Báo cáo khoa học: Study of synthetic peptides derived from the PKI55 ppt

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH Rita Selvatici 1 , Sofia Falzarano 1 , Lara Franceschetti 2 , Adriano Mollica 3 , Remo Guerrini 4 , Anna Siniscalchi 5 and Susanna Spisani 2 1 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione Genetica Medica, Universita ` degli Studi di Ferrara, Italy 2 Dipartimento di Biochimica e Biologia Molecolare, Universita ` degli Studi di Ferrara, Italy 3 Dipartimento di Studi Farmaceutici, Universita ` di Roma ‘La Sapienza’, Italy 4 Dipartimento di Scienze Farmaceutiche, Universita ` degli Studi di Ferrara, Italy 5 Dipartimento di Medicina Clinica e Sperimentale, Sezione Farmacologia, Universita ` degli Studi di Ferrara, Italy Polymorphonuclear leukocytes (PMNs) play an essen- tial role in innate human immunity, and their primary role in the inflammatory response is to seek, bind, ingest and destroy invading pathogens by phagocyto- sis and oxygen-dependent and independent killing mechanisms. The hydrophobic N-formyl tripeptide Keywords chemotaxis; human neutrophils; lysozyme; PKC; PKI55 Correspondence R. Selvatici, Department of Experimental and Diagnostic Medicine, Medical Genetics Section, via Fossato di Mortara 74, 44100 Ferrara, Italy Fax: +39 0532 236157 Tel: +39 0532 974474 E-mail: svr@unife.it (Received 16 October 2007, revised 23 November 2007, accepted 28 November 2007) doi:10.1111/j.1742-4658.2007.06212.x Elucidation of the involvement of protein kinase C subtypes in several dis- eases is an important challenge for the future development of new drug tar- gets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lym- phocytes and to inhibit the activity of a, b 1 and b 2 protein kinase C iso- forms. The present study aimed to identify the minimal amino acid sequence of PKI55 that is able to inhibit the enzyme activity of protein kinase C. Peptides derived from both C- and N-terminal sequences were synthesized and initially assayed in rat brain protein kinase C to identify which part of the entire protein maintained the in vitro effects described for PKI55, and then the active peptides were tested on the isoforms a, b 1 , b 2 , c, d, e and f to identify their specific inhibition properties. Specific protein kinase C isoforms have been associated with the activation of specific sig- nal transduction pathways involved in inflammatory responses. Thus, the potential therapeutic role of the selected peptides has been studied in poly- morphonuclear leukocytes activated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH to evaluate their ability to modulate chemotaxis, superoxide anion production and lysozyme release. These studies have shown that only chemotactic function is signifi- cantly inhibited by these peptides, whereas superoxide anion production and lysozyme release remain unaffected. Western blotting experiments also demonstrated a selective reduction in the levels of the protein kinase C b 1 isoform, which was previously demonstrated to be associated with the polymorphonuclear leukocyte chemotactic response. Abbreviations fMLP-OMe, methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH; KRPG, Krebs-Ringer-phosphate containing 0.1% w ⁄ v glucose; PKC, protein kinase C; PMN, polymorphonuclear leukocyte. FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS 449 for-Met-Leu-Phe-OH (fMLP) and its methyl ester derivative (fMLP-OMe) are used as chemoattractants due to their high effectiveness in activating all physio- logical functions of human PMNs, such as chemotaxis, superoxide anion production and lysosomal enzyme secretion [1]. The interaction of fMLP ⁄ fMLP-OMe with specific formyl peptide receptors FPR and ⁄ or FPR like-1 expressed on PMNs [2–4] activates the phospholipase C, phospholipase D and phospholi- pase A 2 multiple second messenger pathways and leads to an increase in intracellular cAMP levels. The involvement of kinases, such as protein kinase C (PKC), phosphatidylinositide 3-kinase and mitogen- activated protein kinases has also been demonstrated [5]. We have previously reported that the chemotactic response of the PMNs triggered by fMLP-OMe is associated with specific PKC b 1 isoform translocation and p38 mitogen-activated protein kinase phosphoryla- tion by two independent pathways [6]. PKC is a family of serine-threonine kinases comprised of nine genes that express structurally related phospholipid-depen- dent kinases with distinct means of regulation and tissue distribution. Based on their structures and sensi- tivities to Ca 2+ and diacylglycerol, they have been classified into conventional PKCs (a, b and c), which are dependent on diacylglycerol and Ca 2+ for activity; novel PKCs (d, e, g and h), which are insensitive to Ca 2+ ; and atypical PKCs (f, and k ⁄ s), which require neither diacylglycerol nor Ca 2+ for their activation. PKC isoforms have different and often overlapping expression patterns, and most small molecule activa- tors and inhibitors used to probe PKC function lack isoform specificity [7]. PKC inhibitors, including peptides [8,9], have been extensively used to define the role of PKC and its iso- forms in signalling studies, and the large number of signal transduction events mediated by PKC suggests endless therapeutic potential for PKC inhibitors [10,11]. However, the usefulness of these inhibitors is limited by their poor pharmacokinetic characteristics and by their toxicity to normal tissues. The PKI55 protein was recently characterized in our laboratory [12] as a specific modulator of PKC that is normally poorly translated in vivo and whose synthesis is stimulated by PKC activation to prevent the over- expression of specific isoforms. We demonstrated that PKI55 and PKC form a complex with 1 : 1 stoichio- metry that can be digested by calpain. PKI55 associa- tes with PKC, but, unlike a great number of PKC inhibitors, it is not ATP-competitive and does not compete with the main C1 and C2 cofactors. PKI55, by promoting PKC degradation, establishes a feedback loop of inhibition. This is the behaviour of a suicidal inhibitor, which is required when a harmful substance (i.e. over-activated PKC) must be removed. Moreover, PKI55 was found to inhibit the recombinant a, b 1 , b 2 , c, d, f and g PKC isoforms in vitro and, when added to peripheral blood mononuclear cells activated with phytohaemagglutinin, was able to down-regulate the PKC enzyme activity of the a, b 1 and b 2 isoforms [13]. The present study aimed to identify peptides derived from the amino acid sequence of the PKI55 protein to be used as pharmacological tools. The effects of the peptides in vitro were studied on recombinant PKCs to identify their inhibitory profile versus specific isoforms. Subsequently, the potential therapeutic role of the active peptides was studied on human PMN inflam- matory responses. Since a fine regulation of such responses occurs through differences in activation of a spectrum of signalling pathways [6], we decided to evaluate which physiological functions (chemotaxis, superoxide anion generation and lysozyme release) were modulated by the selected peptides. The level of PKC a, b 1 , b 2 and f isoforms was also studied. Results Synthesis of peptides derived from PKI55 and their inhibitory effect on rat brain PKC A series of peptides was synthesized in order to iden- tify the minimal amino acid sequence of PKI55 able to inhibit PKC enzyme activity (Table 1). The C-terminal peptide 1 and its fragments 2 and 3 were devoid of inhibitory effects on rat brain PKC enzyme activity tested in vitro up to a concentration of 100 lm. The N-terminal peptide 4 and its derivatives 5, 6, 7, 8, 9 and 10 were then studied. Peptides 5, 8 and 9 dis- played inhibitory action, whereas peptides 6, 7 and 10 were found to be inactive (Table 1). Peptides 5, 8 and 9 were selected for further study to identify their inhib- itory profile versus specific PKC isoforms and to assess their potential anti-inflammatory action. Inhibitory effect of peptides derived from PKI55 on PKC isoforms Results obtained in a previous study of the inhibition properties of PKI55 protein on human recombinant PKC isoforms [13] were confirmed in the present study. PKI55 protein (6 lm) significantly decreased the enzyme activity of a, b 1 , b 2 , c, d and f, but not of e PKC isoforms (Fig. 1). Peptides 5, 8 and 9 were tested in vitro at a concentration of 6 lm on the same recom- binant PKC isoforms. As shown in Fig. 1, peptide 5, in comparison to PKI55, lost the inhibitory effect on Potential therapeutic role of PKI55-derived peptides R. Selvatici et al. 450 FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS c and f but maintained the inhibition on a, b 1 , b 2 and d isoforms. Peptide 8 lost the inhibitory effect on a but acquired the ability to inhibit the e isoform, whereas peptide 9 was only effective on the b 1 , e and f iso- forms. Interestingly, the inhibitory action of peptides 5 and 8 on the b 1 isoform was found to be significantly higher (P < 0.05) compared to the whole PKI55 protein. Effects of selected peptides on PMN inflammatory responses Peptides 5, 8 and 9 were tested for their ability to affect the physiological functions, such as chemotaxis, O 2 ) production and lysozyme release, of PMNs acti- vated with fMLP-OMe. In preliminary experiments, the PMN viability was assessed via the Trypan blue method, 90 min after incubation at 37 °C with peptides 5, 8 and 9 (0.1– 50 lm). Cell survival was not modified compared to untreated cells. The peptides did not display intrinsic agonist activity for human PMN chemotaxis or lyso- zyme assay up to a concentration of 50 lm. As regards O 2 ) production, only concentrations of 0.1, 0.5 and 1 lm were used because higher concentrations inter- fered with cytochrome c (data not shown). Figure 2 shows the effect of increasing concentra- tions (0.1–25 lm) of PKI55 and its derivative peptides 5, 8 and 9 on the chemotactic response triggered by 10 nm fMLP-OMe, which is the optimal concentration for this function [6]. The chemotactic movement was already significantly inhibited by PKI55 at 0.1 lm and by peptides 5, 8 and 9 at 0.5 lm. Peptide 5 was the most effective, reducing chemotaxis by 80%. The effects exerted by peptides 5, 8 and 9 on O 2 ) production and lysozyme release were studied in PMNs stimulated by 1 lm fMLP-OMe, the optimal concentration to activate these functions [14]. As shown in Fig. 3, none of the peptides was able to inhibit O 2 ) production at the tested concentrations. 0 10 20 30 40 50 60 70 80 90 100 * * * * * * * * * * * * * * * * * * Fig. 1. Percentage inhibition of the PKC a, b 1 , b 2, c, d, e and f isoform enzyme activity in the presence of the PKI55 protein and the derived peptides 5, 8 and 9, all tested at a concentration of 6 l M. The data are the mean ± SEM of three separate experi- ments. *P < 0.05 versus the control activity. Table 1. Amino acid sequence of the PKI55 protein and its peptide derivatives. For each peptide, the inhibition constant (IC 50 ) on PKC rat brain activity was assessed, by calculating the sigmoidal dose-dependence curve. Negative signs ()) indicate no activity up to a concentra- tion of 100 l M. The minimum active amino acid sequence is shown in bold. Peptides Amino acid sequence IC 50 on PKC rat brain (lM) PKI55 MLYKLHDV CRQLWFSCPACHHRAMRICCPAQHHRTISVCKTILSSPPPLDSLPCM 6.0 1 PACHHRAMRICCPAQHHRTISVCKTILSSPPPLDSLPCM ) 2 SVCKTILSSPPPLDSLPCM ) 3 PACHHRAMRICCPAQHHRTI ) 4 MLYKLHDV CRQLWFSCPACHHRAMRI 6.92 5 MLYKLHDV CRQLWFSC 9.15 6 MLYKLHDVCR ) 7 QLWFSCPACHHRAMRI ) 8 CRQLWFSC 10.69 9 CRQLW 12.48 10 FSCPACH ) R. Selvatici et al. Potential therapeutic role of PKI55-derived peptides FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS 451 Similarly, they showed no effect on lysozyme release, even at higher concentrations (Fig. 4). Western blotting As PKC-b 1 was previously shown [6] to be involved in chemotactic response, we performed western blotting experiments in activated PMNs to study the changes induced by peptides 5, 8 and 9. Fig. 5 shows the total level of PKC-b 1 in untreated human PMNs (lane 1), in PMNs activated with 10 nm fMLP-OMe for 30 s (lane 2) and in fMLP-OMe-activated PMNs pre-incubated at 37 °C for 10 min with peptides 5, 8 and 9 (at a concentration of 6 lm, lanes 3, 4 and 5, respectively). The levels of the PKC-b 1 isoform were significantly reduced in the PMNs treated with the peptides com- pared to fMLP-OMe-activated PMNs, as shown by the absorbance values of the corresponding autoradio- graphic bands (Fig. 5). The lack of an effect on the a, b 2 and f isoforms is also shown in Fig. 5. Discussion In the present study, selected peptides derived from the amino acid sequence of the PKI55 protein [12] are shown: (a) to inhibit specific PKC isoforms; (b) to Fig. 2. Chemotactic assays in presence of PKI55 or its derivative peptides 5, 8 and 9. The chemotactic index toward 10 n M fMLP- OMe was calculated in PMNs following a 10-min pre-treatment with the peptides. Each value represents the mean ± SEM of six separate experiments. *P < 0.05 versus fMLP-OMe. Fig. 3. Superoxide anion production in the presence of the selected peptides 5, 8 and 9 derived from PKI55. PMNs were pre-trea- ted with the selected peptides 5, 8 and 9 and stimulated with 1 l M fMLP-OMe, and O 2) production (nmol) measured. Each value represents the mean ± SEM of six separate experiments. Fig. 4. Lysozyme release with peptides 5, 8 and 9, derived from PKI55. PMNs were pre- treated with the selected peptides 5, 8 and 9 and stimulated with 1 l M fMLP-OMe, and the lysozyme release was evaluated. Each value represents the mean ± SEM of six separate experiments. Potential therapeutic role of PKI55-derived peptides R. Selvatici et al. 452 FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS selectively inhibit chemotaxis in PMNs activated with fMLP-OMe; and (c) to decrease the total level of the PKC-b 1 isoform. Almost all responses of the living cell, including acute inflammation, involve reversible phosphorylation of proteins. The number of protein kinases encoded by the human genome is estimated to comprise 1.7% of the human genome [15], and these kinases either cross-talk, cooperate, or compete with each other to determine the fate of the cell. Clarifica- tion of the specific role of each protein kinase is essen- tial for a detailed understanding of the signal transduction pathway, and should lead to the develop- ment of new drugs [16]. PKC is an attractive candidate as a therapeutic target, but clinically useful inhibitors need to be iso- form-specific and still retain enough potency to allow a sufficiently broad therapeutic index, given the critical role that PKC plays in many normal cellular signalling events [17]. A fine-tuned mechanism for the regulation of PKC involving a series of intra- and inter-molecular interactions was recently demonstrated [18]. There is currently a limited number of known selective PKC inhibitors. The commonly used pharmacological agents also inhibit other protein kinases (as catalytic domain inhibitors) and usually show no discriminatory activity on individual PKC isozymes [19,20]. The PKI55 protein, an endogenous PKC inhibitor identified and characterised in our laboratory, is not ATP-competitive and does not compete with the main C1 and C2 cofactors [12]. A series of peptides derived from the PKI55 protein was synthesized in order to identify the shortest amino acid sequence able to inhibit rat brain PKC enzyme activity. The results obtained show that: (a) the 39-amino-acid C-terminal peptide 1 and its derivatives 2 and 3 were ineffective; (b) the 26-amino-acid N-ter- minal peptide 4, from whose sequence peptides 5, 6, 7, 8, 9 and 10 were derived, displayed an inhibitory effect; (c) peptides 5, 8 and 9 showed an inhibitory effect on rat brain PKC; and (d) peptides 6, 7 and 10 were inactive. From these findings, it can be estab- lished that the amino acid sequence CRQLW (peptide 9) is necessary to inhibit PKC enzyme activity. The inactive peptides were not studied further. Peptides 5, 8 and 9 (containing the CRQLW amino acid sequence) were selected and further studied on the recombinant PKC isoforms a, b 1 , b 2 , c, d, e, f and their inhibitory profiles were compared with PKI55 protein. PKI55 protein was a broad inhibitor; only the e isoform was not inhibited. The selected peptides showed a more selective inhibiting profile, acquiring or losing the abil- ity to inhibit some isoforms: peptide 5 inhibited PKC a, b 1 , b 2 and d isoforms; peptide 8 inhibited b 1 , b 2 , d, e and f isoforms; and peptide 9 inhibited b 1 , e and f isoforms. Interestingly, the PKC-b 1 isoform was the only one to be significantly inhibited by both PKI55 and peptides 5, 8 and 9. Since we previously reported that specific PKC isoforms are involved in the different PMN responses during acute inflammation [6,14], pep- tides 5, 8 and 9 were tested on PMN functions to investigate their potential as therapeutic agents. The selected peptides displayed no agonist activity towards the responses of PMNs to fMLP-OMe, but signifi- cantly inhibited chemotactic function at concentrations unable to change the cell viability of PMNs. The pep- tides did not modify superoxide production or lyso- zyme release. It should be noted that the O 2 ) production assay was performed only with low peptide concentrations because higher concentrations interfered with the test. Nevertheless, lysozyme release was not modified, even at higher concentrations, suggesting that peptides 5, 8 and 9 had no effect on killing Fig. 5. Representative western blotting of PKC a, b 1 , b 2 and f in human PMNs. Lane 1, untreated PMNs; lane 2, PMNs stimulated with 10 n M fMLP-OMe; and lanes 3, 4 and 5, PMNs pre-treated with 6 l M 5, 8 and 9 peptides, respectively, for 10 min at 37 °C, and then stimulated with 10 n M fMLP-OMe for 2 min. The histo- grams represent the absorbance (A) of PKC-b 1 autoradiographic bands expressed as units mm –2 ; the values are mean ± SEM of three separate experiments. *P < 0.05, significantly different from fMLP-OMe-stimulated PMNs. R. Selvatici et al. Potential therapeutic role of PKI55-derived peptides FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS 453 mechanisms but displayed selective action on chemo- taxis. This peculiar behaviour could be related to the high inhibitory effect on the PKC-b 1 isoform shared by all the selected peptides, as shown by western blot- ting analysis. Activation of PKC in a variety of differ- ent cell types leads to changes in the cell cytoskeleton, including lymphocyte surface receptor capping [21], smooth muscle contraction [22], actin rearrangement and cytoskeletal reorganization in T cells [23] and neu- trophils [24,25]. Given the ubiquitous expression of PKC and the diversity of cytoskeletons in different cell types, it is not surprising that PKC has been shown to phosphorylate or be associated with a wide range of cytoskeletal components [26]. Previously [6], we showed that PKC-b 1 isoform activation was strongly associated with the chemotactic response of fMLP- OMe-activated PMN. In the present study, western blotting experiments showed that the treatment of acti- vated PMNs with the peptides 5, 8 and 9 selectively decreased PKC-b 1 isoform levels. We suggest that the peptides 5, 8 and 9 could either interfere with the link between fMLP-OMe and its receptor or, alternatively, decrease the ability of PKC-b 1 to associate with the some cytoskeletal component, thus also diminishing the chemotactic response. However, a direct relation- ship between a biochemical and functional effect can not be established from the data obtained in the pres- ent study. In conclusion, peptides 5, 8 and 9 behave as PKC inhibitors. Due their ability to inhibit the PKC-b 1 iso- form, they could feasibly be used as pharmacological tools to decrease PMN cell migration [27]. Inhibition of the leukocyte recruitment process has recently been proposed as an important focus in the design of anti- inflammatory drugs for use in diseases such as athero- sclerosis, osteoporosis and Alzheimer’s disease, in which the inflammatory component is inappropriate, serving no host defence function [28]. Further investi- gations are required to determine whether the cellular effects observed in vitro correspond to effects that occur in vivo. The sequence of peptide 9, the minimum required for activity, could comprise the basis for chemical modifications aiming to improve pharmaco- kinetic characteristics. Experimental procedures Reagents Dextran, Ficoll–Paque, [c 32 P]-ATP and ECL western blotting detection reagents were purchased from Amer- sham-Pharmacia Biotech (Milan, Italy) and FMLP-OMe, dimethylsulfoxide, histone type III-S, cytochalasin B, cytochrome c and Micrococcus lysodeikticus were purchased from Sigma-Aldrich (Milan, Italy). Rat brain PKC and the a, b 1 , b 2 , c, d, e and f human recombinant PKC iso- forms were obtained from Calbiochem (Milan, Italy), poly(vinylidene difluoride) membranes were from Bio-Rad Laboratories S.r.l. (Milan, Italy) and PKC a, b 1 , b 2 and f antibodies were from Santa Cruz Biotechnology (Heidel- berg, Germany). All other reagents were of the highest grade commercially available. Synthesis of PKI55 and its fragments Automated protein synthesis and purification of PKI55 was carried out as described previously [12]. The same procedure was used for the synthesis of the PKI55 frag- ments, as described below. Peptides were synthesized by solid-phase method using Fmoc ⁄ tBu chemistry [29] with a SYRO XP synthesizer (MultiSyntech, Witten, Germany). Rink resin (0.65 mmolÆg )1 ) and Wang resin preloaded with Fmoc-Met (0.45 mmolÆg )1 ) (Fluka, Buchs, Switzer- land) were used as a support for the syntheses of peptide amides or free acid, respectively. The resin (0.2 g in all syntheses) was treated with piperidine (20%) in dimethy- formamide, and Fmoc amino acid derivatives (four-fold excess) were coupled to the growing peptide chain using [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa- fluorophosphate] [30] (four-fold excess). Piperidine (20%) in dimethyformamide was used to remove the Fmoc group in all steps. After deprotection of the last Fmoc group, the peptide resin was washed with methanol and dried in vacuo to yield the protected peptide resin. Protected peptides were cleaved from the resin by treatment with Reagent B [31], trifluoro- acetic acid-phenol-triisopropylosilan-H 2 O (88:5:2:5, v ⁄ v), 5 mLÆ0.2Æg )1 of resin at room temperature for 2 h. After filtration of the exhausted resin, the solvent was con- centrated in vacuo and the residue triturated with ether. The crude peptides were then purified by preparative reverse-phase HPLC to yield a white powder after lyophil- ization using a Water Delta Prep 4000 system (Waters, Mil- ford, MA, USA) with a Phenomenex (Torrance, CA, USA) Jupiter C 18 column (250 · 30 mm, 300 A, 15 lm spherical particle size column). The column was perfused at a flow rate of 25 mLÆmin )1 with solvent A (10%, v ⁄ v, acetonitrile in 0.1% aqueous trifluoroacetic acid), and a linear gradient from 0–60% of solvent B (60%, v ⁄ v, acetonitrile in 0.1% aqueous trifluoroacetic acid) over 25 min was adopted for elution of the peptides. Analytical HPLC analyses were per- formed on a Beckman (Fullerton, CA, USA) 125 liquid chromatograph fitted with an Alltech C 18 column (4.6 · 150 mm, 5 lm particle size), and equipped with a Beckman 168 diode array detector. The analytical purity of each peptide was determined using HPLC conditions in the above solvent system (solvents A and B) programmed at a flow rate of 1 mLÆmin )1 with a linear gradient from 5% to Potential therapeutic role of PKI55-derived peptides R. Selvatici et al. 454 FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS 50% B over 25 min. All analogues showed > 95% purity when monitored at 220 nm. The synthesized peptides showed a correct molecular mass as determined by electro- spray MS. PKC activity Rat brain PKC and the human recombinant PKC iso- forms a, b 1 , b 2 , c, d, e and f, were diluted in 20 mm Hepes (pH 7.5 at 30 °C) and 2 mm dithiothreitol immedi- ately prior to assay. Typically, 3 units (10 lL) were assayed in the presence or absence of Ca 2+ by measuring the rate of phosphate incorporation from 6000 CiÆmmol )1 [c 32 P]-ATP into saturating amounts of histone III-S, according to Orr and Newton [32]. The reaction mixture (80 lL) contained 0.1 mm [c 32 P]-ATP, 25 mm MgCl 2 , lipid sonicated dispersion of phosphatidylserine (140 lm) and diacylglycerol (3.8 lm), prepared as described previously [33] and 0.5 mm Ca 2+ or 0.5 mm EGTA. Samples were incubated at 30 °C for 6 min and the reaction was stopped by the addition of 25 lL of a solution containing 0.1 m ATP and 0.1 m EDTA (pH 8). Aliquots (85 lL) were spotted on P81 ion-exchange chromatography paper (Whatman, Springfield, UK) and washed four times with 0.4% (v ⁄ v) phosphoric acid, followed by a 95% ethanol rinse, and 32 P incorporation was detected by liquid scintillation counting in 5 mL of scintillation fluid (Packard, Ramsey, MN, USA). One unit of PKC activity was defined as the amount of enzyme that caused the incorporation of 1 nmolÆmin )1 of phosphate into the sub- strate under these conditions. Formylpeptide dilution A10 )2 m stock solution of fMLP-OMe was prepared in dimethylsulfoxide and diluted in Krebs-Ringer-phosphate containing 0.1% w ⁄ v glucose (KRPG, pH 7.4_ before use. KRPG was made up as a five times working strength stock solution with the following composition: NaCl 40 gÆL )1 ; KCl 1.875 gÆL )1 ;Na 2 HPO 4 .2H 2 O 0.6 gÆL )1 ;KH 2 PO 4 0.125 gÆL )1 ; NaHCO 3 1.25 gÆL )1 ; and glucose 10 gÆL )1 . 1mm MgCl 2 and CaCl 2 supplemented the buffer before biological tests. Purification of human PMNs Cells were obtained from the peripheral blood of healthy subjects, and the PMNs were purified employing the stan- dard techniques of dextran sedimentation, centrifugation on Ficoll–Paque and hypotonic lysis of contaminating red blood cells. The cells were washed twice and resuspended in KRPG, pH 7.4, at a final concentration of 50 · 10 6 cell- sÆmL )1 , and used immediately. The percentage of PMNs was 98–100% pure and ‡ 99% viable, as determined by the Trypan blue exclusion test. No donors had received any medication for 3 days prior to donation and all were non-smokers. The study was approved by the local Ethics Committee, and informed consent was obtained from all participants. Random locomotion and chemotaxis Random locomotion and chemotaxis studies were per- formed with a 48-well microchemotaxis chamber (BioProbe, Milan, Italy), and migration into the filter was evaluated by the leading-front method, according to Zigmond and Hirsch [34]. Untreated PMNs, as control, and PMNs pre-incubated for 10 min at 37 °C with PKI55 protein and the selected peptides were loaded into the higher compartment of the microchemotaxis chamber, whereas fMLP-OMe 10 nm was added to the lower compartment. After 90 min of incuba- tion at 37 °C, the cell migration was evaluated. The random movement, expressed as migration toward the buffer, was used as control. Data were expressed in terms of the chemo- tactic index (CI) ratio as: (migration toward fMLP – Ome migration toward the buffer) ⁄ (migration toward the buffer). Superoxide anion production Superoxide anion production was measured by the super- oxide dismutase-inhibited reduction of ferricytochrome c modified for microplate-based assays [35]. Tests were carried out in a final volume of 200 lL containing 4 · 10 5 PMNs, 100 nmol cytochrome c and KRPG. PMNs were pre-incubated with the selected peptides derived from PKI55 for 10 min at 37 °C. The cells were then incubated with 5 lgÆmL )1 cytochalasin B for 5 min, 1 lm fMLP-OMe was added and the plates were incubated in a microplate reader (Ceres 900; Bio-Tek Instruments, Inc., Winooski, VT, USA) at 37 °C. Absorbance was recorded at wave- lengths of 550 and 468 nm. Differences in absorbance at the two wavelengths were used to calculate the amount O 2) produced (nmol) using a molar extinction coefficient for cytochrome c of 18.5 mm )1 Æcm )1 . Granule enzyme assay The release of PMN granule enzymes was evaluated by determining the lysozyme activity modified for microplate- based assays; 3 · 10 6 cells were pre-incubated with 5 lgÆmL )1 cytochalasin B, with or without the selected peptides derived from PKI55, for 10 min at 37 °C. PMNs were then activated using 1 lm fMLP-OMe for 15 min at 37 °C, and centrifuged for 5 min at 400 g. The lysozyme was quantified nephelometrically by the rate of lysis of a cell wall suspension of Micrococcus lysodeikticus (Sigma- Aldrich). The reaction rate was measured with a micro- plate reader at 465 nm. Enzyme release was expressed as R. Selvatici et al. Potential therapeutic role of PKI55-derived peptides FEBS Journal 275 (2008) 449–457 ª 2007 The Authors Journal compilation ª 2007 FEBS 455 the net percentage of total enzyme content released by 0.1% Triton X-100. Spontaneous release was less than 10%, and total enzyme activity was 85±1 l g Æ1 · 10 7 cells )1 Æ min )1 . Western blotting Suspensions of 1 · 10 7 PMNsÆmL )1 were pre-incubated, with or without the selected peptides derived from PKI55, at 37 °C for 10 min and then stimulated with 10 nm fMLP-OMe for 2 min. The reactions were halted by the addition of ice-cold KRPG, and the cells were pelletted at 6000 g for 5 min at 4 °C. The supernatant was dis- carded and the pellet was suspended in RIPA buffer con- taining 20 mm Tris pH 7.5, 0.25 m saccharose, 2 mm EDTA, 10 mm EGTA, 2 mm phenyl-methylsulfonyl fluo- ride and a protease inhibitor cocktail tablet (Roche, Milan, Italy). Cell lysates were sonicated (6 · 10 s) at 4 °C and centrifuged at 17 000 g for 5 min. The pellet, corresponding to nuclei and unbroken cells, was discarded and the supernatant was recovered in a separate tube, sonicated (6 · 10 s) and used to analyze the total level of PKC a, b 1 , b 2 and f (corresponding to cytosol plus mem- brane). Protein content was determined by bicinchoninic acid method [36]. Equal amounts of proteins (25 lg) were subjected to gel electrophoresis on a 10% gel, and then electrophoretically transferred to poly(vinylidene difluoride) membrane at 100 V for 1 h. Blots were incubated in NaCl ⁄ Tris, pH 7.6, containing 5% non-fat dry milk and 0.1% (v ⁄ v) Tween 20 (NaCl ⁄ Tris-T) for 1 h at room temperature, and then incubated overnight at 4 °C with the PKC a, b 1 , b 2 and f polyclonal antibody isoform (0.3 lgÆmL )1 in NaCl ⁄ Tris-T). After washing with NaCl ⁄ Tris-T buffer, a 1 : 6000 dilution of horseradish peroxidase-labelled anti-rabbit IgG was added at room temperature for 1 h. ECL western blotting detection reagents were used to visualize specific hybridisa- tion signals. The molecular weight was calculated with pre- stained SDS ⁄ PAGE standards (New England Bio-Labs Inc., Milan, Italy) and densitometric analysis of autoradiographic bands was performed with a Bio-Rad densitometer GS700 and expressed as absorbance (A). Statistical analysis Data are given as mean ± SEM. The significance of differ- ences between treated and control samples was assessed with Student’s t test for non-paired data. Differences between treatment groups were judged to be statistically significant at P £ 0.05. For each peptide, the inhibition con- stant (IC 50 ) on rat brain PKC activity was assessed, by calculating the sigmoidal dose-dependence curve, using graphpad prism software (GraphPad Software Inc., San Diego, CA, USA). 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