Báo cáo khoa học: Proteolytic degradation of nitric oxide synthase isoforms by calpain is modulated by the expression levels of HSP90 potx

Proteolytic degradation of nitric oxide synthase isoforms
by calpain is modulated by the expression levels of HSP90
Monica Averna, Roberto Stifanese, Roberta De Tullio, Franca Salamino, Mara Bertuccio,
Sandro Pontremoli and Edon Melloni
Department of Experimental Medicine (DIMES) ) Biochemistry Section and Centre of Excellence for Biomedical Research (CEBR),
University of Genoa, Italy
Nitric oxide (NO) is a gaseous free radical promoting
many biological effects, including the control of micro-
vascular tone, the renin and eicosanoic systems and
other modulators of inflammation [1–4]. Due to its
high chemical reactivity, NO can be harmful through
the nitrosylation of many proteins [1,5]. NO is gener-
ated exclusively by three NO synthase (NOS) isoforms
[3]. Two of them constitutively expressed in cells have
been identified as neuronal NOS (nNOS) and endo-
thelial NOS (eNOS) on the basis of their preferential
expression in neuronal or in endothelial cells, respec-
tively. The expression of the third form, inducible
NOS (iNOS), is induced by various cytokines [1]. All
three isozymes catalyze the formation of NO from
arginine, oxygen and NADPH [1–4]. A number of co-
factors are required for their catalytic activity, includ-
ing tetrahydrobiopterin, FAD and FMN, in addition
to a heme prosthetic group. To acquire the active state
Keywords
Ca
2+
homeostasis; calpain; calpastatin;
HSP90; NOS
Correspondence
S. Pontremoli, University of Genoa,

DIMES ) Bicohemistry Section,
Viale Benedetto XV 1, 16132 Genoa, Italy
Fax: +39 010 518343
Tel: +39 010 3538162
E-mail:
(Received 31 July 2007, revised 12 Septem-
ber 2007, accepted 8 October 2007)
doi:10.1111/j.1742-4658.2007.06133.x
Ca
2+
loading of Jurkat and bovine aorta endothelium cells induces the
degradation of the neuronal and endothelial nitric oxide synthases that are
selectively expressed in these cell lines. For neuronal nitric oxide synthase,
this process involves a conservative limited proteolysis without appreciable
loss of catalytic activity. By contrast, endothelial nitic oxide synthase diges-
tion proceeds through a parallel loss of protein and catalytic activity. The
chaperone heat shock protein 90 (HSP90) is present in a large amount in
Jurkat cells and at significantly lower levels in bovine aorta endothelium
cells. The differing ratios of HSP90 ⁄ nitric oxide synthase (NOS) occurring
in the two cell types are responsible for the conservative or nonconservative
digestion of NOS isozymes. Consistently, we demonstrate that, in the
absence of Ca
2+
, HSP90 forms binary complexes with NOS isozymes or
with calpain. When Ca
2+
is present, a ternary complex containing the three
proteins is produced. In this associated state, HSP90 and NOS forms are
almost completely resistant to calpain digestion, probably due to a struc-
tural hindrance and a reduction in the catalytic efficiency of the protease.

Thus, the recruitment of calpain in the HSP90–NOS complexes reduces the
extent of the proteolysis of these two proteins. We have also observed that
calpastatin competes with HSP90 for the binding of calpain in recon-
structed systems. Digestion of the proteins present in the complexes can
occur only when free active calpain is present in the system. This process
can be visualized as a novel mechanism involving the association of NOS
with HSP90 and the concomitant recruitment of active calpain in ternary
complexes in which the proteolysis of both NOS isozymes and HSP90 is
significantly reduced.
Abbreviations
AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; BAE-1, bovine aorta endothelium; CaM, calmodulin; eNOS, endothelial nitric oxide
synthase; HSP90, heat shock protein 90; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; NO, nitric oxide;
NOS, nitric oxide synthase.
6116 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS
nNOS and eNOS isoforms also requires calmodulin
(CaM) and Ca
2+
ions, indicating that NO synthesis is
triggered, in target tissues or cells, by an elevation of
free [Ca
2+
]
i
.
A number of structural differences characterize the
three NOS isoforms. At the N-terminal region, nNOS
contains a PDZ domain that addresses the enzyme to
specific synaptic sites in brain and muscle [2,3,6]. This
segment is absent in eNOS and iNOS forms and the
latter is insensitive to Ca

2+
ions due to its high affinity
for the CaM binding site also at basal Ca
2+
levels [7].
NO production is highly regulated by many factors
and mechanisms, including protein–protein interactions
involved in allosteric regulation, scaffolding and traf-
ficking [3,8–13]. Of particular interest is the interaction
with the chaperone heat shock protein 90 (HSP90) that
causes the release of the synthase from its association
with caveolin-3, a protein that maintains eNOS in an
inhibited state [14,15]. HSP90 can also favour the
insertion of the heme group in the synthase at its natu-
ral site, promoting the dimerization and thus the
acquirement of the active conformation through the
association with CaM and Ca
2+
ions [16,17]. The loss
of HSP90 or its inhibition by geldanamycin prevents
the onset of the active NOS form and increases its deg-
radation by the ATP–ubiquitin–proteasome pathway
[18–21].
Degradation of NOS has also been proposed as a
regulatory mechanism in conditions of high NO pro-
duction in order to prevent the toxic effects of this
compound [20–22]. It has been suggested that calpain
is the protease involved [23–27] because its activation
occurs in conditions that also cause the production of
NO, and because NOS and HSP90 have been identi-

fied as target substrates of the thiol protease [28–30].
In the present study, we further explored the
involvement of calpain in the regulation of nNOS and
eNOS activity taking into account a possible role of
HSP90 in this process.
In this respect, we have initially observed that, in
Jurkat and in bovine aorta endothelium (BAE-1) cells,
containing different amounts of HSP90, the extent of
degradation of nNOS and eNOS by calpain was
directly related to the level of the chaperone protein.
In reconstructed systems, we have demonstrated that
HSP90 significantly reduces the extent of NOS proteo-
lysis by calpain through the formation of selective
binary and ternary heterocomplexes containing the
synthases and the protease. Accordingly, we propose
that the protective effect exerted by HSP90 is due to
the recruitment of calpain in a complex form in which
the chaperone protein becomes resistant to proteolysis,
and also due to a concomitant decrease in the Ca
2+
binding capacity of calpain. The physiological rele-
vance of this novel property of HSP90 is also dis-
cussed.
Results
Degradation of NOS isozymes in Ca
2+
loaded
Jurkat and BAE-1 cells
Degradation of NOS isozymes was studied in intact
Jurkat and BAE-1 cells, containing nNOS and eNOS,

respectively. Calpain was activated [31] by increasing
[Ca
2+
]
i
following exposure of cells to the Ca
2+
-iono-
phore A23187. As shown in Fig. 1, in Jurkat cells, the
nNOS native 160 kDa band was progressively reduced
and converted into two new bands showing an approx-
imate molecular mass of 140 and 130 kDa. Because, in
these conditions, more than 50–60% of the 160 kDa
band disappeared, whereas the total catalytic activity
A
B
Fig. 1. Digestion of NOS isozymes in cells loaded with Ca
2+
. Jurkat
and BAE-1 cells (2 · 10
6
for each experiment) were incubated in
2mLof10m
M Hepes, pH 7.4, containing 0.14 M NaCl, 5 mM KCl,
and 2 gÆL
)1
glucose for 30 min at 37 °C in the absence (control) or
in the presence of 1 l
M ionophore A23187 and 1 mM CaCl
2

(Ca
2+
ion). Alternatively, cells were preincubated for 30 min at 37 °C with
50 l
M PD151746 (PD). Cells were then collected by centrifugation
and lysed by freezing and thawing, followed by sonication. (A)
Aliquots (15 lg protein) of cell extracts were submitted to 7%
SDS ⁄ PAGE and immunoblotting. NOS isozymes and b-actin were
detected with the specific mAbs. (B) NOS activity was assayed as
described in the Experimental procedures using an aliquot (200 lL)
of each cell extract. The values reported are the arithmetical
mean ± SD of three different experiments.
M. Averna et al. Degradation of NOS isozymes by calpain
FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6117
of nNOS was only reduced by 15–20%, it was assumed
that the digestion products still retained the ability to
produce NO. In cells pretreated with the synthetic cal-
pain inhibitor PD151746 [22,32], the native 160 kDa
band, as well as the catalytic activity, were completely
preserved whereas no low molecular mass nNOS forms
were accumulated. Identical results were obtained fol-
lowing preincubation of the cells with calpain inhibi-
tor-1 [33,34], known to be highly effective on calpain
although not completely specific (data not shown).
Conversely, in Ca
2+
loaded BAE-1 cells, approxi-
mately 80% of the native eNOS 130 kDa band and
total catalytic activity disappeared, without the appear-
ance of detectable active intermediates. Pre-treatment

with the synthetic calpain inhibitor PD151746 [22,32]
completely prevented the loss of both eNOS protein
and catalytic activity.
Thus, whereas Ca
2+
loading in Jurkat cells pro-
motes a limited proteolysis of nNOS, in BAE-1, cells
Ca
2+
-enrichement induces digestion and inactivation
of eNOS to a large extent.
Expression of HSP90 and NOS isozymes in Jurkat
and in BAE-1 cells
To explain the different vulnerability of the two NOS
isozymes present in Jurkat and BAE-1 cells, and to
explore their relationship with the level of HSP90, the
three proteins were isolated and quantified. By ion
exchange chromatography (Fig. 2A), we separated
NOS from HSP90 and directly measured the amount
of these proteins expressed in both cells. As shown in
Fig. 2B, in Jurkat cells, a large amount of nNOS was
found to be accompanied by an even greater quantity
of HSP90. By contrast, in BAE-1 cells, the levels of
both eNOS and HSP90 were lower than those of
nNOS and HSP90 in Jurkat cells. The quantification
of each protein shown in Fig. 2C established that the
ratio nNOS ⁄ HSP90 in Jurkat cells was largely in
favour of HSP90 whereas, in BAE-1 cells, eNOS
slightly exceeds the level of the chaperone protein
(Fig. 2C). These findings indicate that HSP90 could

exert a protective effect in the digestion of NOS by
calpain.
Interaction between HSP90, NOS isozymes and
l-calpain
To explore this process, we first examined the ability
of HSP90 to associate with NOS isozymes. As previ-
ously reported [8,11,35], it was found that both NOS
isozymes immunoprecipitated from cell lysates with
an antibody-immobilized HSP90 (Fig. 3A). Identical
results were obtained using purified protein prepara-
tions, indicating that the association between NOS and
HSP90 did not require specific factors present in the
crude cell extracts. We further characterized this
A
B
C
Fig. 2. Separation of NOS isozymes from HSP90 in Jurkat and
BAE-1 cells by ion exchange chromatography. (A) Cell extracts from
Jurkat (60 · 10
6
) and BAE-1 cells (10 · 10
6
), obtained as described
in the Experimental procedures, were submitted to ion exchange
chromatography following the procedure described in the Experi-
mental procedures. NOS activity was evaluated on the eluted frac-
tions (100 lL) as reported in the Experimental procedures. s,
nNOS activity; d, eNOS activity. The arrow indicates the position
of HSP90 elution. (B) Aliquots (30 lL) of the fractions eluted from
the ion exchange chromatography described in (A) were submitted

to 7% SDS ⁄ PAGE and Immunoblotting. NOS isozymes and HSP90
were detected with the specific mAbs as reported in the Experi-
mental procedures. (C) The immunoreactive bands of nNOS, eNOS
and HSP90 were scanned and quantified as described in the Exper-
imental procedures. The areas of the peaks were normalized on
the basis of the amount of protein loaded on the column. The val-
ues reported are the arithmetical mean ± SD of three different
experiments.
Degradation of NOS isozymes by calpain M. Averna et al.
6118 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS
association by the gel penetration technique (Fig. 3B)
and established that HSP90 could form a one-to-one
discrete complex with both NO isozymes with a mass
of approximately 500–550 kDa, corresponding to the
association of the two native dimeric proteins
(Fig. 3C).
Digestion of NOS isozymes and HSP90
by l-calpain
On the basis of the results so far described, the suscep-
tibility to digestion by human erythrocyte l-calpain of
purified nNOS, eNOS and HSP90 as single proteins or
in the associated forms was then evaluated. As shown
in Fig. 4A, in the presence of l-calpain, the digestion
of the nNOS native 160 kDa protein band was pre-
ceded by the transient accumulation of a 130 kDa
band. The catalytic activity of nNOS also progressively
disappeared, in parallel with the digestion of the
130 kDa protein. By contrast, eNOS was concomi-
tantly digested and inactivated by calpain (Fig. 4B)
without the appearance of intermediate active frag-

ments.
These digestion patterns are consistent with the
removal of the N-terminal PDZ domain from the
nNOS molecule that converts this enzyme in a molecu-
lar form similar to eNOS [2,4]. Both synthases are then
cleaved in a position close to the CaM binding site
that leads to the loss of catalytic activity in both iso-
forms [2,4]. The addition of CaM has no effect on the
pattern on digestion (data not shown). Our findings
appear to indicate that the digestion process of both
NOS proceeds through the hydrolysis of a very limited
number of peptide bonds; specifically, in the case of
nNOS, degradation can occur with the cleavage of two
peptide bonds and, in the case of eNOS, with the
cleavage of a single bond. HSP90 isolated from Jurkat
cells was also digested by calpain with the transient
accumulation of an 85–86 kDa band that replaces the
native one (Fig. 4C). However, the calpain requirement
for HSP90 digestion was found to be five- to ten-fold
higher than that required for digestion of NOS. In
addition, HSP90 from BAE-1 cells was digested by
A
B
C
Fig. 3. NOS isozyme–HSP90 interaction in Jurkat and BAE-1 cells.
(A) Immunoprecipitation of nNOS and eNOS–HSP90 complexes.
Aliquots (500 lg protein) of Jurkat and BAE-1 cell extracts (C, Ex.)
obtained as described in the Experimental procedures, were incu-
bated overnight at 4 °C with monoclonal anti-HSP90 serum as pre-
viously reported [8,11,35]. The mixtures were then incubated for

1 h at 4 °C with Protein G-Sepharose (30 lL). The particles were
collected, washed three times with the immunoprecipitation buffer,
resuspended in SDS ⁄ PAGE loading solution (30 lL) and submitted
to 7% SDS ⁄ PAGE. The presence of NOS isozymes together with
HSP90 in the solubilized material was established using the specific
mAbs. Alternatively, cell extracts were replaced with purified (Pur.)
NOS isozymes (1 lg) and HSP90 (1 lg). For experimental details,
see the Experimental procedures. (B) Changes in molecular size of
NOS isozymes in the presence of HSP90 detected by gel penetra-
tion technique. Equal amounts (0.5 lg) of nNOS or eNOS isolated
from Jurkat cells and BAE-1 cells, respectively, were diluted alone
or with the indicated amounts of HSP90, isolated from the corre-
sponding cell lines and added to packed Sephacryl S-300. The distri-
bution coefficient of NOS isozymes between the aqueous phase
and the gel fraction was determined as described previously
[36,37]. The values reported are the arithmetical mean ± SD of
three different experiments. (C) The molecular mass of NOS iso-
zymes, HSP90 and NOS isozyme–HSP90 complexes was evaluated
from the distribution coefficient of aldolase (molecular mass ¼
160 kDa) and ferritin (molecular mass ¼ 450 kDa) used as standard
proteins. The distribution coefficient of these proteins between the
aqueous phase and the gel fraction was determined as described
previously [36,37]. The values reported are the arithmetical
mean ± SD of three different experiments.
M. Averna et al. Degradation of NOS isozymes by calpain
FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6119
human erythrocyte l-calpain in an identical manner
(data not shown).
Comparing the amount of l-calpain required to
reduce the native bands of nNOS, eNOS and HSP90,

it was thereby established that nNOS was the most
sensitive substrate; eNOS was slightly more resistant,
whereas HSP90, independently from its source, was
five- to ten-fold less susceptible (Fig. 4D). A similar
degradation pattern was obtained with m-calpain iso-
lated from rat brain (data not shown).
When HSP90 was added to the nNOS digestion mix-
ture (Fig. 5A), the catalytic activity of the synthase
was almost completely preserved in spite of the disap-
pearance of the native band, which was completely
converted into the 130 kDa protein species. These
results confirm the previous assumption that the
130 kDa nNOS form retained full catalytic activity. In
the case of eNOS, the addition of HSP90 prevented its
calpain-mediated degradation as well as its inactivation
(Fig. 5B). Because HSP90 produces an identical pro-
tective effect regardless of whether it is isolated from
Jurkat or BAE-1 cells, it can be assumed that the
effect of the chaperone is not restricted to a single cell
type.
Thus, the present findings suggest that HSP90 can
prevent the degradation by calpain of both NOS by
protecting the cleavage of the peptide bond close to
the CaM binding site. This explains why both NOS
are inactivated by calpain in the absence of HSP90.
The removal of the PDZ domain by calpain, which
occurs without loss of catalytic activity in NOS even in
the presence of HSP90, provides additional evidence
that the function of this domain is probably related to
changes in intracellular localization of the active syn-

thase [6]. This novel protective effect exerted by
HSP90, as well as its higher resistance to calpain pro-
teolysis, was then further explored utilizing purified
A
B
C
D
Fig. 4. Susceptibility of nNOS, eNOS and HSP90 to calpain diges-
tion. nNOS (A), eNOS (B) and HSP90 (C) were incubated (1 lg
each) with increasing amounts of human erythrocyte calpain as
described in the Experimental procedures. The insets in (A), (B) and
(C) are representative of the immunoblots carried out to detect
NOS isozymes or HSP90 digested by calpain. NOS isozymes
activity was evaluated on aliquots of each incubation (50 lL) as
described in the Experimental procedures and is reported as a per-
centage of NOS activity assayed in the absence of calpain. The
amount of native band of HSP90 shown in (C) was quantified as
described in the Experimental procedures and is reported as a per-
centage of the protein level measured in the absence of calpain.(D)
Summary of the susceptibility of the three proteins to calpain diges-
tion shown in (A), (B) and (C). The values reported are the arithmet-
ical mean ± SD of three separate experiments.
Degradation of NOS isozymes by calpain M. Averna et al.
6120 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS
immunocomplexes (see Experimental procedures) to
avoid the contamination by free proteins and possible
artefacts.
Susceptibility of the ternary HSP90
⁄
NOS

⁄
calpain
complex to proteolysis
For the first time, we were able to show that the anti-
body-immobilized HSP90 was capable of binding NOS
isozymes or calpain in the absence of Ca
2+
ions, thus
forming alternative binary complexes (Fig. 6A, lanes 1,
2 and 4). When eNOS or nNOS isozymes were sepa-
rately added to the HSP90 ⁄ calpain immunoprecipitated,
no ternary complexes were formed because calpain was
completely displaced by NOS (Fig. 6A, lanes 3 and 5).
However, in the presence of Ca
2+
ions, each NOS iso-
zyme and the protease could still be recruited and
A
B
Fig. 6. Isolation of HSP90 ⁄ NOS ⁄ calpain complexes. (A) HSP90
(5 lg) was immobilized to Protein G-Sepharose resin using mono-
clonal anti-HSP90 serum as reported in the Experimental proce-
dures. The immunoprecipitated material was then incubated in the
presence of: human erythrocyte calpain (lane 1), nNOS (lane 2),
nNOS together with human erythrocyte calpain (lane 3), eNOS (lane
4), eNOS together with human erythrocyte calpain (lane 5) in the
conditions described in the Experimental procedures. Equal
amounts (30 lL) of each sample were submitted to SDS ⁄ PAGE fol-
lowed by immunoblot analysis (see Experimental procedures). The
formed immunocomplexes were revealed with the specific mAbs

against each of the proteins added to the samples containing the
immobilized HSP90. (B) The same experiments described in (A)
were carried out replacing EDTA with 1 m
M CaCl
2
. The immunopre-
cipitated material was then incubated in the presence of human
erythrocyte calpain (lane 1), nNOS (lane 2), nNOS together with
human erythrocyte calpain (lane 3), eNOS (lane 5), eNOS together
with human erythrocyte calpain (lane 6), in the conditions described
in the Experimental procedures. The same experiments reported
in lanes 3 and 6 were also performed with the addition to the
immunoprecipitated material of RNCAST600 (0.1 lg) [38] and are
reported in lanes 4 and 7, respectively. Equal amounts (30 lL) of
each sample were submitted to SDS ⁄ PAGE followed by immuno-
blot analysis (see Experimental procedures). The formed immuno-
complexes were revealed with the specific mAbs against each of
the proteins added to the samples containing the immobilized
HSP90.
A
B
Fig. 5. Calpain digestion of NOS isozymes in the presence of
HSP90. (A) nNOS (1 lg) or (B) eNOS (1 lg) were mixed with 1 lg
of HSP90 and incubated in the presence of the indicated amounts
of human erythrocyte calpain in the conditions reported in the
Experimental procedures. At the end of the incubation, aliquots of
each sample (50 lL) were utilized to assay NOS activity, which is
expressed as a percentage of the activity detected in the absence
of calpain. Insets in (A) and (B) represent the immunoblotting car-
ried out on aliquots (30 lL) of the same incubations, to detect

nNOS and eNOS, respectively. The values reported are the arith-
metical mean ± SD of three separate experiments.
M. Averna et al. Degradation of NOS isozymes by calpain
FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6121
remained associated with HSP90, resulting in the
formation of ternary complexes (Fig. 6B). These
protein–protein interactions were highly specific, as
indicated by the finding that the addition of calpastatin
removed calpain from the ternary complex (Fig. 6B,
lanes 4 and 7). These results may be of physiological
relevance because they indicate that the formation of
the ternary complex is correlated with the level of cal-
pastatin present in the cytosol. By quantification anal-
ysis of the immunoblots, it has been established that
the amount of calpain retained by immobilized HSP90
is almost equimolar to that of NOS isozymes. Thus,
the ternary complexes may contain a copy of each
enzyme protein. Altogether, these results can fit into
Scheme 1, which summarizes the type of protein–
protein interaction that can occur and their intercon-
version.
We then tested whether calpain could still express
catalytic activity when inserted in these binary or
ternary complexes. It was found that calpain, once
associated with HSP90, was unable to digest the
chaperone protein (Fig. 7, upper panel), probably
because, following interaction, the susceptible peptide
bonds in HSP90 are no longer accessible to the
protease. This hypothesis was confirmed by the
observation that calpain in its HSP90-associated

form can still digest exogenous substrates, such as
human denatured globin (Table 1). However, the cat-
alytic properties of the associated calpain differ from
that of the native enzyme because its efficiency is
reduced by 50%, probably due to a lower Ca
2+
-
binding capacity. This explains why higher amounts
of calpain are required for digestion of HSP90
(Fig. 4D). The changes in catalytic properties of cal-
pain provide an explanation of the mechanism by
which, in the ternary complex form, both HSP90
and NOS isozymes are protected from calpain diges-
tion. This protection, however, was complete for
eNOS, whereas the nNOS native 160 kDa band was
still partially converted into the 130 kDa band
(Fig. 7). Degradation of the chaperone protein and
the NOS isozymes became detectable only when the
calpain concentration exceeded that of HSP90, a
condition in which free active calpain molecules are
now present (Fig. 7).
Taken together, these findings strongly support the
idea that the protective effect of HSP90 can represent
a novel mechanism allowing the production of NO
even in conditions in which isolated forms of NOS
could be rapidly degraded by calpain.
This protection is mediated by HSP90, on the basis
of a dual mechanism: binding to NOS, which favours
Fig. 7. Calpain digestion of isolated HSP90 ⁄ NOS ⁄ calpain complex-
esThe ternary complexes containing HSP90, NOS and calpain were

purified as described in the Experimental procedures and incubated
with 1 m
M CaCl
2
in the absence or in the presence of 2 lg of exo-
genous human erythrocyte calpain. Equal amounts (30 lL) of each
incubation were then submitted to SDS ⁄ PAGE electrophoresis fol-
lowed by immunoblot analysis (see Experimental procedures). The
immunoreactive material was revealed using the specific mAbs.
Scheme 1. Binary and ternary complexes generated by HSP90,
NOS and calpain. CLP, calpain; CST, calpastatin.
Table 1. Effect of HSP90 on the catalytic efficiency of human
erythrocyte l-calpain. Calpain was purified from human erythro-
cytes as reported previously [39]. HSP90 was isolated from Jurkat
or BAE-1 cells as described in the Experimental procedures. Calpain
activity was assayed using human denatured globin as a substrate,
as previously reported [39], in the absence or in the presence of
equimolar amounts of HSP90 chaperone protein. Inhibition is
expressed as a percentage of the total calpain activity. The activity
measured in the absence of HSP90 was taken as 100%. K
0.5
refers
to the [Ca
2+
] ions required by calpain to express 1 ⁄ 2 V
max
. The val-
ues reported are the arithmetical means of three different experi-
ments ± SD.
Addition

Calpain catalytic properties
V
max
(unitsÆmg
)1
) Inhibition (%) K
0.5
(lM)
None 1125 ± 60 0 20 ± 5
Jurkat HSP90 545 ± 35 52 45 ± 5
BAE-1 HSP90 560 ± 40 50 45 ± 5
Degradation of NOS isozymes by calpain M. Averna et al.
6122 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS
the acquirement of the active conformation and the
fully functional state of the synthases [14–17], and
recruitment of active calpain, which prevents the inac-
tivation of NOS.
Discussion
In the present study, we describe the digestion pattern
of NOS isozymes and HSP90 both in reconstructed
systems and in intact cells. Our data consistently indi-
cate that the degradation of NOS is a highly regulated
process under the control of different mechanisms
and factors [2,18,20,26,28,35,40]. Activation of NOS
requires an increase in [Ca
2+
]
I
, a condition promoting
also the activation of calpain. If we consider the sus-

ceptibility of the isolated NOS forms to calpain diges-
tion, NO production in cells and organisms should be
very limited both in extent and time. However, in stim-
ulated cells, activation of NOS isozymes is sustained
for too long a time period compared to the resistance
of these proteins to calpain proteolysis. Thus, the
digestion must be controlled and degradation should
occur only when NO becomes a possible toxic agent
[41–44].
The findings reported in the present study represent
an answer to this question. We have demonstrated
that, in addition to the well-known calpain modula-
tors, HSP90 is directly involved in this regulatory pro-
cess. This chaperone binds to calpain and, when
associated with the protease, becomes resistant to
digestion. In addition, the calpain present in the com-
plex maintains the ability to degrade exogenous sub-
strates, but with a reduced capacity that is also due to
a decreased ability to bind Ca
2+
ions. When nNOS or
eNOS associates to the binary HSP90–calpain com-
plex, they are also protected from digestion by the
endogenous calpain. Moreover, the amount of calpain
present in such a ternary complex is under the control
of calpastatin. If the active calpain species increases
over the binding capacity of HSP90, the complex and
obviously the isolated proteins are degraded by the
protease. On the basis of these observations, we can
explain not only the protective effect exerted by

HSP90 on the digestion of NOS isozymes, but also the
requirement of high levels of active calpain for the
chaperone proteolysis. The inhibitory effect of HSP90
could derive from structural constraints on the flexibil-
ity of the calpain molecule, which reduces its proteo-
lytic efficiency but still allows the removal of the
PDZ domain from nNOS. This proteolytic step does
not modify the overall catalytic efficiency of nNOS,
but it might produce a change in intracellular localiza-
tion of the synthase.
The physiological relevance of these findings
becomes particularly evident on the basis of the results
observed in both cell models utilized in the present
study (Fig. 1). Thus, when Jurkat and BAE-1 cells
were stimulated in identical conditions, the different
patterns of nNOS and eNOS digestion can be attrib-
uted to the different amounts of HSP90 expressed in
the two cell lines. Thus, the limited digestion of nNOS
observed in Jurkat cells is due to the high levels of
HSP90, which can trap part of the active calpain on
one side and protect nNOS on the other. In BAE-1
cells, this process is less efficient due to the low level
of HSP90, resulting in a high degree of eNOS diges-
tion. This new function of HSP90 in the control of
NO production might also be relevant in nervous and
vascular tissues in which this free radical plays a par-
ticularly important role in vessel relaxation.
Experimental procedures
Materials
Leupeptin, calpain inhibitor 1 [33,34], NADPH, Ca

2+
-iono-
phore A23187, calmodulin, FAD, FMN, tetrahydrobiopter-
in, l-arginine. l-[
14
C]arginine (25 nCi; specific activity
308 CiÆmol
)1
), Source 15Q Sephacryl S-300, phenyl sepha-
rose, Sephadex G-200 resins and protein G-Sepharose were
obtained from GE Healthcare (Milan, Italy). Dowex 50W8
Na
+
form resin was obtained from Bio-Rad (Milan, Italy).
Monoclonal antibodies against nNOS (catalogue number
611852), eNOS (catalogue number 610427) and HSP90 (cat-
alogue number 610419) were purchased from BD Trans-
duction Laboratories (Milan, Italy). Monoclonal b-actin
antibody (catalogue number A-5441) was obtained from
Sigma Aldrich (Milan, Italy). 4-(2-Aminoethyl)benzene-
sulfonylfluoride (AEBSF) and calpain inhibitor 3-(5-fluoro-
3-indoyl)-2-mercapto-(Z)-2-propenoic acid (PD151746)
[22,32] were obtained from Calbiochem (Mississauga, Can-
ada). Monoclonal anti-l-calpain serum (mAb 56.3) was
produced as indicated previously [45]. Human erythrocyte
calpain was purified as reported previously [39]. Rat brain
m-calpain was purified as described previously [46]. The
ECLÒ Detection System was obtained from GE Health-
care.
Cell culture

BAE-1 cells were purchased from cell bank Interlab Cell
Line Collection (accession no ICLCAL 00004) and main-
tained in continuous culture at 37 °C (5% CO
2
) with
DMEM (Sigma Aldrich) growth medium containing 10%
fetal bovine serum and 2 m ml-glutamine; Jurkat (T cell
leukaemia) cells were kindly provided by C. Mingari
(DIMES, University of Genoa, Italy) and maintained in
M. Averna et al. Degradation of NOS isozymes by calpain
FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6123
continuous culture at 37 °C (5% CO
2
) with RPMI 1640
(Sigma Aldrich) growth medium containing 10% foetal
bovine serum, 10 UÆmL
)1
penicillin (Sigma Aldrich),
100 lgÆmL
)1
streptomycin (Sigma Aldrich) and 4 mml-glu-
tamine (Sigma Aldrich).
Purification of nNOS, eNOS and HSP90 from
different sources
BAE-1 cells (10 · 10
6
) were collected, lysed by sonication
in three volumes of ice-cold 50 mm sodium borate buffer,
pH 7.5, containing 1 mm EDTA, 0.5 mm 2-mercaptoetha-
nol, 0.1 mgÆmL

)1
leupeptin and 2 mm AEBSF. The particu-
late material was discarded by centrifugation (100 000 g for
10 min) and the soluble fraction (cell extract) was collected
and 1 mg protein was loaded onto a ion-exchange Source
15Q column (1.5 · 3 cm) previously equilibrated in 50 mm
sodium borate buffer, pH 7.5, containing 0.1 mm EDTA
and 0.5 mm 2-mercaptoethanol (buffer A). The protein con-
centration was determined with the Bradford method [47],
using purified BSA as standard. The adsorbed proteins
were eluted with a linear gradient (20 mL) 0–0.6 m NaCl
and collected in 1 mL fractions. Aliquots of each eluted
fraction (30 lL) were resuspended in Laemmli loading
buffer [48] and submitted to 7% SDS ⁄ polyacrylamide gel
electrophoresis followed by immunoblotting performed as
described in the section ‘Immunoprecipitation and immuno-
blotting’. The immunoreactive material was detected using
the specific mAbs directed against HSP90 and eNOS. The
eluted fractions containing the two proteins were separately
collected, concentrated by ultrafiltration and loaded onto
Phenyl Sepharose column (1.5 · 3 cm) equilibrated in
buffer A containing 0.3 m NaCl. HSP90 and eNOS were
retained by the resin whereas unabsorbed proteins were
washed out. The adsorbed proteins were eluted with
buffer A, collected, concentrated and loaded onto Sephadex
G-200 column (1.8 · 160 cm) equilibrated in the same
buffer A containing 0.15 m NaCl. Proteins were collected in
1 mL fractions and 30 lL of each fraction were utilized to
detect HSP90 and eNOS by immunoblot analysis. HSP90
was now separated from eNOS and the two proteins were

separately collected and concentrated by ultrafiltration. The
same procedure was applied to obtain HSP90 and nNOS
from Jurkat cells (60 · 10
6
).
Alternatively, aliquots of eluted fractions (100 lL) from
the ion exchange Source 15Q chromatography previously
described were utilized to assay NOS isozymes activity as
described above.
Assay of NOS activity
NOS activity was measured by detecting the production
of citrulline from l-[
14
C]arginine as previously reported
[26] with the following modifications: aliquots of Jurkat
and BAE-1 cell extracts (100 lg) or of the fractions
(100 lL) eluted from the ion exchange chromatography
described above, were incubated in buffer A (250 lL)
containing 1 mm NADPH, 200 mm calmodulin, 20 lm
tetrahydrobiopterin, 1 lm FAD, 1 lm FMN and 5 lm
l-arginine and 25 nCi of l-[
14
C]arginine (specific radio
activity 308 CiÆmol
)1
)at37°C. After 30 min, the reaction
was stopped with ice-cold 50 mm Hepes, pH 5.5, contain-
ing 5 mm EDTA (2 mL). The samples were then submit-
ted to anion exchange chromatography using 2 mL of
packed Dowex 50W8 Na

+
form resin pre-equilibrated
with stop buffer. l-citrulline was eluted by washing the
resin with 3 mL of stop buffer and the radioactivity
present was counted in a liquid scintillation counter.
One unit of NOS activity is defined as the amount of
enzyme producing 1 pmol citrullineÆmin
)1
in the specified
conditions.
Immunoprecipitation and immunoblotting
Jurkat (50 · 10
6
) or BAE-1 cells (5 · 10
6
) were lysed in
ice-cold 20 mm Tris ⁄ HCl, pH 7.4, containing 2.5 mm
EDTA, 2.5 mm EGTA, 0.14 m NaCl, 1% Triton X-100,
10 lgÆmL
)1
aprotinin, 20 lgÆmL
)1
leupeptin, 10 lgÆmL
)1
AEBSF and 10 lgÆmL
)1
phosphatases inhibitor cocktail I
and II, followed by brief sonication. Cell lysates were cen-
trifuged (12 000 g for 15 min at 4 °C) and protein quantifi-
cation of the supernatants was performed using the Lowry

assay. The immunoprecipitation was performed as previ-
ously described using 500 lg of detergent-soluble protein
(cell extract) and 3 lg of monoclonal anti-HSP90 serum
[8,11,35].
Alternatively, nNOS (1 lg) isolated from Jurkat cells
or eNOS (1 lg) isolated from BAE-1 cells, as previously
described, were incubated with HSP90 (1 lg) isolated from
the corresponding cell types. The mixtures were immobi-
lized to Protein G-Sepharose resin using monoclonal anti-
HSP90 serum (1 lg) in 300 lL (final volume) of 50 mm
sodium borate buffer, pH 7.5, containing 1 mm EDTA
(buffer B), following a previously reported procedure [38].
After incubation, the different samples were centrifuged
and the pellet was resuspended in 30 lL SDS ⁄ PAGE load-
ing buffer [48], heated for 5 min and submitted to 7% poly-
acrylamide gel electrophoresis in the presence of SDS.
Proteins were blotted to a nitrocellulose membrane (Bio-
Rad) and probed with specific mAbs, followed by a peroxi-
dase-conjugated secondary antibody as described [49]. The
immunoreactive bands were developed with an ECL detec-
tion system, detected with a Bio-Rad Chemi Doc XRS
apparatus and quantified using the Quantity One software,
release 4.6.1 (Bio-Rad). To quantify proteins from bands
revealed by western blotting, known amounts of protein
were submitted to SDS ⁄ PAGE and stained with the appro-
priate antibody. The bands were then scanned and the area
of the peaks obtained was used to create a calibration
curve.
Degradation of NOS isozymes by calpain M. Averna et al.
6124 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS

Equilibrium distribution experiments
in sephacryl S-300
Equilibrium gel distribution (gel penetration) experiments
with samples containing different mixtures of NOS and
HSP90 were carried out as previously described [38].
Briefly, nNOS (0.5 lg) or eNOS (0.5 lg) isolated from Jur-
kat cells or BAE-1 cells, respectively, were diluted alone or
with increasing amounts (0–1 lg) of HSP90 isolated from
the corresponding cell types in buffer B (0.5 mL). The solu-
tions were added to 0.5 mL of packed Sephacryl S-300 pre-
viously equilibrated with buffer B and rotated end-over-end
for 2 h at 4 °C. The resin was packed for 15–20 min at
4 °C and NOS activity was assayed as previously described
in this section using aliquots (0.2 mL) of the clear aqueous
phase.
Isolation of the NOS
⁄
HSP90
⁄
calpain complexes
Isolated HSP90 (5 lg) from Jurkat or BAE-1 cells was
incubated with monoclonal anti-HSP90 serum at 4 °C for
2 h, in buffer B (300 l L final volume). Protein G-Sepharose
(30 lL) was then added to the samples and the mixtures
were rotated end-over-end for 2 h at 4 °C. Sepharose beads
were collected, washed three times with buffer B (500 lL)
to discard proteins not specifically bound. Human erythro-
cyte calpain (0.5 lg in 300 lL of buffer B) was added to
the pellet and incubated for 2 h at 4 °C. Sepharose immu-
noprecipitated material was collected, washed three times

with buffer B (500 lL) and exposed to nNOS (1 lg) or
eNOS (1 lg) isolated from Jurkat or BAE-1 cells, respec-
tively. Alternatively, the EDTA present in buffer B was
replaced with 1 mm CaCl
2
(final concentration). In these
conditions, human erythrocyte calpain was maintained in
its inactive state by the addition in these mixtures of
0.1 mgÆmL
)1
(final concentration) leupeptin.
In vitro digestion of NOS isozymes and HSP90
with human erythrocyte calpain
HSP90, nNOS, eNOS (1 lg each) isolated from Jurkat or
BAE-1 cells as reported above, were incubated (100 lL
final volume) with human erythrocyte calpain [39] in buf-
fer B for 1 h at 37 °C, in the presence of 1 mm CaCl
2
.
Digestion of the ternary complex
HSP90
⁄
NOS
⁄
calpain
nNOS, eNOS, HSP90 and calpain, coimmunoprecipitated
as previously described, were incubated (30 lL final vol-
ume) in 50 mm sodium borate buffer, pH 7.5, containing
1mm CaCl
2

for 1 h at 37 °C in the absence or in the pres-
ence of exogenous purified calpain (0.1 lg). Reactions were
stopped with 0.1 m EDTA (2 lL). The samples were sub-
mitted to 7% SDS ⁄ PAGE and, following blotting, the
nitrocellulose membrane was probed with monoclonal anti-
HSP90, nNOS and eNOS sera.
Assay of calpain activity
Calpain activity was assayed as previously described [39].
One unit was defined as the amount of enzyme causing the
release from the substrate of 1 nmol of free NH
2
groups.
The specific activity of human erythrocyte calpain and of
rat brain m-calpain was 1075 unitsÆmg
)1
and 655 unitsÆ
mg
)1
, respectively.
Acknowledgements
This work was supported in part by grants from
MIUR, FIRB and PRIN projects, and from the Uni-
versity of Genoa.
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