10 lM). The protein kinase A inhibitor KT5720 (1 lM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels Keywords: Oligopeptidase of specific intracellular peptides and isoproterenol signal transduction. G-protein coupled receptor Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. siRNA Intracellular peptide Ubiquitinproteasome system

1. Introduction Point mutations in EP24.15 leading to inactivation of its catalytic properties were generated, and the inactive form of EP24.15 Thimet oligopeptidase (EC3.4.24.15; EP24.15) is a characteristic was used in a substrate capture assay that identiﬁed several novel metalloendopeptidase with a HEXXH catalytic motif containing peptides [10]. Several of these intracellular peptides isolated from zinc that belongs to the family of M3 metallopeptidases. EP24.15 the rat brain using the inactive EP24.15 substrate capture assay is designated as an oligopeptidase, as it only degrades short pep- actively affected the signal transduction of G protein-coupled tides (5–17 amino acids). Because EP24.15 is activated by thiol receptors (GPCR) when introduced into CHO-S-AT1 and HEK293 compounds, it is referred to as thimet (for thiol-activated metallo- cells [11]. Overexpression of EP24.15 in these cells also alters sig- protease) [1,2]. Maximal enzymatic activity of EP24.15 is main- nal transduction induced by GPCR agonists, such as angiotensin II tained by partial S-glutathionylation, which is a mechanism that and isoproterenol [11], and leads to a marked alteration of the regulates protein oligomerization and enzymatic activity [3]. intracellular peptide content [12]. The mechanisms by which Despite being described as a secreted neuropeptide-metabolizing EP24.15 and/or its peptide substrates/products exert their effects enzyme [4], experimental evidences accumulated over the past on intracellular signal transduction of GPCRs are not fully under- years shows that EP24.15 is predominately expressed in the cyto- stood; however, peptides that alter the signal transduction of plasm and nucleus of neuronal cells [5]. Several studies have sug- GPCRs have been shown to bind to speciﬁc proteins involved in cell gested intracellular functions for EP24.15 [6–9]. In macrophages, signaling [11]. Recently, intracellular peptides have been shown to EP24.15 inhibition reduces the presentation of heat shock protein modulate protein–protein interactions, which could be a mecha- 65 antigens associated with the major histocompatibility complex nism related to their physiological function [13]. The overexpres- class I (MHC-I) while the addition of recombinant EP24.15 increases sion of EP24.15 in HEK293 cells transfected with the bradykinin hsp65 presentation [9]. EP24.15 was also shown to contribute to C- B2 receptor markedly changes the production of inositol phosphate terminal trimming of antigenic peptides, after the processing activ- and increases the levels of intracellular free calcium [14]. Thus, ity of the proteasome [6]. EP24.15 intracellular function and intracellular peptides may be related to signal transduction. ⇑ In this study, we inhibited the expression of EP24.15 in HEK293 Corresponding author. Address: Department of Cell Biology and Development, cells using siRNAs and examined changes in the intracellular Biomedical Sciences Institute, Av. Prof. Lineu Prestes 1524, Sala 431, São Paulo 05508-000, SP, Brazil. Fax: +55 11 3091 7402. peptide content and isoproterenol GPCR signal transduction in a E-mail address: [email protected] (E.S. Ferro). luciferase gene reporter assay.

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2012.07.002 3288 L.C. Russo et al. / FEBS Letters 586 (2012) 3287–3292

2. Materials and methods multiple-charged protonated peptides generated by electrospray ionization (ESI) were automatically mass selected and dissociated 2.1. Cell culture and EP24.15 suppression in MS/MS by 10–30-eV collisions with argon. Typical LC and ESI conditions consisted of a flow rate of 600 nL/min, capillary voltage HEK293 cells were cultured in Dulbecco’s modified Eagle’s med- of 3.5 kV, block temperature of 100 °C, and cone voltage of 100 V. ium (DMEM) containing 10% fetal bovine serum, penicillin (100 U/ ml) and streptomycin (100 lg/mL) (Life Technologies Co., Carlsbad, 2.5. MS data analyses CA, USA) and were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. The suppression of EP24.15 expres- To identify peptides, the raw data files were converted to a peak sion was performed using RNA interference (siRNA) in HEK293 list format (mgf) by Mascot Distiller version 2.1.1 (Matrix Science cells. siRNAs targeting human EP24.15 were purchased commer- Ltd., London, UK) and analyzed using the search engine MASCOT cially (IDT, Integrated DNA Technologies Inc., Coralville, IA, USA) version 2.2 (Matrix Science Ltd.). Selected variable modifications as a kit that contained three target sequences to EP24.15. We chose on lysine residues (K) and the N-terminus (N-term) included TMAB the sequence responsible for the greatest inhibition of EP24.15 GIST-Quat (K) and GIST-Quat (N-term), for labels with nine hydro- (Supplemental Material) and also used a control sequence (scram- gen, and GIST Quat: 2H (9) (K) and GIST-Quat: 2H (9) (N-term), for bled) that contains no homology to any existing protein. Briefly, labels with nine deuterium. The NCBI database (from 01 Jul 2009; HEK293 cells were plated in 35 mm plates (0.4 Â 106 cells/well) 9,298,190 sequences) was used for searches with taxonomy Homo overnight and were transiently transfected with siRNA sequences sapiens, ‘‘no enzyme’’, and 0.1 Da of mass tolerance for MS and MS/ using Lipofectamine RNAiMaxÒ (Life Technologies Co., Calsbad, MS precursor ions. Mascot searches were followed by manual inter- CA, USA). Enzymatic activity assays and western blot analysis were pretation to eliminate false positives. Several criteria were used to performed as previously described in our laboratory [15,16] to ver- accept or decline the peptides that were identified by MASCOT as ify the time at which there was the greatest decrease in EP24.15 follows: (1) the majority (>80%) of the major MS/MS fragment ions expression. matched predicted a, b, or y ions or parent ions with loss of trimethylamine; (2) a minimum of five fragment ions matched b or y ions; 2.2. EP24.15 enzymatic assay and Western blot (3) the number of tags incorporated into the peptide matched the number of free amines (N terminus and side chains of Lys). Quanti- The enzymatic activity of EP24.15 was determined in triplicate fication was performed by measuring the ratio of peak intensity for using a continuous assay with a quenched fluorescent substrate the various TMAB-labeled peptide pairs in the MS spectra. For this (QFS; 7-methoxycoumarin-4-acetyl-P-L-G-P-dK-(2,4-dinitrophenyl) analysis, the monoisotopic peak and the peaks containing one or as previously described [17]. The EP24.15 specific inhibitor N-[1- two atoms of 13C were used. Multiple scans of the MS spectra were (R,S)-carboxy-3-phenylpropyl]-Ala-Aib-Tyr-p-aminobenzoate (JA2; combined prior to quantification. 1 lM) was used to discern the peptidolytic activity attributed exclu- sively to EP24.15 [18]. The results were expressed as arbitrary units 2.6. Luciferase reporter gene assay of fluorescence (UAF) per minute normalized by protein concentra- tion. Western blotting was conducted using anti-EP24.15-specific To analyze the possible effects of the peptides on GPCR intracel- antiserum (1:3000; Proteimax Biotech, São Paulo, SP, Brazil) diluted lular signaling, a reporter gene construct composed of three copies in 25 mM Tris–HCl, pH 7.4 and 125 mM NaCl containing 0.1% Tween of the IL-6 multiple response element in tandem with a cAMP- 20 (TTBS) in 5% fat-free dry milk with a 2-h incubation time at room responsive element (CRE) that drives luciferase expression in temperature. Goat anti-rabbit IgG conjugated to peroxidase (1:2000; response to Gi, Gs, or Gq stimulation was constructed as previously GE Healthcare Co., Waukesha, WI, USA) was used as the secondary described [11]. To assess promoter activity, a dual luciferase repor- antibody. Blots were developed using the Super Signal Chemilumi- ter assay system (Promega Co., Fitchburg, WI, USA) was used. In nescent Substrate and high sensitivity photographic film (Thermo brief, 24 h after the siRNA transfection, HEK293 cells were tran- Scientific Inc., Rockford, IL, USA). siently transfected with pEGFP (1 lg; codes for the pGL3-CRE- MRE promoter luciferase construct) and the pRL-CMV plasmid 2.3. Peptide extraction, quantification and isotopic labeling (16 ng; encodes the Renilla luciferase and is used to normalize transfection efficacy) using HEKFectin™ Cell Line Specific Lipid Peptide extraction from HEK293 cells and quantification was (Bio-Rad Laboratories Inc., Hercules, CA, USA) for 6 h. Transfected conducted as previously described [12,19]. The peptide quantifica- cells were incubated in Dulbecco’s modified Eagle’s medium con- tion reaction was performed at pH 6.8 to ensure that only the ami- taining 10% fetal bovine serum overnight and were subsequently no groups of peptides but not free amino acids react with seeded (0.1 Â 106 cells/well) onto a 96-well plates. Forty-eight fluorescamine [20]. The isotopic labeling of peptides for semi- hours after transfection, HEK293 cells were stimulated with either quantification was performed as originally described [21–24]. 1or10lM isoproterenol for 6 h at 37 °C. Firefly and Renilla luciferase activities in cell lysates were measured using a LMax lumino- 2.4. Liquid chromatography and tandem mass spectrometry meter (Molecular Devices, LLC., Sunnyvale, CA, USA) according to (LC–MS/MS) analyses the manufacturer’s instructions. The relative luciferase activity was calculated by normalizing firefly luciferase activity to Renilla LC–MS/MS experiments were performed on a Synapt G1 mass luciferase. Spectrometer (Waters Co., Milford, MA, USA). The peptide mixture was desalted on line for 15 min using a Symmetry C18 trapping 3. Results column (5 lm particles, 180 lm inner diameter, 20 mm length; Waters Co., Milford, MA, USA). The mixture of trapped peptides One of the three siRNA sequences initially evaluated in this was subsequently separated by elution with a water/acetonitrile/ study was able to cause a higher reduction on the EP24.15 0.1% formic acid gradient through a BEH 130-c18 column (1.7 lm enzymatic activity and was selected for further experiments particles, 100 lm inner diameter, 100 mm length; Waters Co., Mil- (Supplemental Fig. 1). The HEK293 cells transfected with EP24.15 ford, MA, USA). Data were acquired in data-dependent mode and siRNA showed an approximately 75% reduction of EP24.15 L.C. Russo et al. / FEBS Letters 586 (2012) 3287–3292 3289 enzymatic activity compared with HEK293 cells that were trans- peak containing one atom of 13C were measured. In this analysis, fected with the control siRNAs containing no homology to any about half of the peptides identified had altered levels in cells existing protein (scrambled siRNA; Fig. 1A). In western blot assays, transfected with siRNA to suppress EP24.15 expression compared EP24.15 immunoreactivity was absent in cells transfected with with control cells (Supplemental Table 1). Twenty peptides were EP24.15 siRNA (Fig. 1B). The slight discrepancy between the degree increased in the experimental samples (ratio P 1.80) and may rep- of siRNA-mediated inhibition of EP24.15 expression (100%) and resent substrates of the enzyme (Fig. 3, A, D and G). Six peptides activity (75%) may be related to the higher sensitivity of the enzy- decreased in the experimental samples (ratio 6 0.70) and may rep- matic assays. resent products of this enzyme (Fig. 4, B, E and H). Eighty-seven HEK293 cells transfected with EP24.15 siRNA (Fig. 1) were sub- peptides were considered unchanged (ratios between 0.7 and sequently used in mass spectrometry experiments. A total of 113 1.80) and thus are unlikely to serve as substrates or products of distinct peptides could be reproducibly detected in all three exper- EP24.15 (Fig. 3, C, F and I). For all of these analyses, the forward iments, while the amino acid sequence of 53 peptides were identi- and reverse labeling generally produced similar results (Fig. 3). fied after MS/MS sequencing analysis using the MASCOT search The variations between the triplicates are shown in Supplemental program followed by manual interpretation of the respective spec- Table 1. tra. Manual interpretation of the Mascot search results was neces- In addition, a luciferase gene reporter assay was employed to sary because the program does not consider the trimethylamine analyze the effects of EP24.15 inhibition on isoproterenol signal lost from the parent ion, and these ions are often the major ions transduction (Fig. 4; Supplemental Fig. 2). It has been previously in the spectra. Fig. 2 shows a representative MS/MS spectrum used shown that HEK293 cells endogenously express the b2-adrenergic in the analysis of the 3+ ion with an m/z of 502.74 for the peptide receptors coupled to Gs proteins and upon stimulation activate from heat shock 10-kDa protein. In addition to many fragments adenylate cyclase to raise intracellular cAMP levels and activate identified as a, b, or y ions, there were two additional intense frag- protein kinase A, as well as other downstream molecules [27,28], ments not identified by Mascot. The 651.38 m/z with 2+ represents which results in increased luciferase expression through multiple the parent ion with the trimethylamine and the 480.70 and response elements in the gene reporter assays [11]. HEK293 cells 457.32 m/z fragments represent the parent ion lacking one and were transfected with scrambled siRNA, EP24.15 siRNA or no two TMAB, respectively. The peptides identified here ranged in size siRNA (mock). After 24 h, the cells were transfected with the lucif- from 7 to 22 amino acids and were derived from 21 different pro- erase plasmids (pEGFP coding for the pGL3-CRE-MRE promoter teins. Approximately half of the peptides identified here have been luciferase construct and the pRL-CMV plasmid). The cells were identified in previous peptidomic studies using HEK293 cells treated with the b-adrenergic agonist isoproterenol 72 h after the [12,25,26], further validating the present analyses. transfection with siRNA. In all experiments, a significant increase Quantitative analyses were performed, and the relative peptide in luciferase activity was observed following stimulation of levels were quantified in each LC/MS runs by measurement of the HEK293 cells with either 1 or 10 lM isoproterenol (Supplemental peak intensities for each isotopic form detected in the MS spectra. Fig. 2). HEK293 cells transfected with EP24.15 siRNA exhibited For this analysis, the intensity of the monoisotopic peak and the additional increase in luciferase activity that was statistically sig-

Fig. 1. Effect of siRNA on the expression and enzymatic activity of EP24.15 in HEK293 cells. Cells were transiently transfected with EP24.15 siRNA or scrambled siRNA (control). Enzymatic activity and western blot assays were performed to confirm that cells transfected with EP24.15 siRNA, which would later be used for mass spectrometry assay, showed a significant decrease in EP24.15 expression. (A) EP24.15 enzymatic activity was determined using a continuous assay and is shown as the JA2-sensitive fraction of QFS hydrolyzing activity. Activity was inhibited by approximately 75% in cells that were transfected with EP24.15 siRNA compared to scrambled siRNA. Statistical comparisons were performed using analysis of variance (ANOVA) followed by the Tukey test. p < 0.0001 (⁄⁄⁄) indicate a significant difference between cells transfected with EP24.15 siRNA compared with cells transfected with scrambled siRNA. The experiments were performed in triplicate. (B) Crude cell extracts (100 lg) from control cells and from cells transfected with EP24.15 siRNA were separated by SDS–PAGE on an 8% polyacrylamide gel, transferred to nitrocellulose membranes, and incubated with specific rabbit antiserum against EP24.15 (1:3000). After incubation with an anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibody (1:3000), the immunoreactive bands were visualized by chemiluminescence. 3290 L.C. Russo et al. / FEBS Letters 586 (2012) 3287–3292

Fig. 2. Representative peptide sequencing by ESI-MS/MS. MS/MS analysis of the peptide KVGDKVLLPE 3+ ion with an m/z of 502.74 that eluted from the reverse-phase column at 16.7 min and was derived from Heat shock 10 kDa protein 1 (monoisotopic mass of the unprotonated and untagged peptide = 1096.65 Da) is shown. This peptide represents the D9-TMAB-labeled peptide shown in Fig. 4, A, D and G. After collision-induced dissociation, a, b, and y fragments that contain TMAB tags lost 68 Da (due to neutral loss of (C2H3)3 N from the tag). In addition, the parent ion [M] also shows neutral loss of 136 Da (2 Â 68 Da) to produce the 3+ ion with m/z = 457.32 and neutral loss of 2+0 2+ 68 Da to produce the 3+ ion with m/z = 480.03. b3 =b3 À H2O.

nificant compared with mock or scrambled siRNA-transfected con- signal transduction, suggesting a physiological significance for trols (Supplemental Fig. 2). The presence of the protein kinase these original findings. The mechanism by which intracellular pep- inhibitor KT5720 (1 lM) significantly reduced the stimulating tides modulate GPCR signaling appears to involve their ability to effects of the isoproterenol treatment on the luciferase activity, interact with EP24.15 and other specific proteins [11]. However, both in EP24.15 siRNA and scrambled siRNA cells (Fig. 4). the direct use of intracellular peptides for therapeutic purposes can be challenging, as the internalization of peptides or protein 4. Discussion into cells is not a trivial task. Conversely, the use of siRNA is considered a promising therapeutic option for the treatment of a vari- Endogenous intracellular peptides have now been described in ety of diseases, including genetic and viral diseases, as well as different human cell lines and in mouse brain tissue [12,25,26]. cancer. RNA interference (RNAi) is a post-transcriptional gene Although the cellular function of these peptides has not been silencing mechanism that involves the degradation of messenger well-established, evidence suggests that they may play a role in RNA in a highly sequence-specific manner. Double-stranded small GPCR signal transduction [11], insulin-induced glucose uptake interfering RNA (or simply siRNA), consisting of 21–25 nucleotides, [29,30] and protein–protein interaction [13]. The metabolism of can induce RNAi and inhibit the expression of target proteins. these intracellular peptides has recently been examined and stud- Therefore, siRNA therapeutics represent a fundamental new way ies suggest that both oligopeptidase EP24.15 [12] and the protea- to treat human disease by addressing targets that are otherwise some [25] can regulate specific intracellular peptides in HEK293 ‘undruggable’ with existing treatments. The discovery of long dou- cells. In this study, we have shown that EP24.15 enzymatic activity ble-stranded RNA-mediated RNAi in the worm [31] and the subse- can be inhibited by a specific siRNA, which, in parallel, changed the quent demonstration that RNAi, mediated by siRNA, operates in levels of specific intracellular peptides and potentiated the stimu- mammalian cells, sparked an explosion of research to uncover latory effects of isoproterenol on GPCR signal transduction. new mechanisms of gene silencing, which revolutionized our Previous studies have shown that oligopeptides isolated from understanding of endogenous mechanisms of gene regulation rat brain tissue using the inactive EP24.15 and introduced into and provided powerful new tools for biological research and drug CHO-S or HEK293 cells are capable of potentiating both angioten- discovery [32]. From the first in vivo evidence of RNAi-based ther- sin II and isoproterenol signal transduction [11]. EP24.15 apeutic efficacy in an animal disease model in 2003 [33], the pace overexpression decreased both angiotensin II and isoproterenol of drug development has been rapid. Three Phase I studies to inves- L.C. Russo et al. / FEBS Letters 586 (2012) 3287–3292 3291

Fig. 3. Representative MS spectras of TMAB-labeled peptide extracts from cells transfected with EP24.15 siRNA (experimental) or SCB siRNA (control). (A, D and G) MS spectra of the peptide fragment from heat shock 10-kDa protein 1, subsequently identified by MS/MS with the peptide sequence KVGDKVLLPE; this peptide was elevated in experimental samples relative to control samples and was considered a optimal substrate of EP24.15. (B, E and H) MS spectra of a peptide decreased in experimental samples and therefore was considered a product of EP24.15. This peptide was not identified by MASCOT and manual sequencing. (C, F and I) MS spectra of the peptide fragment from Histidine triad nucleotide-binding protein 1 identified as Ac-ADEIAKAQVAR. This peptide was not altered between experimental samples and control samples.

Fig. 4. EP24.15 siRNA activation of isoproterenol signal transduction is inhibited by the protein kinase inhibitor KT5720. HEK293 cells were transfected with a siRNA control sequence (scrambled), which contains no homology to any existing protein (scrambled siRNA) or with a siRNA sequence for suppression of EP24.15 (EP24.15 siRNA). After 24 h, cells were transfected with the luciferase plasmid vectors, as detailed in ‘‘Section 2’’. After 48 h, cells were stimulated with 1 lM isoproterenol, in the presence or absence of 1 lM KT5720, for 20 min, and luciferase activity was subsequently measured after 6 h. While all cells showed increased luciferase activity when stimulated with isoproterenol, the cells with decreased expression of EP24.15 (EP24.15 siRNA) showed a larger increase of luciferase activity when compared with scrambled siRNA cells (#p < 0.001; unpaired t test). The protein kinase inhibitor KT5720 signiﬁcantly decreased the isoproterenol effect on luciferase activity both in EP24.15 siRNA and scrambled siRNA treated cells (⁄⁄⁄p < 0.001; ⁄p < 0.05).

tigate RNAi-based drugs for age-related macular degeneration produced and investigated to improve in vivo stability; these stud- (AMD), a leading cause of blindness, and for respiratory syncytial ies have involved modification of the siRNA backbone, the sugar virus (RSV), the leading cause of pediatric hospitalizations in the moiety, and the nucleotide bases of antisense and/or sense strands United States, have already been completed without unfavorable [35]. Therefore, it is exciting that EP24.15 inhibition by siRNA can toxicity [34]. Several types of chemically modified siRNA have been alter isoproterenol signal transduction in cell culture. In the future, 3292 L.C. Russo et al. / FEBS Letters 586 (2012) 3287–3292 in vivo studies should be conducted to evaluate the effects of [13] Russo, L.C., Asega, A.F., Castro, L.M., Negraes, P.D., Cruz, L., Gozzo, F.C., et al. EP24.15 siRNA inhibition on the physiology of the sympathetic (2012) Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin. nervous system. Proteomics, 12, http://dx.doi.org/10.1002/pmic.201200032. [14] Sanden, C., Enquist, J., Bengtson, S.H., Herwald, H. and Leeb-Lundberg, L.M. Acknowledgments (2008) Kinin B2 receptor-mediated bradykinin internalization and metalloendopeptidase EP24.15-dependent intracellular bradykinin degradation. J. Pharmacol. Exp. Ther. 326, 24–32. The authors are thankful to Prof. Lloyd D. Fricker from the [15] Carreno, F.R., Goni, C.N., Castro, L.M. and Ferro, E.S. (2005) 14–3-3 epsilon Albert Einstein College of Medicine of Yeshiva University, Bronx, modulates the stimulated secretion of endopeptidase 24.15. J. Neurochem. 93, 10–25. NY, USA, for providing the TMAB isotope labels that were used in [16] Russo, L.C., Goni, C.N., Castro, L.M., Asega, A.F., Camargo, A.C., Trujillo, C.A., this work. This work was primarily supported by the Brazilian Na- et al. (2009) Interaction with calmodulin is important for the secretion of tional Research Council (CNPq; Grant 559698/2009-7, Rede GENO- thimet oligopeptidase following stimulation. FEBS J. 276, 4358–4371. [17] Rioli, V., Kato, A., Portaro, F.C., Cury, G.K., te Kaat, K., Vincent, B., et al. (1998) PROT) and partially supported by the University of São Paulo Neuropeptide specificity and inhibition of recombinant isoforms of the (Grants 2011.1.9333.1.3, NAPNA). ESF, FCG and LMC were funded endopeptidase 3.4.24.16 family: comparison with the related recombinant by CNPq research fellowships. LCR was supported by a post-doc- endopeptidase 3.4.24.15. Biochem. Biophys. Res. 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