Soluble Guanylyl Cyclase Requires Heat Shock Protein 90 for Heme Insertion During Maturation of the NO-Active Enzyme

Soluble guanylyl cyclase requires heat shock protein 90 for heme insertion during maturation of the NO-active enzyme

Arnab Ghosh and Dennis J. Stuehr1

Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195

Edited by Ruma Banerjee, University of Michigan, Ann Arbor, MI, and accepted by the Editorial Board June 26, 2012 (received for review April 6, 2012) Heme insertion is key during maturation of soluble guanylyl 32) that allowed us to follow heme insertion into transiently cyclase (sGC) because it enables sGC to recognize NO and trans- transfected and endogenously expressed apo-sGC. We used duce its multiple biological effects. Although sGC is often associ- pharmacological hsp90 inhibitors, an ATPase-inactive hsp90 ated with the 90-kDa heat shock protein (hsp90) in cells, the mutant, and heme-dependent or heme-independent sGC activa- implications are unclear. The present study reveals that hsp90 is tors as tools to decipher the role of hsp90. Our findings reveal that required to drive heme insertion into sGC and complete its hsp90 plays a key role in promoting heme insertion into sGC. This maturation. We used a mammalian cell culture approach and hsp90-dependent process, which is essential for sGC maturation, followed heme insertion into transiently and endogenously may be a previously unknown but important feature to control sGC expressed heme-free sGC. We used pharmacological hsp90 inhib- activity in cells, and has important implications regarding hsp90 itors, an ATP-ase inactive hsp90 mutant, and heme-dependent or inhibitor-based cancer therapy. heme-independent sGC activators as tools to decipher the role of hsp90. Our findings suggest that hsp90 complexes with apo-sGC, Results drives heme insertion through its inherent ATPase activity, and Heme Insertion into apo-sGC Requires Active hsp90. COS-7 cells then dissociates from the mature, heme-replete sGC. Together, were used to examine a possible role for hsp90 in heme insertion this improves our understanding of sGC maturation and reveals into apo-sGC. Normal or heme-deficient cells were transfected to a unique means to control sGC activity in cells, and it has impor- express sGC-α1 (Myc-tagged) and sGC-β1 (V5-tagged) subunits, tant implications for hsp90 inhibitor-based cancer therapy. and then we compared cellular sGC expression levels and sGC activities in response to S-nitroso-N-acetyl-DL-penicillamine NO-dependent | cGMP-dependent | vasorelaxation (SNAP), which releases NO and can activate only the heme-replete form of sGC. The normal and heme-deficient cell groups he molecular chaperone 90-kDa heat-shock protein (hsp90) expressed similar amounts of sGC proteins (Fig. S1), indicating Thelps orchestrate fundamental processes including gene ex- that apo-sGC-β1 was stable and able to accumulate in the heme- pression, signal transduction, innate immunity, oncogenesis, and deficient cells. The cells transfected under normal conditions influencing evolutionary phenotypes (1–5). Hsp90 functions displayed good sGC activation by SNAP, whereas the transfected through its subdomain molecular motions and an inherent heme-deficient cells showed much less SNAP activation (Fig. 1). ATPase activity to help control client protein maturation, traf- However, subsequent addition of hemin for 3 h converted the apo- ficking, and lifetime in cells (2, 6, 7). The molecular-level impacts sGC-β1 in the cells to the heme-replete form, as judged by their of hsp90 on various client proteins are just beginning to be recovering a normal sGC activation by SNAP (Fig. 1). When hsp90 elucidated (8–13). inhibitors radicicol or novobiocin were included during the hemin Soluble guanylyl cyclase (sGC) is the primary intracellular treatment, they prevented recovery of the SNAP response (Fig. 1). receptor for the signal molecule NO and is often found associ- This suggests the hsp90 inhibitors may have prevented the cells ated with hsp90 in cells (14–17). The NO-active sGC is a heter- from inserting heme into the apo-sGC-β1. odimer of α1 and β1 subunits and contains a heme prosthetic To further explore this possibility, we used sGC activators group axially ligated to His105 in the β1 subunit (18). NO acti- BAY 41-2272 and BAY 60-2770, which do not release NO but vates sGC by binding to its heme group, which enables sGC to still activate the heme-containing sGC or the apo-sGC, re- catalyze conversion of GTP to cGMP and mediate many of the spectively (33, 34). Fig. 2 shows that both compounds stimulated biological actions of NO (18–21). Accordingly, the pathogenesis comparable sGC activation in sGC-transfected cells cultured of several diseases appears linked to insufficient sGC activity under normal conditions, but, in cells made heme-deficient, the (22), which may be linked to oxidation of the sGC heme and/or degree of sGC activation by BAY 60-2770 increased whereas buildup of heme-free sGC (23, 24). However, our understanding that by BAY 41-2272 decreased by fourfold. This is consistent of sGC maturation in cells, processes regulating the levels of with apo-sGC being predominant in the heme-deficient cells, and active sGC, and the potential roles of hsp90 is incomplete. Thus BAY 60-2770 activating the apo-sGC (33, 34). Hemin re- far, hsp90 has been reported to improve sGC NO response (25), constitution of the heme-deficient cells allowed BAY 41-2272 to and pharmacologically inhibiting hsp90 over a period of several markedly recover its sGC activation and diminished somewhat days was shown to lower sGC activity by increasing its protea- the ability of BAY 60-2770 to activate sGC (Fig. 2). Together, somal degradation (26–29). these data suggest a majority of the sGC in the heme-deficient Recently, we reported that maturation of the hemeprotein-inducible NO synthase (iNOS) is hsp90-dependent (30). Here, the hsp90 associates with the heme-free (i.e., apo) iNOS in cells and Author contributions: A.G. and D.J.S. designed research; A.G. performed research; A.G. then helps to drive heme insertion into iNOS through an inherent and D.J.S. analyzed data; and A.G. and D.J.S. wrote the paper. ATPase activity. Heme insertion into the related neuronal NOS The authors declare no conflict of interest. also requires hsp90 (31). Given the pervasive nature of the sGC– This article is a PNAS Direct Submission. R.B. is a guest editor invited by the Editorial Board. hsp90 association, we wondered if hsp90 might play a similar role 1To whom correspondence should be addressed. E-mail: [email protected]. in enabling heme insertion into sGC during its maturation. To This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. address this, we adopted a mammalian cell culture approach (30, 1073/pnas.1205854109/-/DCSupplemental.

12998–13003 | PNAS | August 7, 2012 | vol. 109 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1205854109 Downloaded by guest on October 1, 2021 25 needed for cells to insert heme into apo-sGC. Inhibiting hsp90 *** *** *** prevented heme insertion but otherwise did not hinder the apo-

20 sGC from being activated by the heme-independent pathway.

Hsp90 Interacts with apo-sGC and Dissociates upon Heme Insertion. 15 We next sought to document the hsp90–sGC interaction in cells and determine whether it depends on the heme content of sGC- β α β 10 1. Because sGC is a heterodimer of 1/ 1 subunits, we also examined the importance of sGC α1 by transfecting cells with sGC α1/β1 together or with sGC-β1 alone. The cells were or were cGMP (pmol protein) mg / 5 not made heme-deﬁcient and then were transfected to express sGC α1/β1 together or only sGC-β1 under various conditions,

0 and then we examined the hsp90-sGC protein interaction by SNAP -----+++ ++immunoprecipitation by using anti-V5 (sGC-β1) antibody. Fig. α1β1 α1β1 +SA α1β1 +SA α1β1 +SA α1β1 +SA 3A and Fig. S3 compare the amount of hsp90–sGC complex +Hemin +Hemin +Rad +Hemin +Novo isolated from normal vs. heme-depleted cells, and also show the Fig. 1. Effect of hsp90 inhibition during heme reconstitution of NO-de- effect of incubation with hemin in the presence or absence of two pendent sGC activity. COS-7 cells were pretreated with or without SA for 48 different hsp90 inhibitors. There was fourfold greater hsp90-sGC h and then cotransfected with sGC α1 and β1 constructs for 42 h. This was association in heme-depleted cells compared with normal cells followed by addition of vehicle or hemin (5 μM) to cells and hsp90 inhibitors that were overexpressing sGC α1/β1 (Fig. 3A and Fig. S3B), radicicol or novobiocin (added for 30 min before hemin). After 3 h, cells were μ despite their expressing equal levels of the sGC proteins. The treated with SNAP (50 M) for 5 min to activate sGC and then harvested, and hsp90–sGC interaction was greatly decreased after incubating sGC protein expression and cGMP product were measured. Bar graph shows ± the heme-deficient cells with hemin for 3 h to reconstitute their GMP concentration in supernatants determined by ELISA. Values are mean β SD of three independent experiments, each containing five replicates (***P < apo-sGC- 1 [i.e., plus succinyl acetone (SA) plus hemin]. The 0.001 by one-way ANOVA). hsp90–sGC interaction was partly retained if the hsp90 inhibitor radicicol was present during the hemin incubation, but this was not the case with novobiocin. We observed similar results for the cells was in the apo form, and confirm that functional heme in- hsp90–sGC interactions in cells that overexpressed sGC-β1 alone sertion into the apo-sGC population is achieved upon exogenous (Fig. S3A), indicating that overexpression of the α-subunit is not hemin treatment. required. Together, our data suggest that hsp90 associates pre- In this context, we added the hsp90 inhibitor radicicol to the dominantly with apo-sGC-β1, and this association weakens after heme-deficient cells during their hemin treatment to see if it heme insertion occurs. would alter the subsequent sGC activation in response to the two To independently test if the hsp90–sGC interaction depends drugs. Radicicol still allowed good sGC activation by the heme- on sGC heme content, we separately expressed V5-tagged ver- independent activator BAY 60-2770, but it inhibited the recovery sions of the WT sGC-β1 and the heme-free mutant sGC-β1H105F of sGC activation by the heme-dependent activator BAY 41- in COS-7 cells and then immunoprecipitated by using anti-V5 2272 (Fig. 2). Western analysis showed that none of the activity antibody to compare their levels of hsp90 binding. As shown in differences could be ascribed to unequal sGC protein expression Fig. 3B, the extent of hsp90 binding to the H105F mutant was (Fig. S2). Together, our findings demonstrate that active hsp90 is more than threefold greater compared with WT sGC-β1. This result matches our seeing a greater hsp90–sGC association in heme-depleted cells, and argues that greater association is di- fi β 40 rectly related to the level of heme de ciency of sGC- 1, rather than to any indirect effects of cellular heme depletion. ** – 35 ** *** *** Finally, we examined the dynamics of the hsp90 sGC interaction in relation to the change in sGC heme content by 30 monitoring the kinetics of both facets in heme-deficient cells that * 25 were transfected to express apo-sGC-β1 and then had hemin added. At each time point, we added SNAP for 5 min before cell 20 harvest and then measured cell cGMP levels as an indicator of 15 heme-dependent sGC activity (Fig. S4), which served to in-

directly determine the heme content of sGC. [The time point of CELL BIOLOGY

cGMP (pmol protein) mg / 10 SNAP treatment was done for 5 min as it correlated with max- imum cGMP accumulation (Fig. S4).] As shown in Fig. 3C and 5 Fig. S5, there was a strong hsp90–sGC-β1 association at the 0 initial time point followed by a sharp decrease within the 15-min BAY 60-2770 - + - - + - - + - - + - period following hemin addition. The decrease in sGC–hsp90 BAY 41-2272 - - + - - + - - + - - + interaction was somewhat greater for cells undergoing the SNAP α1β1 α1β1+ SA α1β1+ SA +Hemin α1β1 +SA D +Hemin +Rad treatment. The corresponding cGMP measures (Fig. 3 ) showed that the apo-sGC became enzymatically active over the same Fig. 2. Effect of inhibiting hsp90 during heme reconstitution on heme-de- time course after hemin was added, consistent with it in- pendent and heme-independent sGC activity. COS-7 cells were pretreated ﬁ α β corporating heme within the rst 15 min. The time courses show with or without SA for 48 h and then cotransfected with sGC 1 and 1 that the hsp90–apo-sGC association is relatively strong before constructs, followed by hemin treatment with/without hsp90 inhibitor rad- heme insertion and then decreases rapidly with the onset of icicol (added for 30 min before hemin). After 3 h, the cells were treated with β a heme-independent (BAY 60-2770, 10 μM) or heme-dependent (BAY 41- heme insertion into the sGC- 1 subunit. 2272, 10 µM) sGC activator for 30 min and then harvested. Bar graph shows cGMP concentration in supernatants determined by ELISA. Values are mean ± D88N-hsp90 Down-Regulates sGC Activity by Blocking Heme Insertion. SD of three independent experiments, each containing ﬁve replicates (*P < To further examine the importance of hsp90 ATPase activity in 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA). enabling heme insertion into apo-sGC-β1, we used the D88N

Ghosh and Stuehr PNAS | August 7, 2012 | vol. 109 | no. 32 | 12999 Downloaded by guest on October 1, 2021 A IP: Anti-V5 B IB: Hsp90 20 18 * )

-3 16 IP: Anti-V5 14 IB: Hsp90 12 +SA +SA +Hemin +SA +Rad +SA +Hemin +SA +Novo +SA +Hemin +SA α1β1(sGC)

10 H105F 1

Hsp90 8 β β1 6 Hsp90 4 Anti-V5 (x 10 Band Intensity Anti-V5 (sGC- β1) 2 0 Anti-Myc β1(WT) β1H105F (sGC-α1) Bound Hsp90 C D 14 16 ns

) * 14 **

-2 12

12 10 10 8 * 8 6 6 4 4 Band Intensity(X 10 2 2 cGMP (pmol / mg protein) 0 - - 0 +++----- + ++SNAP -+--+ ----+++ ++ 0 15 30 60 90 120 180 min 0 15 30 60 90 120 180 min Bound Hsp90 β1 +SA +Hemin

Fig. 3. Hsp90-sGC interactions before and after heme reconstitution of sGC-β1 and effect of hsp90 inhibition. (A) COS-7 cells were or were not made heme- deﬁcient (deﬁciency indicated by +SA) and then were transfected 42 h to express sGC α1 and β1. Cells were then incubated 3 h with or without hemin (5 μM) and hsp90 inhibitors radicicol or novobiocin, and the supernatants were prepared, subjected to immunoprecipitation with anti-V5 (sGC-β1) antibody, and analyzed by SDS/PAGE and Western blotting. (A) Immunoprecipitated hsp90 with associated sGC-β1 and α1 subunits (input 10%) froms GCα1 plus β1 transfected cells. (B)

COS-7 cells were transfected with sGC-β1 or sGC-β1H105F for 42 h and then harvested. Right: Immunoprecipitation shows hsp90 associated with sGC-β1 or sGC- β1H105F (input 10%). Bar graph shows densitometric quantification of the associated hsp90 bands. Data are mean of three independent experiments. (C and D) Heme-deficient COS-7 cells were transfected to express sGC-β1 for 42 h, followed by addition of hemin (5 μM) at various time points between 0 and 180 min, and then treated with or without SNAP (50 μM) for 5 min before harvesting. (C) Bar graph compares the level of hsp90 associated with immunoprecipitated sGC-β1 (input 10%) in each sample (data are mean of three independent experiments). (D) Supernatant cGMP concentration as determined by ELISA. Values are mean ± SD of three independent experiments, with each containing three replicates (*P < 0.05 and **P < 0.01, by one-way ANOVA; ns, not statistically significant).

hsp90 mutant, which has no ATPase activity but is known to form (RLF-6) that expresses high levels of endogenous sGC (36). in-cell complexes with client proteins such as endothelial NOS RLF-6 cells were cultured for 3 d with SA to make them heme- and iNOS (30, 35). We coexpressed the D88N mutant or WT deficient. This did not lower sGC protein expression (Fig. S7)or hsp90β (both HA-tagged) with sGC-β1 in COS-7 cells for 42 h, its activation by heme-independent activator BAY 60-2770, but and then activated the sGC by SNAP before cell harvest to greatly lowered sGC activation by SNAP (Fig. 5A), indicating measure heme-dependent sGC activation. As shown in Fig. 4A, apo-sGC had built up in the heme-deficient RLF-6 cells. Further cells expressing D88N-hsp90 had a 70% reduction in cGMP culture for 1 h with hemin restored sGC activation by SNAP accumulation after SNAP treatment compared with cells (Fig. 5A), consistent with heme insertion taking place during this expressing WT hsp90, without impacting the cell sGC-β1 protein time period, and radicicol inhibited this recovery. In related level (Fig. S6A). This shows D88N-hsp90 acts as a dominant- experiments, we transfected RLF-6 cells with WT or D88N negative inhibitor of sGC activation during its expression in cells, hsp90β and then compared their protein expression, association confirming the original observation by Miao et al. (35). We next with native sGC, and sGC activation by SNAP. Both hsp90 studied if D88N-hsp90 expression would impact heme insertion proteins were expressed well in RLF-6 cells (Fig. 5B). Pull-down into apo-sGC-β1. In this case, heme-deficient COS-7 cells were studies using an anti-sGC antibody showed that the HA-tagged cotransfected with sGC-β1 and the WT hsp90 or D88N mutant WT and D88N hsp90 proteins both associated with sGC-β1in and cultured for an additional 48 h, followed by hemin addition the RLF-6 cells (Fig. 5C). There was a decrease in hsp90–sGC- for 1 h, SNAP treatment for 5 min, and then cell harvest. Heme β1 interaction during sGC activation by SNAP that was not ev- incorporation into apo-sGC was assessed by cGMP accumulation ident for the D88N hsp90–sGC-β1 complex, which may indicate in response to SNAP. Coexpression of D88N-hsp90 completely a more stable complex formed between D88N hsp90 and sGC- inhibited heme incorporation into apo-sGC relative to the con- β1. D88N-hsp90 expression diminished SNAP activation of sGC trol without impacting the cell sGC protein level (Fig. 4B and (Fig. 5D). Our results show that hsp90 and its ATP-ase activity Fig. S6B). These results confirm that the ATP-ase activity of are also required for heme insertion into the sGC-β1 that is hsp90 is critical for heme insertion into apo-sGC-β1. endogenously expressed in lung fibroblast cells.

Hsp90 Drives Heme Insertion into Native sGC Expressed in Lung Discussion Fibroblasts. To test if hsp90 is required for heme insertion in Heme insertion into apo-sGC-β1 is essential for maturation of a more natural setting, we used a rat lung ﬁbroblast cell line the NO-responsive enzyme. We found that heme insertion into

13000 | www.pnas.org/cgi/doi/10.1073/pnas.1205854109 Ghosh and Stuehr Downloaded by guest on October 1, 2021 A WT. Although their association did not require hsp90 to have an 16 intact ATPase activity, as judged from our results with the use of ** radicicol, novobiocin, and an ATPase-defective hsp90 mutant, an 14 intact ATPase activity was essential to drive heme insertion into “ 12 * ** apo-sGC. The fact that radicicol or novobiocin had no down- stream” impact on the NO-dependent activation of mature sGC 10 or on activation of apo-sGC by the heme-independent stimulator BAY 60-2770, implies that hsp90 restricts its participation to the 8 heme insertion step during the protein maturation phase and is 6 not involved in activating the catalysis of heme-replete or heme- free sGC. Together, our data suggest a model for hsp90 function 4 that is illustrated in Fig. 6. Interestingly, this model for hsp90 mimics its proposed role in driving heme insertion into apo- 2 iNOS (30). The similarity implies hsp90 may operate through cGMP (pmol / mg protein) 0 a common mechanism to target and stabilize heme-free forms of SNAP -+-++ - client heme proteins, and then enable their maturation by driving WT Hsp90 D88N heme-insertion in an ATP-dependent process. Some apo-sGC appeared to be present in our cell cultures COS-7 cells under normal conditions, as judged by BAY 60-2770 causing B significant additional stimulation of sGC activity relative to 18 SNAP alone (Fig. 2). Indeed, our study suggests that observing ns any hsp90-sGC association in cells may itself indicate that heme- 16 ** free sGC is present. Interestingly, Ignarro et al. purified sGC 14 ** from bovine lung in a heme-deficient form (39). Overall, these concepts support the claim by Roy et al. (40) that heme-free sGC 12 ** normally exists in cells and that BAY 60-2770 can activate the 10 heme-free sGC (34).

8 Implications of hsp90 Inhibition on sGC Activity. Our cell culture 6 systems were purposely designed to test how short-term hsp90 inhibition (0–3 h) impacts a critical posttranslational step in 4 sGC maturation, namely heme insertion into apo-sGC-β1.

cGMP (pmolprotein) mg / 2 However, in regard to hsp90 inhibitor therapy, one must also consider longer-term effects. Previous studies that investigated 0 how hsp90 inhibitors impact sGC activity all incubated cells with SNAP -+-+-+-+-+the inhibitors overnight to 1-d time periods (25, 26, 28). Inhib- -SA +SA +SA +SA +SA iting hsp90 over these longer periods diminished sGC activity +Hemin +Hemin +Hemin mainly by shunting sGC toward the proteasomal degradation +Hsp90 +D88N pathway, thus lowering its overall expression level in cells. In- COS-7 cells creased degradation may also explain why long-term hsp90 inhibition diminishes the expression levels of several hsp90 client Fig. 4. ATPase-defective D88N-hsp90 down-regulates NO activation of proteins (41). In contrast, our study reveals a more immediate sGC by antagonizing heme insertion. (A) COS-7 cells were cotransfected mechanism by which hsp90 inhibition can diminish sGC activity, with sGC-β1 and hsp90β or D88N-hsp90β for 42 h, given SNAP (50 μM) for namely by preventing heme insertion into apo-sGC-β1. Thus, 5 min, and then harvested. (A) cGMP concentration in the supernatants over long periods of hsp90 inhibition, we surmise that any newly as assayed by ELISA. (B) COS-7 cells were made heme deﬁcient with SA β β β translated sGC- 1 protein would fail to mature and would re- before 42 h cotransfection with sGC- 1plushsp90 or D88N-hsp90, fol- β lowed by 1 h hemin addition (5 μM)andthen5minSNAPtreatment.(B) main in its heme-free form. Whether apo-sGC- 1 is inherently cGMP concentrations in supernatants as assayed by ELISA. Values are shorter-lived and degraded faster over a period of days in cells, mean ± SD of three independent experiments, with each containing three or whether hsp90 inhibition works in a less direct manner to replicates (*P < 0.05 and **P < 0.01, one-way ANOVA; ns, not statistically alter the sGC-β1 level, are important questions that can now signiﬁcant). be addressed. CELL BIOLOGY

Implications for hsp90 Inhibitor-Based Cancer Therapy. There is apo-sGC-β1 depends on hsp90. An hsp90 requirement was ap- a tremendous interest in developing hsp90-targeted cancer parent whether we followed incorporation of exogenously added therapeutic agents (42). Whether newly developed hsp90 inhib- heme or endogenous cell-derived heme into apo-sGC-β1, and it itors would block heme insertion into apo-sGC-β1 or the NOS held for sGC that was transiently expressed and for sGC that was enzymes is still untested, but the fact that most drugs are expressed naturally in a lung fibroblast cell line. sGC is the third designed to target the hsp90 ATPase activity (shown here to be protein whose heme insertion has now been found to be hsp90- essential for heme insertion) suggests the possibility is real and dependent (the others are iNOS and neuronal NOS) (30, 31). may have important implications for the development of hsp90 Given that sGC and NOS enzymes have markedly different inhibitors for cancer therapy. We predict that inhibiting hsp90- protein structures and heme environments (37, 38), our findings based heme insertion into both the sGC and NOS enzymes suggest that hsp90 may have a more general role in heme protein would cause a synergistic lowering of cGMP levels because it maturation than was previously realized. would diminish production of the signal molecule (i.e., NO) and In the present study, hsp90 associated most strongly with apo- diminish the responsiveness of the amplifier (i.e., sGC) toward sGC-β1 in cells and the association weakened or fell apart when NO. Our study shows that one could circumvent the potential heme had become inserted. Likewise, there was greater hsp90 impact of hsp90 inhibitors by administering heme-independent association with heme-free mutant sGC-β1H105F compared with sGC activators like BAY 58-2667, which can still activate heme-

Ghosh and Stuehr PNAS | August 7, 2012 | vol. 109 | no. 32 | 13001 Downloaded by guest on October 1, 2021 A 14 ** 12 ** ** ** ** ** 10 * 8

6 Fig. 5. Hsp90 drives heme insertion into endogenous sGC expressed in RLF-6 cells. (A) RLF-6 cells 4 were pretreated with or without SA for 72 h, in-

cGMP (pmol protein) mg / cubated 3 h with hemin (5 μM) with or without 2 hsp90 inhibitors radicicol or novobiocin, and then treated with SNAP (50 µM) for 5 min or with heme- 0 dependent (BAY 41-2272, 10 μM) or heme-in- SNAP - +++- + - - - + + --+++ --BAY 60-2770 --+ - ++- + BAY 41-2272 dependent (BAY 60-2770, 10 µM) sGC activators for - SA + SA + SA +SA +Rad +SA +Novo + SA +SA +SA +Rad 30 min before being harvested. (A) Cell supernatant +Hemin +Hemin +Hemin - SA +Hemin +Hemin cGMP concentrations determined by ELISA. (B–D) B C D RLF-6 cells were transfected with WT or D88N-hsp90 IP:sGC-β1 10 for 42 h, treated with SNAP for 5 min, and har- RLF-6 cells IB:HA ** vested. (B) Representative Western blot shows 8 * ** RLF-6 cells protein expression levels (sGC-β1, hsp90β or D88N- +D88N +Hsp90 6 hsp90, and total hsp90) in supernatants. (C) Immu- - ++-- +SNAP

+D88N β β +Hsp90 noprecipitations depict hsp90 or D88N-hsp90 as- 4 β sGC-β1 --++SNAP sociated with sGC- 1 (input 10%) with or without 2 HA- WT SNAP treatment. (D) cGMP concentration in super- HA- WT /D88N cGMP (pmol / mg protein) natants as determined by ELISA. Data are mean ± /D88N 0 sGC-β1 SNAP -+- ++- SD of three independent experiments, with each Hsp90 WT Hsp90 D88N containing three replicates (*P < 0.05 and **P < RLF-6 cells 0.01, one-way ANOVA).

free sGC and thus allow it to function in the signal cascades, or Fisher. Stock solutions of novobiocin were prepared in water whereas leading to vasorelaxation or other cGMP-dependent processes. 3-isobutyl-1-methylxanthine (IBMX) and radicicol were dissolved in DMSO. Stock solutions of hemin were freshly prepared in 0.1 N NaOH (32). cDNAs Our work provides a platform to explore these concepts. for human hsp90β and D88N-hsp90β mutant were gifts from Bill Sessa (Yale α β β Materials and Methods University, New Haven, CT). cDNAs for sGC 1, 1, and sGC-H105F 1 mutant were gifts from Andreas Papapetropoulos (University of Patras, Patras, Antibodies and Reagents. BAY 60-2770 and BAY 41-2272 were provided by Greece). Monkey COS-7 and rat RLF-6 cells were purchased from American Bayer, SNAP was purchased from Cayman Chemicals, and lipofectamine was Type Culture Collection. Rabbit polyclonal hsp90 antibody was purchased purchased from Invitrogen. All other chemicals were purchased from Sigma from Cell Signaling Technology. Anti-V5 antibody was purchased from Invitrogen, and anti-Myc and anti-HA tag antibodies were purchased from Sigma. A cGMP ELISA kit was obtained from Cell Signaling Technology.

apo- sGC (heme free) Cell Culture and Preparation of Supernatants. All cell lines were grown in 100 mm tissue culture dishes. COS-7 cells were cultured in DMEM containing 10% Hsp90 (vol/vol) FBS and 5,000 U of penicillin-streptomycin. RLF-6 cells were cultured HCP in Ham F-12 K media containing L-glutamine and pyruvate, 5,000 U/L of Heme penicillin/streptomycin and 20% (vol/vol) FBS. Heme depletion was achieved α1 β1 HCP by culturing cells with 400 μM SA for 48 h before transfection or activation of sGC (30, 32). RFL-6 cells expressing endogenous sGC were activated by NO Heme- HCP donor SNAP (50 μM) or with heme-dependent (BAY 41-2272; 10 μM) or independent GTP heme-independent activators (BAY 60-2770; 10 μM) from 0 to 30 min as Hsp90 indicated. At the point of cell harvest, the monolayers were washed twice l cGMP o n with 4 mL cold PBS solution plus 1 mg/mL glucose, collected by scraping in ic ci ic io 500 μL lysis buffer [40 mM 3-(4-[2-hydroxyethyl]-1-piperazinyl)propane- Heme- ad b α1 β1 R vo dependent o sulfonic acid buffer, pH 7.6, 10% glycerol, 3 mM DTT, 150 mM NaCl, and .5% NO N ATP Nonidet P-40). Cells were lysed by three cycles of freeze/thawing, the lysates 90 ADP p were centrifuged for 30 min at 4 °C, and the resulting supernatants were x s H assayed for total protein content by using a Bio-Rad protein assay kit. Hsp90 N 8 D8 Heme Depletion and Cell Transfection. Cultures (50–60% conﬂuent) of COS-7 α1 β1 α1 β1 or RLF-6 cells or SA-pretreated COS-7 cells were transfected for 42 h with sGC heterodimer Hsp90 expression constructs of sGC α1, β1, sGC-H105F β1, hsp90β, or D88N-hsp90β in various combinations using Lipofectamine [5 μg DNA was used for single Fig. 6. Model for hsp90 function during maturation of active sGC. Hsp90 transfections and 10 μg DNA (i.e., 5µgplus5μg) was used for cotransfec- binds to heme-free sGC in cells, and this complex likely interacts with a heme tions]. Cells were then treated with 0.5 mM IBMX for 10 min followed by carrier protein (HCP). Hsp90 then uses its ATPase activity to help drive heme SNAP or with Bayer compounds (i.e., BAY 60-2770 or BAY 41-2272) for insertion into apo-sGC-β1. This process is blocked by hsp90 inhibitors radi- various times (0–30 min) before being harvested. In some cases, cells were cicol or novobiocin and antagonized by the ATPase-defective D88N-hsp90. pretreated with SA (400 μM) before transfection with sGC expression Hsp90 then dissociates from the heme-replete sGC, whose catalysis can now constructs. After 42 h, cells were given radicicol (20 μM), novobiocin (500 be activated by NO. In contrast, sGC activation by heme-independent sGC μM), or vehicle for 30 min and then treated with hemin for 3 h. Cell sGC activators does not require hsp90. Further details are provided in the text. was then activated by adding SNAP or Bayer compounds at the indicated

13002 | www.pnas.org/cgi/doi/10.1073/pnas.1205854109 Ghosh and Stuehr Downloaded by guest on October 1, 2021 time points before harvesting. In all cases, the cells were treated with cy- then added and incubated for 1 h at 4 °C. The beads were microcentrifuged cloheximide (10 mg/mL) for 30 min before hemin addition. Transfections (3,220 × g), washed three times with wash buffer (50 mM Hepes, pH 7.6, 100 were carried out in duplicate plates, and the experiments were repeated at mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and then boiled with loading least three times. buffer for SDS-PAGE gels and subsequent Western blotting.

Western Blots and Immunoprecipitation. For Western blotting, 30 μgofcell cGMP Assay. sGC activity in cell supernatants was measured as a function of supernatants were loaded on 8% SDS/PAGE gels. Proteins were electro- cGMP concentration by using a cGMP assay kit (Cell Signaling Technology). blotted to PVDF membrane and probed with the respective antibodies. For immunoprecipitation, 500 μg of the total cell supernatants was precleared ACKNOWLEDGMENTS. We thank Drs. W. Sessa and A. Papapetropoulos for µ – providing hsp90 and sGC constructs and Dr. Hans-Peter Stasch (Bayer, with 20 L of protein G sepharose beads (Amersham) for 1 h at 4 °C, beads Leverkusen, Germany) for providing sGC activators BAY 60-2770 and BAY were pelleted, and the supernatants were incubated overnight at 4 °C with 41-2272. This work was supported by National Institutes of Health Grants 3 μg of anti-HA or anti-iNOS antibody. Protein G–sepharose beads (20 μL) were GM51491 and HL076491 (to D.J.S.).

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