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 Keywords Ca2+ 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: pontremoli@unige.it (Received 31 July 2007, revised 12 September 2007, accepted October 2007) doi:10.1111/j.1742-4658.2007.06133.x Ca2+ 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 digestion 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 Ca2+, HSP90 forms binary complexes with NOS isozymes or with calpain When Ca2+ 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 structural 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 reconstructed 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 Nitric oxide (NO) is a gaseous free radical promoting many biological effects, including the control of microvascular 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 generated 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, respectively 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 cofactors are required for their catalytic activity, including tetrahydrobiopterin, FAD and FMN, in addition to a heme prosthetic group To acquire the active state 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 M Averna et al nNOS and eNOS isoforms also requires calmodulin (CaM) and Ca2+ ions, indicating that NO synthesis is triggered, in target tissues or cells, by an elevation of free [Ca2+]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 Ca2+ ions due to its high affinity for the CaM binding site also at basal Ca2+ levels [7] NO production is highly regulated by many factors and mechanisms, including protein–protein interactions involved in allosteric regulation, scaffolding and trafficking [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 natural site, promoting the dimerization and thus the acquirement of the active conformation through the association with CaM and Ca2+ ions [16,17] The loss of HSP90 or its inhibition by geldanamycin prevents the onset of the active NOS form and increases its degradation by the ATP–ubiquitin–proteasome pathway [18–21] Degradation of NOS has also been proposed as a regulatory mechanism in conditions of high NO production 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 identified 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 proteolysis 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 Ca2+ Degradation of NOS isozymes by calpain binding capacity of calpain The physiological relevance of this novel property of HSP90 is also discussed Results Degradation of NOS isozymes in Ca2+ 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 [Ca2+]i following exposure of cells to the Ca2+-ionophore 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 approximate 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 Digestion of NOS isozymes in cells loaded with Ca2+ Jurkat and BAE-1 cells (2 · 106 for each experiment) were incubated in mL of 10 mM Hepes, pH 7.4, containing 0.14 M NaCl, mM KCl, and gỈL)1 glucose for 30 at 37 °C in the absence (control) or in the presence of lM ionophore A23187 and mM CaCl2 (Ca2+ ion) Alternatively, cells were preincubated for 30 at 37 °C with 50 lM 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 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6117 Degradation of NOS isozymes by calpain M Averna et al 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 calpain 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 following preincubation of the cells with calpain inhibitor-1 [33,34], known to be highly effective on calpain although not completely specific (data not shown) Conversely, in Ca2+ loaded BAE-1 cells, approximately 80% of the native eNOS 130 kDa band and total catalytic activity disappeared, without the appearance 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 Ca2+ loading in Jurkat cells promotes a limited proteolysis of nNOS, in BAE-1, cells Ca2+-enrichement induces digestion and inactivation of eNOS to a large extent A B C 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 previously reported [8,11,35], it was found that both NOS isozymes immunoprecipitated from cell lysates with an antibody-immobilized HSP90 (Fig 3A) Identical 6118 Fig Separation of NOS isozymes from HSP90 in Jurkat and BAE-1 cells by ion exchange chromatography (A) Cell extracts from Jurkat (60 · 106) and BAE-1 cells (10 · 106), obtained as described in the Experimental procedures, were submitted to ion exchange chromatography following the procedure described in the Experimental procedures NOS activity was evaluated on the eluted fractions (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 Experimental procedures (C) The immunoreactive bands of nNOS, eNOS and HSP90 were scanned and quantified as described in the Experimental procedures The areas of the peaks were normalized on the basis of the amount of protein loaded on the column The values reported are the arithmetical mean ± SD of three different experiments results were obtained using purified protein preparations, indicating that the association between NOS and HSP90 did not require specific factors present in the crude cell extracts We further characterized this FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS M Averna et al Degradation of NOS isozymes by calpain Fig 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 incubated overnight at °C with monoclonal anti-HSP90 serum as previously reported [8,11,35] The mixtures were then incubated for h at °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 penetration 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 corresponding cell lines and added to packed Sephacryl S-300 The distribution 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 isozymes, 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 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 susceptibility 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 preceded 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 concomitantly digested and inactivated by calpain (Fig 4B) without the appearance of intermediate active fragments A B C These digestion patterns are consistent with the removal of the N-terminal PDZ domain from the nNOS molecule that converts this enzyme in a molecular 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 isoforms [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 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6119 Degradation of NOS isozymes by calpain A B C D 6120 M Averna et al Fig Susceptibility of nNOS, eNOS and HSP90 to calpain digestion 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 percentage 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 percentage of the protein level measured in the absence of calpain.(D) Summary of the susceptibility of the three proteins to calpain digestion shown in (A), (B) and (C) The values reported are the arithmetical mean ± SD of three separate experiments 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 isolated from rat brain (data not shown) When HSP90 was added to the nNOS digestion mixture (Fig 5A), the catalytic activity of the synthase was almost completely preserved in spite of the disappearance 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 protective 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 synthase [6] This novel protective effect exerted by HSP90, as well as its higher resistance to calpain proteolysis, was then further explored utilizing purified FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS M Averna et al A Degradation of NOS isozymes by calpain A B B Fig Calpain digestion of NOS isozymes in the presence of HSP90 (A) nNOS (1 lg) or (B) eNOS (1 lg) were mixed with 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 carried out on aliquots (30 lL) of the same incubations, to detect nNOS and eNOS, respectively The values reported are the arithmetical mean ± SD of three separate experiments 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 antibody-immobilized HSP90 was capable of binding NOS isozymes or calpain in the absence of Ca2+ ions, thus forming alternative binary complexes (Fig 6A, lanes 1, and 4) When eNOS or nNOS isozymes were separately added to the HSP90 ⁄ calpain immunoprecipitated, Fig Isolation of HSP90 ⁄ NOS ⁄ calpain complexes (A) HSP90 (5 lg) was immobilized to Protein G-Sepharose resin using monoclonal anti-HSP90 serum as reported in the Experimental procedures 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 followed 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 mM CaCl2 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 5), eNOS together with human erythrocyte calpain (lane 6), in the conditions described in the Experimental procedures The same experiments reported in lanes and were also performed with the addition to the immunoprecipitated material of RNCAST600 (0.1 lg) [38] and are reported in lanes and 7, respectively Equal amounts (30 lL) of each sample were submitted to SDS ⁄ PAGE followed 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 no ternary complexes were formed because calpain was completely displaced by NOS (Fig 6A, lanes and 5) However, in the presence of Ca2+ ions, each NOS isozyme and the protease could still be recruited and FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6121 Degradation of NOS isozymes by calpain M Averna et al Scheme Binary and ternary complexes generated by HSP90, NOS and calpain CLP, calpain; CST, calpastatin 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 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 calpastatin present in the cytosol By quantification analysis 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 interconversion 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 catalytic properties of the associated calpain differ from that of the native enzyme because its efficiency is reduced by 50%, probably due to a lower Ca2+binding capacity This explains why higher amounts of calpain are required for digestion of HSP90 (Fig 4D) The changes in catalytic properties of calpain provide an explanation of the mechanism by which, in the ternary complex form, both HSP90 and NOS isozymes are protected from calpain digestion 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 6122 Fig Calpain digestion of isolated HSP90 ⁄ NOS ⁄ calpain complexesThe ternary complexes containing HSP90, NOS and calpain were purified as described in the Experimental procedures and incubated with mM CaCl2 in the absence or in the presence of lg of exogenous human erythrocyte calpain Equal amounts (30 lL) of each incubation were then submitted to SDS ⁄ PAGE electrophoresis followed by immunoblot analysis (see Experimental procedures) The immunoreactive material was revealed using the specific mAbs Table Effect of HSP90 on the catalytic efficiency of human erythrocyte l-calpain Calpain was purified from human erythrocytes 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% K0.5 refers to the [Ca2+] ions required by calpain to express ⁄ Vmax The values reported are the arithmetical means of three different experiments ± SD Calpain catalytic properties Addition Vmax (unitsỈmg)1) Inhibition (%) K0.5 (lM) None Jurkat HSP90 BAE-1 HSP90 1125 ± 60 545 ± 35 560 ± 40 52 50 20 ± 45 ± 45 ± 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 FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS M Averna et al the acquirement of the active conformation and the fully functional state of the synthases [14–17], and recruitment of active calpain, which prevents the inactivation 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 indicate 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 [Ca2+]I, a condition promoting also the activation of calpain If we consider the susceptibility of the isolated NOS forms to calpain digestion, NO production in cells and organisms should be very limited both in extent and time However, in stimulated 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 modulators, HSP90 is directly involved in this regulatory process This chaperone binds to calpain and, when associated with the protease, becomes resistant to digestion In addition, the calpain present in the complex maintains the ability to degrade exogenous substrates, but with a reduced capacity that is also due to a decreased ability to bind Ca2+ ions When nNOS or eNOS associates to the binary HSP90–calpain complex, 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 flexibility of the calpain molecule, which reduces its proteolytic 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 localization of the synthase Degradation of NOS isozymes by calpain 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 attributed 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 digestion 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 particularly important role in vessel relaxation Experimental procedures Materials Leupeptin, calpain inhibitor [33,34], NADPH, Ca2+-ionophore A23187, calmodulin, FAD, FMN, tetrahydrobiopterin, l-arginine l-[14C]arginine (25 nCi; specific activity 308 CiỈmol)1), Source 15Q Sephacryl S-300, phenyl sepharose, 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 (catalogue number 610419) were purchased from BD Transduction Laboratories (Milan, Italy) Monoclonal b-actin antibody (catalogue number A-5441) was obtained from Sigma Aldrich (Milan, Italy) 4-(2-Aminoethyl)benzenesulfonylfluoride (AEBSF) and calpain inhibitor 3-(5-fluoro3-indoyl)-2-mercapto-(Z)-2-propenoic acid (PD151746) [22,32] were obtained from Calbiochem (Mississauga, Canada) 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 Healthcare Cell culture BAE-1 cells were purchased from cell bank Interlab Cell Line Collection (accession no ICLCAL 00004) and maintained in continuous culture at 37 °C (5% CO2) with DMEM (Sigma Aldrich) growth medium containing 10% fetal bovine serum and mm l-glutamine; Jurkat (T cell leukaemia) cells were kindly provided by C Mingari (DIMES, University of Genoa, Italy) and maintained in FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS 6123 Degradation of NOS isozymes by calpain M Averna et al continuous culture at 37 °C (5% CO2) with RPMI 1640 (Sigma Aldrich) growth medium containing 10% foetal bovine serum, 10 mL)1 penicillin (Sigma Aldrich), 100 lgỈmL)1 streptomycin (Sigma Aldrich) and mm l-glutamine (Sigma Aldrich) Purification of nNOS, eNOS and HSP90 from different sources BAE-1 cells (10 · 106) were collected, lysed by sonication in three volumes of ice-cold 50 mm sodium borate buffer, pH 7.5, containing mm EDTA, 0.5 mm 2-mercaptoethanol, 0.1 mgỈmL)1 leupeptin and mm AEBSF The particulate material was discarded by centrifugation (100 000 g for 10 min) and the soluble fraction (cell extract) was collected and mg protein was loaded onto a ion-exchange Source 15Q column (1.5 · 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 concentration 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 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 immunoblotting’ 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 · 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 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 · 106) 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-[14C]arginine as previously reported [26] with the following modifications: aliquots of Jurkat and BAE-1 cell extracts (100 lg) or of the fractions 6124 (100 lL) eluted from the ion exchange chromatography described above, were incubated in buffer A (250 lL) containing mm NADPH, 200 mm calmodulin, 20 lm tetrahydrobiopterin, lm FAD, lm FMN and lm l-arginine and 25 nCi of l-[14C]arginine (specific radio activity 308 CiỈmol)1) at 37 °C After 30 min, the reaction was stopped with ice-cold 50 mm Hepes, pH 5.5, containing mm EDTA (2 mL) The samples were then submitted to anion exchange chromatography using mL of packed Dowex 50W8 Na+ form resin pre-equilibrated with stop buffer l-citrulline was eluted by washing the resin with 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 pmol citrullinmin)1 in the specified conditions Immunoprecipitation and immunoblotting Jurkat (50 · 106) or BAE-1 cells (5 · 106) 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 lgỈmL)1 aprotinin, 20 lgỈmL)1 leupeptin, 10 lgỈmL)1 AEBSF and 10 lgỈmL)1 phosphatases inhibitor cocktail I and II, followed by brief sonication Cell lysates were centrifuged (12 000 g for 15 at °C) and protein quantification of the supernatants was performed using the Lowry assay The immunoprecipitation was performed as previously described using 500 lg of detergent-soluble protein (cell extract) and 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 immobilized to Protein G-Sepharose resin using monoclonal antiHSP90 serum (1 lg) in 300 lL (final volume) of 50 mm sodium borate buffer, pH 7.5, containing 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 loading buffer [48], heated for and submitted to 7% polyacrylamide gel electrophoresis in the presence of SDS Proteins were blotted to a nitrocellulose membrane (BioRad) and probed with specific mAbs, followed by a peroxidase-conjugated secondary antibody as described [49] The immunoreactive bands were developed with an ECL detection 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 appropriate antibody The bands were then scanned and the area of the peaks obtained was used to create a calibration curve FEBS Journal 274 (2007) 6116–6127 ª 2007 The Authors Journal compilation ª 2007 FEBS M Averna et al 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 Jurkat 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 solutions were added to 0.5 mL of packed Sephacryl S-300 previously equilibrated with buffer B and rotated end-over-end for h at °C The resin was packed for 15–20 at °C and NOS activity was assayed as previously described in this section using aliquots (0.2 mL) of the clear aqueous phase Degradation of NOS isozymes by calpain nitrocellulose membrane was probed with monoclonal antiHSP90, 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 nmol of free NH2 groups The specific activity of human erythrocyte calpain and of rat brain m-calpain was 1075 unitsỈmg)1 and 655 unitsỈ mg)1, respectively Acknowledgements This work was supported in part by grants from MIUR, FIRB and PRIN projects, and from the University of Genoa 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 °C for h, in buffer B (300 lL final volume) Protein G-Sepharose (30 lL) was then added to the samples and the mixtures were rotated end-over-end for h at °C Sepharose beads were collected, washed three times with buffer B (500 lL) to discard proteins not specifically bound Human erythrocyte calpain (0.5 lg in 300 lL of buffer B) was added to the pellet and incubated for h at °C Sepharose immunoprecipitated 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, respectively Alternatively, the EDTA present in buffer B was replaced with mm CaCl2 (final concentration) In these conditions, human erythrocyte calpain was maintained in its inactive state by the addition in these mixtures of 0.1 mgỈmL)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 buffer B for h at 37 °C, in the presence of mm CaCl2 Digestion of the ternary complex HSP90 ⁄ NOS ⁄ calpain nNOS, eNOS, HSP90 and calpain, coimmunoprecipitated as previously described, were incubated (30 lL final volume) in 50 mm sodium borate buffer, pH 7.5, containing mm CaCl2 for h at 37 °C in the absence or in the presence of exogenous purified calpain (0.1 lg) Reactions were stopped 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Ca2+ Degradation of NOS isozymes by calpain binding capacity of calpain The physiological relevance of this novel property of HSP90 is also discussed Results Degradation of NOS isozymes in Ca2+... intracellular localization of the synthase Degradation of NOS isozymes by calpain The physiological relevance of these findings becomes particularly evident on the basis of the results observed in... Involvement of the proteasome in activation of endothelial nitric oxide synthase Life Sci 73, 2225–2236 Musial A & Eissa T (2001) Inducible nitric- oxide synthase is regulated by the proteasome degradation