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

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

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-

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

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%.

*
*
*
*
*
*
*
*
*
*
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

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

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

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

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,

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

)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

)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

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
.

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

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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