Báo cáo khoa học: In vivo degradation of nitric oxide synthase (NOS) and heat shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS–HSP90 heterocomplex pot

A Aliquots 50 lg protein of NMS rat brain soluble material, obtained as described in Experimental procedures, were submitted to 6% SDS-PAGE and blotted as described previously.. B Aliquo

shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS–HSP90 heterocomplex

Monica Averna, Roberto Stifanese, Roberta De Tullio, Franca Salamino, Sandro Pontremoli

and Edon Melloni

Department of Experimental Medicine (DIMES)-Biochemistry Section, and Centre of Excellence for Biomedical Research (CEBR),

University of Genoa, Italy

The interaction of nitric oxide synthase (NOS) with a

variety of proteins plays an important role in the

regu-lation of NO production [1–4] Of these interacting

proteins, heat shock protein 90 (HSP90) has been

pro-posed to exert a relevant role for both NOS function

and stability [1,5–7] Thus, HSP90 may serve as an

allosteric positive modulator of NOS isozymes by

inducing the acquisition of the active conformation or

by enhancing the affinity of NOS for the Ca2+sensor

calmodulin [8] It has also been proposed that the

association of NOS with HSP90 favours the correct

insertion of the haem group into apo-NOS and the formation of stable NOS dimers [9,10] As the haem-deficient monomeric NOS form following treatment with HSP90 inhibitors is rapidly polyubiquitinated and degraded by the proteasome pathway, HSP90 has been considered to be indirectly involved in the selective proteolytic degradation of NOS [11–16] In addition to proteasome degradation, several reports have indicated that, in extreme cytotoxic conditions, calpain becomes uncontrollably activated, producing extensive degrada-tion of NOS and HSP90 [17–26]

Keywords

Ca2+homeostasis; calpain; calpastatin; heat

shock protein 90; nitric oxide synthase

Correspondence

S Pontremoli, Department of Experimental

Medicine (DIMES)-Biochemistry Section,

University of Genoa, Viale Benedetto XV,

1-16132 Genoa, Italy

Fax: +39 010 518343

Tel: +39 010 3538128

E-mail: pontremoli@unige.it

(Received 8 October 2007, revised

19 February 2008, accepted 11 March 2008)

doi:10.1111/j.1742-4658.2008.06394.x

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs In addition, the extent of NOS degradation is a func-tion of the level of HSP90 expression Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecu-lar-mass (130 kDa) products takes place In contrast, in aorta, which con-tains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity On the basis of these findings, we pro-pose a novel role of the HSP90–NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain The efficiency of this effect

is directly related to the level of intracellular HSP90 expression, generating

a high HSP90 to NOS ratio, which favours both the formation and stabil-ization of the HSP90–NOS heterocomplex This condition seems to occur

in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS

Abbreviations

[Ca 2+ ]i, intracellular Ca 2+ concentration; C.I.1, synthetic calpain inhibitor-1; eNOS, endothelial nitric oxide synthase; HMS, hypertensive Milan strain; HSD, high-sodium diet; HSP90, heat shock protein 90; iNOS, inducible NOS; NMS, normotensive Milan strain; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase.

We have recently demonstrated that the

suscep-tibility to calpain degradation of purified endothelial

NOS (eNOS) and neuronal NOS (nNOS) is

signifi-cantly reduced in the presence of equimolar amounts

of HSP90 [27] Using immunoprecipitation studies, it

has also been established that the protective effect is

caused by HSP90-specific recruitment by active calpain

molecules In this associated form, HSP90 becomes

resistant to digestion, although the protease still retains

50% of its proteolytic activity against external

sub-strates Furthermore, when NOS isozymes are

associ-ated with this binary complex, they also become

resistant to proteolytic degradation These

observa-tions imply a correlation between the vulnerability of

NOS isozymes and the availability of HSP90 to

gener-ate stable ternary complexes This relationship is

strongly supported by the different digestibility of

NOS in Jurkat and BAE-1 cells, expressing high and

low levels of HSP90, respectively

To verify the occurrence of such a protective effect

in vivo, we used normotensive Milan strain (NMS)

rats as a model Thus, we induced a mild elevation of

intracellular Ca2+ concentration ([Ca2+]i) by the

administration of a high-sodium diet (HSD) [28], and

studied calpain degradation of NOS and HSP90 in

brain and aorta To amplify the range of fluctuations

in [Ca2+]i, hypertensive Milan strain (HMS) rats were

also used, as they are characterized by a constitutive

elevation in [Ca2+]i and a higher responsiveness to

HSD

We report here that, in the brain and aorta of

HSD-treated rats, the extent and patterns of proteolytic

deg-radation of NOS isozymes and HSP90 are similar to

those previously detected in Jurkat and BAE-1 cells

loaded with Ca2+[27] As the differences in expression

of HSP90 in the two rat tissues are similar to those

present in these cell models [27], we propose that the

occurrence of conditions which favour the formation

and stabilization of proteolytically resistant complexes

of NOS with HSP90 are crucial in determining the

in vivoresistance of NOS and HSP90 to calpain

degra-dation

Results

Levels of HSP90 and NOS isozymes in rat brain

and aorta

The level of HSP90 and the type of NOS isoform

pres-ent in rat brain and aorta were determined by

immu-noblotting (Fig 1) In brain, nNOS was the most

preferentially expressed isoform, together with traces

of eNOS (Fig 1A) In aorta, only eNOS isozyme was

detectable (Fig 1B) In both tissues, no expression of inducible NOS (iNOS) was found (Fig 1A,B) HSP90 was present in rat brain in amounts six- to sevenfold

A

B

C

Fig 1 NOS isozymes and HSP90 expressed in brain and aorta of NMS rats (A) Aliquots (50 lg protein) of NMS rat brain soluble material, obtained as described in Experimental procedures, were submitted to 6% SDS-PAGE and blotted as described previously NOS isozymes were detected with the specific mAbs (B) Aliquots (50 lg protein) of NMS rat thoracic aorta total lysate, obtained as described in Experimental procedures, were submitted to 6% SDS-PAGE and blotted as described previously NOS isozymes were detected with specific mAbs (C) HSP90 levels were detected from the same samples as reported in (A) and (B) using the specific mAb The immunoreactive bands detected in (A–C) were quantified (see bars) as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five dif-ferent experiments carried out on five difdif-ferent animals of each strain.

higher than in aorta, resulting in a much higher

HSP90 to NOS ratio in brain (Fig 1C)

Calpain activation in rat brain and aorta

following HSD treatment

To promote in vivo calpain activation, NMS rats were

treated with HSD, which has been established

previ-ously to induce a mild elevation in [Ca2+]i, slightly

higher in aorta than in brain [28] To amplify the

range of elevation in [Ca2+]i, HMS rats were also

used, as a limited increase in [Ca2+]iin both aorta and

brain has been found to be constitutively present in

these animals

To assess the in vivo activation of calpain, we relied

on the following well-established methods: (a) the

occurrence of calpain consumption [26,29–31]; (b) a

specific pattern of calpastatin digestion, resulting in an

imbalance within the proteolytic system [32]; and (c)

the degradation of calpain target proteins [26,30] As

shown in Fig 2A, following HSD treatment, the levels

of both l- and milli-calpain isoforms were reduced to

a limited extent in brain, whereas, in aorta (Fig 2B),

the decrease in the two protease isoforms was more

pronounced

Moreover, in brain, the natural inhibitor of calpain,

calpastatin, was preferentially converted into still

active 15 kDa fragments (Table 1), whereas, in aorta,

the inhibitor was predominantly inactivated As both

the inactivation and fragmentation of calpastatin are

known to be produced by active calpain [32], these

observations further indicate that calpain is activated

in both tissues, although at a higher rate in aorta

Fur-ther direct evidence in support of calpain activation in

aorta was provided by the degradation of talin and

desmin in HSD-treated rats (Fig 3A) Indeed, this

process was completely prevented (Fig 3B) by the

administration to the animals of synthetic calpain

inhibitor-1 (C.I.1) [33,34]

Digestion of HSP90 and NOS in brain and aorta

of normotensive and hypertensive rats treated

with HSD

Following HSD treatment, no appreciable changes in

NOS activity occurred in the brain of NMS rats,

although a small fraction of the native 160 kDa

syn-thase was converted into the still active 130 kDa form

(Fig 4A) The level of HSP90 remained unchanged

during the period of treatment (Fig 4A) By contrast,

in aorta, more than 50% of native eNOS progressively

disappeared (Fig 4B,D), together with a significant

degradation of HSP90, which was only partially

replaced by an 84 kDa form (Fig 4B,D) The involve-ment of calpain in these digestion processes was dem-onstrated by the protective effect on both HSP90 and NOS degradation of the administration to the HSD-treated NMS animals of C.I.1 (Fig 5)

In the brain of hypertensive rats, in spite of a pre-existing condition of altered Ca2+ homeostasis, no appreciable changes in total nNOS activity or the level

of HSP90 were observed (Fig 6A,C) By contrast with NMS rats, a small fraction of a still active 130 kDa form was already present in the brain of untreated HMS rats and increased following HSD treatment (Fig 6A) However, in aorta, the digestion of eNOS and HSP90 appeared to be more extensive (Fig 6B) Indeed, approximately 80–90% of eNOS protein and

A

B

Fig 2 Levels of calpain isoforms and calpain substrates in the aorta

of NMS and HMS rats treated with HSD Aliquots (100 lg protein) of brain soluble material (A) and aorta total lysate (B), prepared as described in Experimental procedures, from untreated or 4-week HSD-treated NMS and HMS rats, were submitted to 8% SDS-PAGE, followed by immunoblotting revealed with serum-l-calpain mAb 56.3 [36] and monoclonal IgG milli-calpain The immunoreactive material was detected and quantified as described in Experimental proce-dures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain.

activity, together with 60–70% of HSP90, were lost

(Fig 6B,D)

The degradation pattern of nNOS in the brain of

HSD-treated rats, resulting in the accumulation of the

still active 130 kDa form, can be reproduced in in vitro

conditions if nNOS digestion by calpain is carried out

in the presence of HSP90 [27] This finding can also

explain the large extent of digestion of eNOS in aorta,

in which, in association with a higher degree of calpain

activation, a lower level of HSP90 is also present

Identification of HSP90–NOS heterocomplexes in

aorta and brain lysates

In order to explore the relationship between the

HSP90 to NOS ratio and the formation of

calpain-resistant heterocomplexes, we first studied, by

immu-noprecipitation analysis, the association of the two

proteins in brain and aorta As shown in Fig 7A,

fol-lowing the addition of IgG1-HSP90 antibody to crude

extracts of brain or aorta, NOS was

immunoprecipitat-ed, indicating a specific association of the two proteins

We then determined the amount of each enzyme

involved in such complexes by submitting samples of crude extracts of rat brain and aorta to gel filtration chromatography As shown in Fig 7B, in brain, nNOS eluted entirely as a single peak at a volume corre-sponding to a molecular mass higher than that of the free native enzyme HSP90 was eluted in two peaks: the first coincident with that of nNOS, and the second containing more than 60% of total chaperone protein, with an elution volume identical to that of free HSP90 Thus, all nNOS appeared to be engaged in a complex with HSP90, whereas the major fraction of the chaper-one was present in the free form

In aorta (Fig 7C), approximately 85–90% of eNOS was recovered in association with HSP90 and the remaining 10–15% was found in the free form; how-ever, the amount of HSP90 recovered as free protein was much lower than that engaged in the complex The large difference in the amount of free chaperone observed in the two tissues is indicative of the existence

Table 1 Levels of native and 15 kDa calpastatin species in brain

and aorta of NMS and HMS rats treated with HSD for 4 weeks.

The data reported are the arithmetical means ± standard deviation

of five different experiments carried out on five different animals of

each strain.

Animal Treatment a

Total calpastatin activity (%) b

15 kDa fragment activity (%) c

Loss of total calpastatin activity (%) d

Brain

Aorta

a

NMS and HMS rats were fed for 4 weeks with HSD as described

in Experimental procedures. bTotal calpastatin activity was

mea-sured as described in Experimental procedures and [28,43] c

Aliqu-ots (1.5 mg protein) of the soluble material, obtained as described

previously from untreated or 4-week HSD-treated NMS and HMS

rat brain and thoracic aorta homogenates, were submitted to 12%

SDS-PAGE divided into 10 lanes [28] The 15 kDa calpastatin

spe-cies were identified on the basis of their electrophoretic mobility,

and quantified following extraction from the gel by measuring their

inhibitory activity as described previously [28,42] d The loss of

calpastatin activity was calculated by subtracting the sum of the

percentage of the active calpastatin species from 100.

A

B

Desmin

Desmin Talin

Talin

+HSD

HMS +HSD

+ HSD + C.I 1 + HSD

Control

Fig 3 Levels of calpain substrates in aorta of NMS and HMS rats treated with HSD and C.I.1 Aliquots (50 lg protein) of aorta total lysate (A), prepared as described in Experimental procedures, from untreated and HMS rats, were submitted to 8% SDS-PAGE fol-lowed by immunoblotting Samples (50 lg protein) of aorta total lysate (B) from untreated or 4-week HSD-treated NMS rats, in the absence (+HSD) or presence (+HSD+C.I.1) of 25 l M C.I.1, were submitted to 8% SDS-PAGE followed by immunoblotting Desmin and talin were detected with specific mAbs.

of conditions favouring and stabilizing the

heterocom-plex much more efficiently in brain than in aorta This

could explain the higher susceptibility of eNOS to

calpain digestion

Discussion

Although several reports [11–26] have indicated that

calpain and the proteasome pathway are the two

major systems responsible for the proteolytic

degrada-tion of NOS, some pertinent quesdegrada-tions still remain

unsolved Indeed, although it has been established,

especially by the use of NOS and HSP90 inhibitors,

that proteasome-promoted degradation selectively

removes inactive structurally damaged NOS forms, or

monomeric haem-deficient isozyme species [11–16],

the precise molecular events that trigger the

proteo-lytic degradation of NOS in vivo still remain to be

defined One of these molecular signals could be

altered or decreased HSP90 function, favouring the

accumulation of abnormal or monomeric NOS

mole-cules and their degradation by the proteasome system

[1,12] Furthermore, proteolytic degradation of NOS

by calpain has been described in conditions of

extreme cytotoxicity [17,19,21,23,26] In these

experi-ments, as a result of high Ca2+ overload, several

calpain targets, including NOS, can undergo proteo-lytic digestion For this reason, the degradation of NOS can be attributed to an overactivation of cal-pain rather than to a selective regulated proteolytic mechanism

In previous studies, we have observed that HSP90 is five- to tenfold less susceptible than nNOS and eNOS

to calpain degradation [27] as a result of the formation

of a calpain–HSP90 complex in which the protease can

no longer degrade the bound chaperone NOS iso-zymes, once recruited into the HSP90–calpain binary complex, also become resistant to calpain digestion This protective effect may be of physiological rele-vance, as conditions promoting NO production also induce calpain activation Thus, the formation of NOS–HSP90 complexes may provide a new insight into the understanding of the mechanisms involved in modulating NO production In such a case, the avail-ability of adequate amounts of HSP90 becomes the limiting factor

Our study poses new important questions that need

to be addressed The first question concerns the vul-nerability of different NOS isoforms to proteolysis

in vivo under conditions of small changes to [Ca2+]i The second question concerns the capacity of HSP90

to protect NOS in vivo against proteolytic

Fig 4 In vivo digestion of NOS and HSP90 in NMS rats during HSD treatment Aliquots (20 lg protein) of rat brain soluble material (A) and aliquots (50 lg protein) of rat aorta total lysate (B), obtained as described in Experimental procedures, from untreated or HSD-treated NMS rats, were submitted to 6% SDS-PAGE and blotted as described previously nNOS, eNOS and HSP90 were detected with specific mAbs (C) nNOS (open circles) and HSP90 (filled circles) immunoreactive materials detected in (A) and the corresponding nNOS activity (open squares) were quantified as described in Experimental procedures (D) eNOS (open circles) and HSP90 (filled circles) immunoreactive materi-als detected in (B) and the corresponding eNOS activity (open squares) were quantified as described in Experimental procedures The values reported are the arithmetical means ± standard deviation of five different experiments carried out on five different animals of each strain.

tion Finally, a third question involves the

possi-ble relationship between such protection and the

well-known different expression of HSP90 in various

tissues

To answer these questions, we have used animals

treated with HSD, which has been shown previously

to induce an increase in the level of [Ca2+]i and a

correlated calpain activation [28] This increase in

[Ca2+]i is more intense in aorta than in brain

Under these conditions, in brain, no change in the

level of HSP90 was observed, although a limited and

conservative degradation of nNOS occurred without

a loss of catalytic activity In contrast, in aorta, both eNOS and HSP90 were highly degraded The differ-ent vulnerability of the two NOS isoforms to proteo-lytic degradation is strictly related to the availability

of HSP90, which is expressed in higher concentra-tions in the brain than in the aorta Furthermore, the patterns of digestion of eNOS and nNOS observed in HSD-treated animals are identical to those previously obtained in reconstructed systems containing the synthases together with different levels

of HSP90

Our data suggest that a large reservoir of HSP90 maintains all NOS engaged in a calpain-resistant het-erocomplex, which is protected from proteolysis, even under conditions of prolonged protease activation This conclusion is further supported by the finding reported here that, in brain, the nNOS–HSP90 com-plex is in equilibrium with a large amount of stabiliz-ing free chaperone, a condition that does not occur in aorta The reduced availability of HSP90 in aorta can thus explain the increased vulnerability of eNOS rela-tive to nNOS to proteolysis On the basis of these find-ings, we propose a novel mechanism in which HSP90 can provide functional stability of NOS isozymes under conditions characterized by an alteration in intracellular Ca2+homeostasis

Experimental procedures Materials

Leupeptin C.I.1, aprotinin, phosphatase inhibitor cocktail I and II, NADPH, calmodulin, FAD, FMN, tetrahydrobiop-terin, l-arginine and aldolase were purchased from Sigma Aldrich, Milan, Italy l-[14C]arginine (925 Bq; specific activ-ity, 1Æ14 · 1011

BqÆmol)1), Sephacryl S-300, Sephadex

G-200 resins, Superose 12 10⁄ 300 GL column and protein G-Sepharose were obtained from GE Healthcare, Milan, Italy Ferritin was purchased from Boehringer Mannheim, Mannheim, Germany Dowex 50W8 resin (Na+form) was obtained from Bio-Rad Laboratories, Milan, Italy 4-(2-Aminoethyl)benzenesulfonylfluoride (AEBSF) was obtained from Calbiochem (Missiagua, Canada) The ECL Detec-tion System was obtained from GE Healthcare

Monoclonal antibodies (mAbs) nNOS, eNOS, iNOS and HSP90 antibodies were purchased from BD Transduction Laboratories, Milan, Italy b-Actin and milli-calpain antibodies were obtained from Sigma Aldrich, Milan, Italy Desmin and talin antibodies were purchased from Novus Biologicals, Littleton, CO, USA IgG1-calpastatin (mAb 35.23) and serum l-calpain (mAb

A

B

Fig 5 Levels of NOS isozymes and HSP90 in brain and aorta of

NMS rats treated with HSD and C.I.1 Aliquots of brain soluble

material (20 lg protein) and of aorta total lysate (50 lg protein),

obtained as described in Experimental procedures, from untreated

or 4-week HSD-treated NMS rats, in the absence (+HSD) or

pres-ence (+HSD+C.I.1) of 25 l M C.I.1, were submitted to 6%

SDS-PAGE followed by immunoblotting, revealed with IgG1-eNOS or

IgG1-nNOS mAbs (A) or IgG1-HSP90 mAb (B) The immunoreactive

material of eNOS, nNOS and HSP90 was detected and quantified

as described in Experimental procedures The values reported are

the arithmetical means ± standard deviation of five different

experi-ments carried out on five different animals of each strain.

56.3) mAbs were produced as indicated in [35] and [36],

respectively

Animals

NMS and HMS rats [37] were housed in controlled

condi-tions (22 ± 1C; humidity, 50 ± 5%; lighting, 8–20 h)

Systolic blood pressure was measured by tail-cuff

plethys-mography [W&W Electronic, BP recorder 8005 (Huntsinlle,

AL, USA)] on prewarmed (37C) rats, following the

procedure originally described by Byrom and Wilson [38]

Normotensive and hypertensive rats showed mean arterial

blood pressures of 100 ± 5 and 145 ± 10 mmHg,

respec-tively

Experimental hypertension

Experimental hypertension was induced in 60-day-old rats

by feeding ad libitum with a standard rat chow and

provid-ing NaCl dissolved in tap water at a concentration of

10 gÆL)1 for a period of time ranging from 15 to 30 days

Each animal received approximately 0.7 gÆday)1 of NaCl

Where indicated, 25 lm C.I.1 was dissolved in tap water in

the presence of 10 gÆL)1 NaCl, and administered to NMS

and HMS rats for 4 weeks [28] Each rat received 0.5–

0.7 mgÆday)1of C.I.1 Experiments were carried out

follow-ing the institution’s ethical guidelines Durfollow-ing the course of the experiments, no appreciable changes were observed in food consumption and body weight

Preparation of tissue homogenates NMS and HMS rats were sacrificed by decapitation; the brain was immediately removed, minced, homogenized in a Potter–Elvehjem homogenizer and sonicated in three vol-umes of 50 mm sodium borate buffer, pH 7.5, containing

1 mm EDTA, 0.5 mm 2-mercaptoethanol, 0.1 mgÆmL)1 leupeptin and 2 mm AEBSF (buffer A) The particulate material was discarded by centrifugation (100 000 g for

10 min) Thoracic aorta was rapidly excised from the same animals After the removal of the adhering connective tissue, the tissue was cut into several segments (approxi-mately 2 mm each), homogenized in a Potter–Elvehjem homogenizer and lysed by sonication in three volumes of buffer A The protein concentration was determined follow-ing the procedure of Bradford [39]

Immunoblot Rat brain and aorta lysates (20–50 lg) were diluted in a final volume of 100 lL of the SDS-PAGE loading buffer and submitted to 6% SDS-PAGE [40] The protein bands

Fig 6 In vivo digestion of NOS and HSP90 in HMS rats during HSD treatment Aliquots (20 lg protein) of rat brain soluble material (A) and aliquots (50 lg protein) of rat aorta total lysate (B), obtained as described in Experimental procedures, from untreated or HSD-treated HMS rats, were submitted to 6% SDS-PAGE and blotted as described previously nNOS, eNOS and HSP90 were detected with specific mAbs (C) nNOS (open circles) and HSP90 (filled circles) protein detected in (A) and the corresponding nNOS activity (open squares) were quantified

as described in Experimental procedures (D) eNOS (open circles) and HSP90 (filled circles) immunoreactive materials detected in (B) and the corresponding eNOS activity (open squares) were quantified as described in Experimental procedures The values reported are the arith-metical means ± standard deviation of five different experiments carried out on five different animals of each strain.

were then blotted onto a nitrocellulose membrane and

saturated with a NaCi/Pisolution, pH 7.5, containing 5%

powered milk The blots were probed with specific

antibod-ies, followed by a peroxidase-conjugated secondary

anti-body as described previously, and then developed with the

ECL Detection System [41] The immunoreactive material

was detected with a Bio-Rad Chemi Doc XRS apparatus

and quantified using quantity one 4.6.1 software

(Bio-Rad Laboratories) The procedure was made quantitative

by the use of known amounts of proteins submitted to

SDS-PAGE and staining with the appropriate antibody

The bands were then scanned, and the areas of the peaks

obtained were used to create a calibration curve

Immunoprecipitation

Brain and thoracic aorta, excised from NMS rats, were

lysed in ice-cold 20 mm Tris⁄ HCl, 2.5 mm EDTA, 2.5 mm

EGTA, 0.14 m NaCl, pH 7.4 (immunoprecipitation buffer), containing 1% Triton X-100, 10 lgÆmL)1 aprotinin,

20 lgÆmL)1leupeptin, 10 lgÆmL)1AEBSF and phosphatase inhibitor cocktail I and II (10 lgÆmL)1), followed by brief sonication Cell lysates were centrifuged at 12 000 g for

15 min at 4C, and protein quantification of the superna-tants was performed using the Lowry assay For the immu-noprecipitations,  500 lg of detergent-soluble protein (crude extract) was previously precleared with protein G-Sepharose, and then incubated in the presence of 2 lg of IgG1-HSP90 mAb at 4C overnight Protein G-Sepharose was then added and incubated for an additional hour The immunocomplexes were washed three times with immuno-precipitation buffer, heated in SDS-PAGE loading buffer for 5 min [40] and submitted to 6% SDS-PAGE Proteins were then transferred by electroblotting onto a nitro-cellulose membrane, and immunoblotting analysis was performed as described above

Identification of NOS–HSP90 association by gel filtration

Aliquots (0.5 mg protein) of the soluble material of brain homogenate and thoracic aorta total lysate, obtained from NMS rats as described previously, were submitted to gel filtration chromatography on a Superose 12 10⁄ 300 GL column (total volume, 24 mL) equilibrated in buffer A con-taining 50 mm NaCl using an FPLC system The flow rate was 100 lLÆmin)1and the eluted proteins were collected in

500 lL fractions The molecular weights of the eluted pro-teins were calculated from the elution volumes of ferritin (Mr= 450 kDa) and aldolase (Mr= 160 kDa), utilized as standard proteins

A

B

C

Fig 7 Identification of NOS–HSP90 association in rat brain and aorta (A) Aliquots (500 lg protein) of brain and aorta crude extract, prepared as described in Experimental procedures, were incubated overnight at 4 C with IgG1-HSP90 antibody (see Experimental procedures), as reported also in [7,44,45] The mixtures were then incubated for 1 h at room temperature with 50 lL of protein G-Sepharose The particles were collected and washed three times with immunoprecipitation buffer The particles were then suspended

in SDS-PAGE loading solution, heated for 5 min at 90 C and submit-ted to 6% SDS-PAGE NOS isozymes and HSP90 were identified with specific mAbs (see Experimental procedures) The values reported are the arithmetical means ± standard deviation of five dif-ferent experiments carried out on five difdif-ferent animals of each strain (B, C) Aliquots (500 lg protein) of the soluble material of brain homogenate and thoracic aorta total lysate, obtained from NMS rats

as described previously, were submitted to gel filtration chromatog-raphy (see Experimental procedures) Aliquots (30 lL) of each eluted fraction were suspended in SDS-PAGE loading solution [40] and sub-mitted to 6% SDS-PAGE, followed by immunoblotting HSP90 (filled circles) and NOS isoforms (open circles) were probed with the appro-priate antibody The immunoreactive material was quantified as described in Experimental procedures.

Aliquots (30 lL) of each eluted fraction were suspended

in SDS-PAGE loading buffer [40] and submitted to 6%

SDS-PAGE Proteins were then transferred to a

nitrocellu-lose membrane by electroblotting, and immunoblotting

analysis was performed as described above The

immunore-active material was detected and quantified as described

above

Assay of NOS activity

NOS activity was assayed by detecting the production of

citrulline from l-[14C]arginine, as reported previously [23]

with the following modifications Aliquots (100 lg protein)

of the crude homogenate were incubated in a total volume

of 250 lL in buffer A containing 1 mm NADPH, 200 mm

calmodulin, 20 lm tetrahydrobiopterin, 1 lm FAD, 1 lm

FMN, 5 lm l-arginine and 925 Bq of l-[14C]arginine

(spe-cific radioactivity, 1Æ14 · 1011 BqÆmol)1) at 37C After

30 min, 2 mL of ice-cold stop buffer (50 mm Hepes,

pH 5.5, containing 5 mm EDTA) was added These

incuba-tions were then submitted to anion exchange

chromatogra-phy 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

radio-activity present was counted in a liquid scintillation

coun-ter One unit of NOS activity was defined as the amount of

enzyme producing 1 pmol citrullineÆmin)1 in the specified

conditions

Separation and quantification of calpastatin

species in rat brain and aorta

Aliquots of the soluble material (10 lanes with 100 lg

pro-tein each), prepared as described above from untreated or

treated NMS and HMS rat brain and thoracic aorta

homo-genates, were submitted to 12% SDS-PAGE [28]

Calpasta-tin species were identified following protein extraction from

the gel, as described previously [42] Calpastatin activity

was measured as described in [43]

Acknowledgements

This work was supported in part by grants from

Min-istero Haliano per I’Universita` e la Ricerca, Fondo per

gli Investimenti della Ricerca di Base and Progetti di

Ricerca di Interesse Nazionale projects, and from the

University of Genoa

References

1 Kone BC, Kuncewicz T, Zhang W & Yu Z (2003)

Pro-tein interaction with nitric oxide synthases: controlling

the right time, the right place, and the right amount of

nitric oxide Am J Physiol Renal Physiol 285, 178–190

2 Kone BC (2000) Protein–protein interactions controlling nitric oxide synthases Acta Physiol Scand 168, 27–31

3 Gratton J, Fontana J, O’Connor D, Garcia-Cardena

G, McCabe T & Sessa C (2000) Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro J Biol Chem 275, 22268– 22272

4 Garcia-Cardena G, Martasek P, Masters BS, Skidd

PM, Conet J, Lisanti MP & Sessa WC (1997) Dissecting the interaction between nitric oxide synthase (NOS) and caveolin Functional significance of the NOS caveolin binding domain in vivo J Biol Chem 272, 25437– 25440

5 Piech A, Dessy C, Havaux X, Feron O & Balligand J (2003) Differential regulation of nitric oxide synthases and their allosteric regulators in heart and vessels of hypertensive rats Cardiovasc Res 57, 456–467

6 Bender AT, Silverstein AM, Demady DR, Kanelakis

KC, Noguchi S, Pratt WB & Osawa Y (1999) Neuro-nal nitric-oxide synthase is regulated by the HSP90-based chaperone system in vivo J Biol Chem 274, 1472–1478

7 Papapetropoulos A, Fulton D, Lin MI, Fontana J, McCabe TJ, Zoellner S, Garcia-Cardena G, Zhou Z, Gratton J & Sessa WC (2004) Vanadate is a potent acti-vator of endothelial nitric-oxide synthase: evidence for the role of the serine⁄ threonine kinase akt and the

90 kDa heat shock protein Mol Pharmacol 65, 407– 415

8 Song Y, Zweier JL & Xia Y (2001) Heat-shock protein augments neuronal nitric oxide synthase activity by enhancing Ca2+⁄ calmodulin binding Biochem J 355, 357–360

9 Minami Y, Kimura Y, Kawasaki H, Suzuki K &

Yaha-ra I (1994) The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function

in vivo Mol Cell Biol 14, 1459–1464

10 Billecke SS, Bender AT, Kanelakis KC, Murphy PJM, Lowe ER, Kamada Y, Pratt WB & Osawa Y (2002) HSP90 is required for heme binding and activation of apo-neuronal nitric-oxide synthase J Biol Chem 277, 20504–20509

11 Dunbar AY, Kamada Y, Jenkins GJ, Lowe ER, Bille-cke SS & Osawa Y (2004) Ubiquitination and degrada-tion of neuronal nitric-oxide synthase in vitro: dimer stabilization protects the enzyme from proteolysis Mol Pharmacol 66, 964–969

12 Osawa Y, Lowe ER, Everett AC, Dunbar AY & Bille-cke SS (2003) Proteolytic degradation of nitric oxide synthase: effect of inhibitors and role of HSP90-based chaperones J Pharmacol Exp Ther 304, 493–497

13 Govers R, de Bree P & Rabelink TJ (2003) Involvement

of the proteasome in activation of endothelial nitric oxide synthase Life Sci 73, 2225–2236

14 Kolodziejski PJ, Musial A, Koo JS & Eissa NT (2002)

Ubiquitination of inducible nitric oxide synthase is

required for its degradation Proc Natl Acad Sci USA

99, 12315–12320

15 Musial A & Eissa T (2001) Inducible nitric-oxide

synthase is regulated by the proteasome degradation

pathway J Biol Chem 276, 24268–24273

16 Bender A, Demady DR & Osawa Y (2000)

Ubiquitina-tion of neuronal nitric-oxide synthase in vitro and

in vivo J Biol Chem 275, 17407–17411

17 Gamerdinger M, Manthey D & Behl C (2006)

Oestro-gen receptor subtype-specific repression of calpain

expression and calpain enzymatic activity in neuronal

cells – implications for neuroprotection against

Ca-med-iated excitotoxicity J Neurochem 97, 57–68

18 Araujo IM & Carvalho CM (2005) Role of nitric oxide

and calpain activation in neuronal death and survival

Curr Drug Targets CNS Neurol Disord 4, 319–324

19 Stalker TJ, Gong Y & Scalia R (2005) The

calcium-dependent protease calpain causes endothelial

dysfunc-tion in type 2 diabetes Diabetes 54, 1132–1140

20 Stalker TJ, Skvarka CB & Scalia R (2003) A novel role

for calpains in the endothelial dysfunction of

hypergly-cemia FASEB J 17, 1511–1513

21 Araujo IM, Ambrosio AF, Leal EC, Santos PF,

Carv-alho AP & CarvCarv-alho CM (2003) Neuronal nitric oxide

synthase proteolysis limits the involvement of nitric

oxide in kainate-induced neurotoxicity in hippocampal

neurons J Neurochem 85, 791–800

22 Walker G, Pfeilschifter J, Otten U & Kunz D (2001)

Proteolytic cleavage of inducible nitric oxide synthase

(iNOS) by calpain I Biochim Biophys Acta 1568, 216–

224

23 Su Y & Block ER (2000) Role of calpain in hypoxic

inhibition of nitric oxide synthase activity in pulmonary

endothelial cells Am J Physiol Lung Cell Mol Physiol

278, 1204–1212

24 Bellocq A, Doublier S, Suberville S, Perez J, Escoubet

B, Fouqueray B, Puyol DR & Baud L (1999)

Somato-statin increases glucocorticoid binding and signalling in

macrophages by blocking the calpain-specific cleavage

of HSP90 J Biol Chem 274, 36891–36896

25 Laine´ R & Ortiz de Montellano PR (1998) Neuronal

nitric oxide synthase isoforms a and l are closely

related calpain sensitive proteins Mol Pharmacol 54,

305–312

26 Hajimohammadreza I, Raser KJ, Nath R, Nadimpalli

R, Scott M & Wang KKW (1997) Neuronal nitric oxide

synthase and calmodulin-dependent protein kinase IIa

undergo neurotoxin-induced proteolysis J Neurochem

69, 1006–1013

27 Averna M, Stifanese R, DeTullio R, Salamino F,

Bertuc-cio M, Pontremoli S & Melloni E (2007) Proteolytic

deg-radation of NOS isoforms by calpain is modulated by the

expression levels of HSP90 FEBS J 274, 6116–6127

28 Averna M, Stifanese R, DeTullio R, Passalacqua M, Defranchi E, Salamino F, Melloni E & Pontremoli S (2007) Regulation of calpain activity in rat brain with altered Ca2+homeostasis J Biol Chem 282, 2656–2665

29 Stifanese R, Averna M, Salamino F, Cantoni C, Min-gari MC, Prato C, Pontremoli S & Melloni E (2006) Characterization of the calpain⁄ calpastatin system in human hemopoietic cell lines Arch Biochem Biophys

456, 48–57

30 Goll DE, Thompson VF, Li H, Wei W & Cong J (2003) The calpain system J Physiol Rev 83, 731–801

31 Melloni E, Pontremoli S, Salamino F, Sparatore B, Michetti M & Horecker BL (1984) Two cytosolic

Ca2+-dependent, neutral proteinases from rabbit liver: purification and properties of the proenzyme Arch Biochem Biophys 232, 505–512

32 De Tullio R, Averna M, Salamino F, Pontremoli S & Melloni E (2000) Differential degradation of calpastatin

by l and m-calpain in Ca2+enriched human neuroblas-toma LAN-5 cells FEBS Lett 475, 17–21

33 Sasaki T, Kishi M, Saito M, Tanaka T, Higuchi N, Kominami E, Katunuma N & Murachi T (1990) Inhibi-tory effect of di- and tripeptidyl aldehydes on calpains and cathepsins J Enzym Inhib 3, 195–201

34 Lu Q & Mellgren RL (1996) Calpain inhibitors and ser-ine protease inhibitors can produce apoptosis in HL-60 cells Arch Biochem Biophys 334, 175–181

35 Melloni E, De Tullio R, Averna M, Tedesco I,

Salami-no F, Sparatore B & Pontremoli S (1998) Properties of calpastatin forms in rat brain FEBS Lett 431, 55–58

36 Pontremoli S, Melloni E, Damiani G, Salamino F, Sparatore B, Michetti M & Horecker BL (1988) Effects

of a monoclonal anti-calpain antibody on responses of stimulated human neutrophils Evidence for a role for proteolytically modified protein kinase C J Biol Chem

263, 1915–1919

37 Bianchi G, Ferrari P & Berber BR (1984) The Milan hypertensive strain In: Handbook of Hypertension, Vol 4 (de Jong W, ed.), pp 328–349 Elsevier Science Publisher

38 Byrom FB & Wilson CA (1938) A plethysmographic method for measuring systolic blood pressure in the intact rat J Physiol 93, 301–304

39 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein–dye binding Anal Biochem 72, 248–254

40 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227, 680–685

41 Palejwala S & Goldsmith LT (1992) Ovarian expression

of cellular Ki-ras p21 varies with physiological status Proc Natl Acad Sci USA 89, 4202–4206

42 Averna M, De Tullio R, Salamino F, Minafra R, Pontremoli S & Melloni E (2001) Age-dependent