Studyofsyntheticpeptidesderivedfromthe 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 ofthe 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 thePKI55 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 ofPKI55 that is able to inhibit the enzyme activity of protein
kinase C. Peptidesderivedfrom both C- and N-terminal sequences were
synthesized and initially assayed in rat brain protein kinase C to identify
which part ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe 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 ofthe a, b
1
and b
2
isoforms [13].
The present study aimed to identify peptides derived
from the amino acid sequence ofthePKI55 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 ofpeptidesderivedfromPKI55 and
their inhibitory effect on rat brain PKC
A series ofpeptides was synthesized in order to iden-
tify the minimal amino acid sequence ofPKI55 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 ofpeptidesderivedfrom PKI55
on PKC isoforms
Results obtained in a previous studyofthe inhibition
properties ofPKI55 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 ofpeptides 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. Thepeptides 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) ofPKI55 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 ofthepeptides 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 ofthe PKC a,
b
1
, b
2,
c, d, e and f isoform enzyme activity
in the presence ofthePKI55 protein and
the derivedpeptides 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 ofthePKI55 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 studythe 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 ofthe PKC-b
1
isoform were significantly
reduced in the PMNs treated with thepeptides com-
pared to fMLP-OMe-activated PMNs, as shown by
the absorbance values ofthe 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 peptidesderivedfrom the
amino acid sequence ofthePKI55 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 ofthe selected peptides 5, 8 and
9 derivedfrom 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, derivedfrom 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 ofthe 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% ofthe human genome [15], and these
kinases either cross-talk, cooperate, or compete with
each other to determine the fate ofthe cell. Clarifica-
tion ofthe specific role of each protein kinase is essen-
tial for a detailed understanding ofthe 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 ofpeptidesderivedfromthePKI55 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 thepeptides 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 fromthe 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 ofthe highest
grade commercially available.
Synthesis ofPKI55 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 ofthePKI55 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 ofthe 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 ofthe 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 ofthe 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 fromthe 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. Thestudy 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 ofthe 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 peptidesderived 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 derivedfrom 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 peptidesderivedfrom 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).
Acknowledgements
This work was supported by grants fromthe 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 ofthe text.
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R. Selvatici et al. Potential therapeutic role of PKI55-derived peptides
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. 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. 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