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

The PKI55 protein is able to penetrate the cell membrane of activated human T-lym-phocytes and to inhibit the activity of a, b1 and b2 protein kinase C iso-forms.. The present study aime

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 Selvatici1, Sofia Falzarano1, Lara Franceschetti2, Adriano Mollica3, Remo Guerrini4,

Anna Siniscalchi5and Susanna Spisani2

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, b1 and b2 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, b1, b2,

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

b1isoform, 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.

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 A2multiple 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 b1isoform 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 Ca2+and diacylglycerol, they have been

classified into conventional PKCs (a, b and c), which

are dependent on diacylglycerol and Ca2+for activity;

novel PKCs (d, e, g and h), which are insensitive to

Ca2+; and atypical PKCs (f, and k⁄ s), which require

neither diacylglycerol nor Ca2+for their activation

PKC isoforms have different and often overlapping

expression patterns, and most small molecule

activa-tors and inhibiactiva-tors 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, b1, b2,

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, b1and b2isoforms [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, b1, b2and 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, b1, b2, 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

c and f but maintained the inhibition on a, b1, b2 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 b1, e and f

iso-forms Interestingly, the inhibitory action of peptides 5

and 8 on the b1 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,

O2 ) 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 37C 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

O2) 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 O2 ) 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 O2) production at the tested concentrations

0 10 20 30 40 50 60 70 80 90 100

*

*

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*

*

*

*

*

*

*

*

*

*

*

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*

*

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.

Similarly, they showed no effect on lysozyme release,

even at higher concentrations (Fig 4)

Western blotting

As PKC-b1was 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-b1 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 37C 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-b1isoform 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,

b2and 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 selecpre-trea-ted peptides 5, 8 and 9 and stimulated with 1 l M fMLP-OMe, and

O2)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.

selectively inhibit chemotaxis in PMNs activated with

fMLP-OMe; and (c) to decrease the total level of the

PKC-b1isoform 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, b1, b2, 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, b1, b2 and d isoforms; peptide 8 inhibited b1, b2, d,

e and f isoforms; and peptide 9 inhibited b1, e and f isoforms Interestingly, the PKC-b1isoform 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 O2 ) 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-b1 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.

mechanisms but displayed selective action on

chemo-taxis This peculiar behaviour could be related to the

high inhibitory effect on the PKC-b1 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-b1isoform 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-b1isoform 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-b1 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-b1

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, [c32P]-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, b1, b2, 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, b1, b2 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-H2O (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 C18 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

(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

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, b1, b2, c, d, e and f, were diluted in 20 mm

Hepes (pH 7.5 at 30C) and 2 mm dithiothreitol

immedi-ately prior to assay Typically, 3 units (10 lL) were

assayed in the presence or absence of Ca2+by measuring

the rate of phosphate incorporation from 6000 CiÆmmol)1

[c32P]-ATP into saturating amounts of histone III-S,

according to Orr and Newton [32] The reaction mixture

(80 lL) contained 0.1 mm [c32P]-ATP, 25 mm MgCl2, lipid

sonicated dispersion of phosphatidylserine (140 lm) and

diacylglycerol (3.8 lm), prepared as described previously

[33] and 0.5 mm Ca2+or 0.5 mm EGTA Samples were

incubated at 30C 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

with 0.4% (v⁄ v) phosphoric acid, followed by a 95%

ethanol rinse, and 32P 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

A 10)2m 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; Na2HPO4.2H2O 0.6 gÆL)1; KH2PO4

0.125 gÆL)1; NaHCO3 1.25 gÆL)1; and glucose 10 gÆL)1

1 mm MgCl2 and CaCl2 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· 106

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 37C 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 37C, 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· 105 PMNs, 100 nmol cytochrome c and KRPG PMNs were pre-incubated with the selected peptides derived from PKI55 for 10 min at 37C The cells were then incubated with 5 lgÆmL)1cytochalasin 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 37C Absorbance was recorded at wave-lengths of 550 and 468 nm Differences in absorbance at the two wavelengths were used to calculate the amount

O2) produced (nmol) using a molar extinction coefficient for cytochrome c of 18.5 mm)1Æcm)1

Granule enzyme assay

The release of PMN granule enzymes was evaluated by determining the lysozyme activity modified for microplate-based assays; 3· 106

cells were pre-incubated with

5 lgÆmL)1 cytochalasin B, with or without the selected peptides derived from PKI55, for 10 min at 37C PMNs were then activated using 1 lm fMLP-OMe for 15 min at

37C, 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

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 lgÆ1· 107cells)1Æ

min)1

Western blotting

Suspensions of 1· 107PMNsÆmL)1 were pre-incubated,

with or without the selected peptides derived from PKI55,

at 37C 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 4C 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

4C 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, b1, b2and 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 4C with the PKC a, b1, b2and 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 (IC50) 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)

Acknowledgements

This work was supported by grants from the Univer-sity of Ferrara; the Associazione Emma e Ernesto

Rul-fo per la Genetica Medica, Parma, Italy; and the Fondazione Cassa di Risparmio di Ferrara, Italy We are grateful to Banca del Sangue of Ferrara for pro-viding fresh blood and Dr Amanda Neville for the English revision of the text

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