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. Author manuscript; available in PMC: 2022 Mar 1.
Published in final edited form as: Nitric Oxide. 2020 Dec 15;108:12–19. doi: 10.1016/j.niox.2020.12.003

Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system

Xiaomin Wu a, Xutong Sun a, Shruti Sharma b, Qing Lu a, Manivannan Yegambaram a, Yali Hou b, Ting Wang c, Jeffrey R Fineman d,e, Stephen M Black a,*
PMCID: PMC8193791  NIHMSID: NIHMS1692463  PMID: 33338599

Abstract

Despite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

Keywords: Arginine recycling, CHIP, hsp90, Protein degradation, Ubiquitin proteasome

1. Introduction

In endothelial cells (EC), nitric oxide (NO) is synthesized from the substrate l-arginine by endothelial NO synthase (eNOS) whereupon it diffuses to the adjacent vascular smooth muscle cell layer to act as an endogenous vasodilator. The endothelial dysfunction associated with impaired NO production is associated with a number of vascular pathologies including hypertension, atherosclerosis and angiogenesis-associated disorders [1,2]. Besides being as a semi-essential amino acid that supports protein synthesis, l-arginine is the precursor in the formation of urea, polyamines, proline, glutamate, creatine and agmatine, it can be obtained from diary diets or synthesized by the body itself. Intracellular levels of l-arginine range from 100 to 800 μmol/L, well above the 5 μmol/L Km value for eNOS [3]. However, NO production can still be stimulated by exogenous l-arginine [4]. This phenomenon, termed the ‘arginine paradox’, suggests the existence of a separate pool of arginine directed to NO synthesis. One important mechanism for controlling this arginine pool is the regeneration of l-arginine from the other product of the eNOS catalyzed reaction, l-citrulline [5]. This regeneration is catalyzed by the enzymes arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL).

The localization of eNOS to the plasmalemmal caveolae sub-compartment is important for the regulation and catalytic efficiency of eNOS [6–8]. The enzymes of the citrulline-arginine cycle (ASS and ASL) are also localized to caveolae and interact with eNOS to form an NO synthetic complex [9]. Disrupting this complex produces NO deficiency even when excess l-arginine is present [9]. This prior study suggested that the heat shock protein (Hsp) 90 was involved in the NO synthetic complex. Hsp90 is a chaperone protein that assists in the proper folding of its client proteins to form the active conformation, and treatment with Hsp90 inhibitors, such as geldanamycin, can stimulate proteins degradation [10]. Another HSP member, Hsp70 facilities protein degradation of Hsp90 client proteins when hsp90 is attenuated and the regulation of the Hsp90/Hsp70 machinery complex appears to be essential for protein proteolysis [11]. Hsp70 triggers the degradation of Hsp90 client proteins through the recruitment of the E3 ubiquitin ligase, C-terminus of Hsp70-interacting protein (CHIP) leading to protein degradation via the ubiquitin proteasome pathway (UPP) [12]. Thus, adequate Hsp90 binding to client proteins is necessary to prevent Hsp70/CHIP-dependent ubiquitination [13]. As we, and others have shown that other members of the NO signaling pathway including eNOS itself [14,15], GTP cyclohydrolase I (GCHI) [16], and soluble guanylate cycle [17] require chaperoning by hsp90, we set out to investigate whether ASS and ASL are also regulated by interactions with hsp90 and to determine if they are susceptible to Hsp70/CHIP-dependent proteasomal degradation.

Using cultured bovine aortic endothelial cells (BAEC), our data indicate that both ASS and ASL form complexes with hsp90. Furthermore, inhibition of hsp90 activity leads to ASS/ASL degradation via UPP, and results in decreases in l-arginine levels in the cell. We were further able to demonstrate that the proteasomal degradation of ASS and ASL involves both Hsp70 and CHIP. Thus, our results suggest targeting the Hsp90/Hsp70/CHIP axis could be used as a therapeutic target to restore NO signaling in pathologic conditions.

2. Materials and methods

2.1. Cell culture and treatment

Bovine aortic endothelial cells (BAEC) and culture medium were purchased from Cell Applications (USA), and maintained at 37 °C in a humidified incubator at 5% CO2. BAEC were grown to 90% confluent prior to exposure to geldanamycin (GA, 2 μM, Sigma) or the proteasome inhibitor, Z-leu-leu-leu-CHO (MG132, 10 μM, Calbiochem) for the required time. BAEC were used up to passage 5 (Cell Applications, USA).

2.2. Expression plasmids and transient transfections

Wild type CHIP and point mutants (K30A [a TPR domain mutant] and H260Q [a UBOX mutant]) expression plasmids were kindly provided by Dr. Cam Patterson [18]. All plasmids were purified using endotoxin free kit (Qiagen, USA). BAEC were cultured to 80% confluence prior to performing plasmid transient transfections using the Effectene Transfection Reagent (Qiagen, USA) according the manufacturers protocol. After incubating the cells for 48 h, cells were harvested using RIPA buffer (Thermo, USA) and then analyzed.

2.3. Immunoprecipitation and western blot analysis

BAEC were harvested in IP buffer (Thermo, USA) supplemented with a protease inhibitor cocktail (Thermo, USA) as user guide described. Cell lysates were then clarified by centrifugation at 20,000 g (20 min at 4 °C), the protein concentrations were determined and 1 mg of each lysate was used to conduct immunoprecipitation anti-ASS and anti-ASL using magnet beads immunoprecipitation kit (Thermo, USA) according to the manual. The immune complexes were eluted using elusion buffer followed by Western blot analysis to detect the pulled down proteins using target antibodies (Hsp90, Hsp70 or CHIP). After transfer, membranes were blocked with 5% nonfat dry milk or 5% BSA in Tris-buffered saline containing 0.1% Tween (TBST). After blocking, the membranes were probed with primary antibodies at 4 °C overnight. Membranes were then probed with horseradish peroxidase conjugated secondary antibodies to either rabbit or mouse (Thermo, USA). Reactive bands were then visualized using chemiluminescence on a Kodak 440CF image station (New Haven, CT) or an Odyssey Fc Imaging System (Li-cor, USA). Immune signals were quantified using Image Studio software or ImageJ. The efficiency of each immunoprecipitation was normalized by reprobing the membranes with the appropriate immunoprecipitation antibody (ASS or ASL). Anti-rabbit serum polyclonal ASS and ASL antibodies were generated as previously described [19]. The following antibodies were purchased commercially: anti-Hsp90 (Catalog # 610,419, BD bioscience, USA), anti-Hsp70 (Catalog #C92F3A-5, Enzo, USA), anti-CHIP (Catalog # 2080, Cell signaling, USA), anti-Ubiquitin (Catalog# 3936, Cell signaling, USA), anti-myc tag (Catalog# 213,316, Thermo, USA) and β-actin (Catalog # A3854, Sigma, USA).

2.4. Ubiquitination assays

Ubiquitinatied ASS and ASL were detected by using Co-immunoprecipitation (CoIP) protocol, 0.1 mg cell lysates of control and treated BAECs were incubated with ASS or ASL antibodies, following processure was performed according to the manual of magnet beads immunoprecipitation kit (Thermo, USA). Ubiquitin (Catalog# 3936, Cell signaling, USA) antibody was probed to test ASS and ASL ubiquitination level. The ubiquitination efficiency was normalized by striping and re-probing with ASS or ASL antibodies.

2.5. Real-time PCR analysis

Total RNA was isolated from BAEC using TRIZOL reagent (Thermo-Fisher) according to the manufacturer’s protocol. First-strand cDNAs were then synthesized from 1 μg of DNase I (Thermo-Fisher)-treated RNA using Anchored Oligo (dT)20 Primer and Maxima H Minus Reverse Transcriptase (Thermo-Fisher) in 20 μl reaction volume. One microliter of the first-strand cDNA reaction was used as a template for real-time PCR using ASS and ASL primers and a SYBR Green Master Mix (Thermo-Fisher). The GAPDH gene was used as an internal control. Real-time PCR assays were performed by using QuantStudio™ 3 Real-Time PCR (Thermo). The primer sequences were: GAPDH Forward 5′- CCT CAT GGT CCA CAT GGC CTC CAA G-3′, Reverse 5′-GGT ACA CAA GGC AGG GCT CCC TAA G-3’; ASS Forward 5′- GGA GAC TAC GAG CCG GTT GAT GC-3′, reverse 5′- TCC CTC CGT CTG TGG ACC ACT TC-3’; ASL forward 5′- ATC GGA GAG CGG GAA GCT ATG GG -3′, reverse 5′- TTC TGC CGC ATC CAC AGC CTG AG -3’. All reactions were performed in triplicate.

2.6. Measurement of l-arginine levels

l-arginine level was analyzed by high-performance liquid chromatography (HPLC) as previously described [20]. l-arginine concentrations were calculated using standards and homoarginine as an internal standard.

2.7. Measurement of ASS and ASL activity

ASS and ASL enzyme activities in BAEC with or without GA were measured using a protocol previously described [19,21].

2.8. Statistical analysis

Statistical analysis was performed using GraphPad Prism. All samples were calculated as Mean ± SEM and significance determined using either the unpaired t-test for two group or ANOVA for more than two groups. P < 0.05 was considered significant.

3. Results

3.1. hsp90 inhibition decreases the activity of ASS and ASL

It has been reported that Hsp90 participates in an NO synthesis complex which includes ASS ASL and NOS [9]. Thus, we first conducted IP studies to determine if there is a direct interaction between hsp90, ASS and ASL in BAEC. Using immunoprecipitation analyses (IP) we were able to show that both ASS and ASL form complexes with Hsp90 (Fig. 1A and B). Furthermore, exposure of BAEC to the Hsp90 inhibitor, Geldanamycin (GA, 2 μM) significantly decreased these interactions (Fig. 1 A & B). Hsp90 inhibition also resulted in a significant decrease in the activities of ASS (Fig. 1C) and ASL (Fig. 1D), leading to decreases in cellular l-arginine levels (Fig. 1E). Together these results suggest that the chaperone activity of hsp90 is essential to maintain ASS and ASL catalytic function.

Fig. 1. Hsp90 inhibition disrupts arginine recycling in bovine aortic endothelial cells.

Fig. 1.

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BAEC were exposed or not to Geldanamycin (GA, 2 μM, 4 h) and then whole cell lysates (1 mg) were subjected to immunoprecipitation (IP) using antibodies specific to ASS or ASL followed by Western blot analysis using a specific antibody against hsp90 (A). Both ASS and ASL form complexes with hsp90 (A) and this interaction is decreased when hsp90 activity is inhibited (B&C). The IP efficiency was normalized by striping and re-probing the membrane with anti-ASS or anti-ASL antibodies. The activity of ASS (D) and ASL (E) are attenuated and cellular l-arginine levels are decreased (F) after exposure to GA (2 μM, 24 h). Values are means ± SEM; n = 3–10. *P < 0.05 vs. control.

3.2. Hsp90 inhibition reduces ASS and ASL protein levels

Since the activities of both ASS and ASL were reduced by hsp90 inhibition, we next explored whether ASS and ASL protein levels were reduced after GA treatment. Our data indicate that GA-induced a time-dependent decrease in the protein levels of both ASS and ASL (Fig. 2A). However, the decrease in protein levels was not associated with reduction in the mRNA levels of either ASS (Fig. 2B) or ASL (Fig. 2C) in the presence of GA. These results implicate a post-translation mechanism, possibly protein degradation via ubiquitintion, is involved in the decrease in ASS and ASL protein levels during hsp90 inhibition.

Fig. 2. hsp90 Inhibition decreases ASS and ASL protein levels in bovine aortic endothelial cells.

Fig. 2.

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BAEC were exposed to Geldanamycin (GA, 2 μM, 0–24 h) and time dependent effects on ASS and ASL protein levels determined. Western blot analyses show a significant decrease in ASS and ASL protein levels after 24 h of GA treatment (A). Blots were first probed with an antibody specific to ASL antibody, then striped and reprobed with and an antibody specific to ASS, then finally striped and reprobed with an antibody for β-actin. The bar graph was generated by normalizing levels to β-actin. hsp90 inhibition (GA, 2 μM, 8 h) does not alter ASS (B) or ASL (C) mRNA levels as determined using qPCR. mRNA levels were normalized to GAPDH. Values are means ± SEM; n = 3–6. *P < 0.05 vs. control.

3.3. Hsp90 inhibition leads to ASS and ASL ubiquitination

To investigate the potential role of ubiquitination in the decrease in ASS and ASL protein levels in response to Hsp90 inhibition, we treated BAEC with GA in the presence or absence of the 26 S proteasome inhibitor, MG132. Our data clearly demonstrate that hsp90 inhibition leads to the poly-ubiquitination of both ASS (Fig. 3A) and ASL (Fig. 3B) in BAEC. Further, blocking the 26 S proteasome with MG132, abolished the GA dependent decrease in both ASS (Fig. 3C) and ASL (Fig. 3D) protein levels.

Fig. 3. ASS and ASL are subject to ubiquitin-dependent proteasomal degradation in bovine aortic endothelial cells.

Fig. 3.

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BAEC were pretreated (2 h) with the proteasomal inhibitor, MG132 (10 μM) or its vehicle (0.1% DMSO) then exposed or not to GA (2 μM, 24 h). Whole cell lysates (1 mg) were subjected to immunoprecipitation using anti-ASS and anti-ASL antibodies followed by immunoblotting with a ubiquitin antibody. The IP efficiency was normalized by striping and reprobing the membrane with anti-ASS or anti-ASL antibodies. Exposure of BAEC to GA or MG132 alone, increases the ubiquitination of ASS (A) and ASL (B). MG132 prevents the GA-mediated decrease in ASS (C) and ASL (D) protein levels. Loading was normalized by re-probing blots with an antibody to β-actin. Values are means ± SEM; n = 3–6. *P < 0.05 vs. Control, †P < 0.05 vs. GA alone, ‡P < 0.05 v. MG132 alone.

3.4. hsp90 inhibition stimulates the interaction of ASS and ASL with hsp70 and the E3 ubiquitin ligase, C-terminus of Hsp70-interating protein (CHIP)

We have previously shown that the proteasomal degradation of GCHI involves the recruitment of hsp70 and CHIP [16]. Thus, we next investigated whether hsp70 and CHIP become associated with ASS and ASL after hsp90 inhibition. Our data indicate that GA treatment significantly increases the interaction of ASS (Fig. 4A and B) and ASL (Fig. 4C and D) with hsp70 and CHIP.

Fig. 4. hsp90 inhibition induces ASS and ASL proteasomal degradation via the recruitment of the hsp70-CHIP complex in bovine aortic endothelial cells.

Fig. 4.

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BAEC were exposed or not to GA (2 μM, 4 h) and then whole lysates (1 mg) were subjected to immunoprecipitation (IP) using antibodies specific to ASS and ASL proteins. This was followed by Western blotting using specific antibodies to either hsp70 or CHIP. There is an increase in the interaction of hsp70 (A) and CHIP (B) with ASS. Similarly, GA increases the interaction of hsp70 (C) and CHIP (D) with ASL. IP efficiency was normalized by striping and re-probing the membrane with antibodies to ASS or ASL. Values are means ± SEM; n = 4–8. *P < 0.05 vs. Control.

3.5. Both the Ubox- and TPR-domains are essential for CHIP-mediated proteasomal degradation of ASS and ASL

To further confirm that CHIP is the selective E3 ubiquitin ligase involved in the proteasomal degradation of ASS and ASL protein levels upon hsp90 inhibition, BAEC were transiently transfected with an expression construct for wild-type CHIP (pcDNA-myc-CHIP) or an empty vector (pcDNA3, as a control), and effects on ASS and ASL protein levels determined by Western blot analysis. Our data demonstrate that CHIP over-expression itself is able to mimic the effect of hsp90 inhibition on the ASS and ASL to produce a dose dependent decrease in both ASS and ASL protein levels (Fig. 5A). CHIP contains N-terminal three tandem tetratricopeptide (TPR) motifs which mediate CHIP binding to hsp70 and hsp90 and a C-terminal U-box domain which contains the E3 ubiquitin ligase activity [22]. To investigate the importance of these domains in the proteasomal degradation of ASS and ASL degradation, we transiently expressed TPR domain (K30A) and U-box domain (H260Q) mutant proteins in BAEC and compared their effect to wild-type CHIP. Unlike wild-type CHIP over-expression (Fig. 5A) neither the TPR domain mutant nor the U-box domain mutant induces the degradation of ASS (Fig. 5B) or ASL (Fig. 5C). Despite this, both the TPR domain mutant (Fig. 5D) and the U-box domain mutant (Fig. 5E) were still able to interact with ASS and ASL. These results confirm the involvement of CHIP in the proteasomal degradation of ASS and ASL and demonstrate that requirement for hsp70-CHIP chaperone complex formation and the E3 ligase activity of CHIP.

Fig. 5. Both the Ubox- and the TPR domains are required for CHIP-mediated proteasomal degradation of ASS and ASL in bovine aortic endothelial cells.

Fig. 5.

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Transiently transfecting BAEC with increasing levels (0, 0.5, 1, 2 μg) of a myc-tagged CHIP expression plasmid significantly decreases the protein levels of ASS and ASL (A) as determined by Western blot analyses. BAEC were also transiently transfected with expression plasmids (2 μg) containing a myc-tagged CHIP TPR domain mutant (TPRm-CHIP) or a CHIP Ubox domain mutant (UBOXm-CHIP). Compared to wildtype CHIP (A) neither TPRm-CHIP (B) nor the UBOXm-CHIP (C) induced ASS and ASL protein degradation as determined using Western blot analyses. In each study, blots were first probed with an antibody specific to ASL antibody, then striped and reprobed with an antibody specific to ASS, then striped and reprobed with an antibody for c-myc to confirm myc-CHIP, mcy-TPRm-CHIP, or myc-UBOXm-CHIP over-expression. Finally, the membrane was striped and reprobed with an antibody to β-actin to normalize for protein loading. IP analyses using an anti-myc antibody indicate that both the TPRm-CHIP (D) and the UBOXm-CHIP (E) were still able to interact with ASS and ASL. In these IP assays, the membranes were initially probed with an anti-ASL antibody, stripped and reprobed with an anti-ASS antibody, before being stripped again and reprobed with an anti-Myc antibody to validate IP efficiency. Values are means ± SEM; n = 3–4. *P < 0.05 vs. no plasmid (A) or empty vector (B &C).

4. Discussion

In endothelial cells, l-arginine metabolism plays critical role in vascular physiology and pathophysiology through its ability to regulate NO signaling via eNOS. Over the past three decades, NO has been identified as the endothelium-derived relaxing factor responsible for smooth muscle relaxation and reducing hypertension [2,23,24]. The upregulation of NO production represents an adaptive response of the blood vessel to maintain a low vascular resistance in response to increased blood flow and pressure [25]. l-arginine is a semi-essential amino acid in mammalian cells and serves as precursor for a number of metabolic products, including protein synthesis, NO generation, polyamine biosynthesis, proline, glutamate, creatine, agmatine, and urea [26]. Among the multiple pathways of l-arginine metabolism, two metabolic circuits have been the most described, the urea cycle which generates urea and ornithine via arginase and the l-citrulline to l-arginine recycling pathway which restores l-arginine levels in cells by converting the NOS byproduct, l-citrulline back to l-arginine. This l-arginine regeneration pathway requires the sequential activity of two enzymes, arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) [1,26,27]. The urea cycle byproduct l-ornithine also can be converted to l-citrulline, allowing for subsequent conversion to l-arginine [28]. In humans, ASS and ASL deficiencies cause citrullinaemia and hyperammonemia respectively [29,30] resulting in urea cycle disorders, neurocognitive deficiencies, hepatic disease, hypertension and electrolyte imbalances [31]. ASL and ASS have been found in a variety of tissues including liver, kidney, heart, brain, muscle, pancreas and red blood cells, suggesting multiple organs in the body are capable of l-arginine biosynthesis [31,32]. The “intestinal-renal axis” generates the major fraction of l-arginine [26]. However, up to 40% of de novo l-arginine synthesized from l-citrulline occurs in extra-renal tissues, including the endothelium [33]. However, the intracellular concentration of l-arginine is up to 20 times greater (0.5–2 mM) than in the blood circulation (~100 μM) and both far exceed the Km for NO synthase (~5 μM) [34]. This “arginine paradox” lead Hecker et al. to demonstrate that cultured EC generate l-arginine through the conversion of l-citrulline by the sequential reactions of ASS and ASL [5] and this is necessary to maintain NO signaling [35]. Further, the disruption of the l-arginine recycling system attenuates NO signaling as ASL deficiency impairs NO production and increases blood pressure in both humans and mice [9, 31]. We have also previously shown that the loss of NO signaling in lambs with increased pulmonary blood flow [36] is associated with decreased activity of ASS and ASL and reductions in l-arginine levels [19]. The importance of this decrease in l-arginine is demonstrated the restoration of NO signaling when these lambs are supplemented with l-arginine [16,37]. DNA microarray screening has revealed that ASS is upregulated in conjunction with increased NO production in EC exposed to shear stress [38]. Blocking ASS using small interfering RNAs decreases NO production even under saturating l-arginine levels in the culture medium, supporting critical role of l-arginine recycling in endothelial NO generation [39]. Similarly, decreasing ASL expression in piglet endothelial cells decreases NO production [9]. Conversely, ASS over-expression stimulates endothelial NO production [38,39] while ASS-mediated increases in l-arginine synthesis in inflammatory macro-phages is involved in host-resistance to mycobacterial infection [40]. Thus l-arginine recycling plays a key role in regulating NO signaling.

It has been previously reported that ASS and ASL are present in a NO synthetic complex along with eNOS and hsp90 [9]. However, the NO synthetic complex has only been demonstrated in a COS-7 cell reconstitution assay [9] and its presence in cultured EC has not been investigated. Thus, our data that both ASS and ASL form complexes with hsp90 are important confirmation that this NO synthetic complex exists in cells that naturally generate NO. Our data also indicate that hsp90 plays an important chaperone role in maintaining the l-arginine recycling system. Structurally, Hsp90 contains three distinct domains: 1) N-terminal domain contains ATP binding site; 2) middle domain, binding sites for client proteins and other co-chaperone proteins; and 3) C-terminal domain, controls client’s interactions and forms Hsp90 dimer. Geldanamycin directly binds to ATP binding site in N-terminal domain of Hsp90, resulting in blocking Hsp90-ATP interaction and affecting its chaperone function. Our data demonstrate that using geldanamycin to inhibit hsp90 also disrupts arginine recycling in BAEC by inhibiting ASS and ASL activity. Further, both ASS and ASL are subject to proteasomal degradation via the ubiquitin-mediated 26 S proteasome complex and this requires interactions of ASS and ASL with hsp70 and the E3 ubiquitin ligase, CHIP. Thus, our results link the citrulline-NO cycle to Hsp70/CHIP system, suggesting that the CHIP-ubiquitin pathway is intimately involved in impairing NO production. Indeed our data add to prior work in which both soluble guanylate cyclase [17] and GCH1 [16], key players in the NO signaling cascade, are hsp90 client proteins and also subject to CHIP-mediated proteasomal degradation. hsp90 is a well characterized ubiquitous molecular chaperone that is essential for maintaining cell signaling including cell cycle, chromatin remodeling and cellular homeostasis [41]. hsp90 is known to chaperone more than 200 clients [42], and extensive evidence supports a role for hsp90 in a number of disease, including cancer, neurodegenerative diseases, and vascular diseases [43,44]. In pulmonary smooth muscle cells, hsp90 accumulates in the mitochondria during the development of pulmonary arterial hypertension, and has been identified as a key regulator of mitochondrial homeostasis contributing to vascular remodeling [44]. During the past decade, our studies have identified a key role for decreased hsp90 activity in the development of pulmonary endothelial dysfunction associated with increased PBF [14,16,37,45]. Secondary to decreases in ATP levels, hsp90 activity is decreased in our lamb model of increased PBF and correlates with decreased NO signaling due to loss of hsp90 mediated interactions with eNOS [14,20] and GCH1, the rate-limiting enzyme in the generation of NOS cofactor, tetrahydrobiopterin (BH4) [16]. The data presented in this study add to our knowledge of how hsp90 regulates NO signaling by demonstrating the formation of a complex consisting of hsp90 with ASS and ASL, and that blocking this binding leads to ASS and ASL proteasomal degradation. Besides decrease in ASS and ASL protein levels, conformational changes in ASS and ASL protein structure after their dissociation form hsp90 complex may also contribute to reductions in ASS and ASL activity. However, further studies will be required to identify the specific domain within ASS and ASL that controls their interaction with hsp90. Thus, it is likely that the decrease in ASS and ASL activity in our lamb model of increased PBF [19] also involves a decrease in their interactions with hsp90. However, further studies will be required to test this possibility.

hsp90 exists widely from eubacteria to all branches of eukaryote, where it is able to assist its client proteins fold into their native conformation [46]. Numerous co-chaperones are also involved forming a dynamic complex termed the hsp90 chaperone machinery [42]. These co-chaperones have diverse effects on hsp90 function, including enhancing the conformational dynamics to maintain client’s stability, transporting specific substrates to the hsp90 complex, and processing ubiquitin-dependent degradation. Two widely studied hsp90 interacting proteins are hsp70 and the C-terminus of Hsp70-interating protein, CHIP. Hsp70 is conserved in eukaryotic cells, and exerts opposing effects to hsp90 leading to client protein degradation while Hsp90 enhances protein stability and imparts resistance to ubiquitination and degradation [13]. CHIP is an ubiquitin E3 ligase and is linked to the degradation of hsp90 client proteins [47]. The ubiquitin-proteasome system is involved in the degradation of a number of cellular proteins. However, it is poorly understood how the substrates are anchored for ubiquitination. CHIP is a U-BOX E3 ligase, and binds to Hsp70 by an amino-terminal TPR domain [12] and we have previously shown that Hsp70 must have interacted with the protein target before CHIP is recruited to the complex to facilitate degradation [16]. In the present study, we demonstrate that ASS and ASL protein degradation is dependent on CHIP as CHIP over-expression on its own is able to induce the degradation of ASS and ASL. Further, the ability of CHIP to interact with ASS and ASL in combination with the ubiquitin ligase activity are both necessary since a CHIP TPR domain mutant [18] and a CHIP U-BOX domain mutant are unable to trigger the proteasomal degradation of ASS and ASL.

l-arginine supplementation has been studied in cardiovascular disease with mixed results. For example, arginine supplementation showed benefits in attenuating the endothelial dysfunction associated with sickle cell disease [48,49] but had no benefit in hypertensive patients [50]. In addition, studies have identified an important issue with l-arginine supplementation: the more arginine is added, the more it is destroyed by arginase [51] increasing the possibility of adverse events via the multiple metabolic roles of arginine [52–54]. The ideal situation would be to develop new therapies that maintain/restore l-arginine levels without exogenous supplementation. The data presented here showing that ASS and ASL are subject to proteasomal degradation means that it is interesting to speculate that proteasomal degradation could be targeted to prevent the loss of l-arginine recycling capacity, thus restoring NO signaling and endothelial function. However, studies in animal models of endothelial dysfunction will be necessary to test this possibility prior to any future clinical trials in humans.

Acknowledgements

This research was supported in part by HL60190 (SMB), HL137282 (SMB/JRF), HL134610 (SMB/TW), HL142212 (SMB), HL146369 (SMB/JRF/TW), HL061284 (JRF), and the Interdisciplinary Training in Cardiovascular Research T32 HL007249 (to XW) all from the National Institutes of Health. The authors have no conflicts of interest to declare.

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