Dynamic cycling with a unique Hsp90/Hsp70-dependent chaperone machinery and GAPDH is needed for heme insertion and activation of neuronal NO synthase.

Heat shock protein 90 (Hsp90) is known to mediate heme insertion and activation of heme-deficient neuronal nitric oxide (NO) synthase (apo-nNOS) in cells by a highly dynamic interaction that has been extremely difficult to study mechanistically with the use of subcellular systems. In that the heme content of many critical hemeproteins is regulated by Hsp90 and the heme chaperone GAPDH, the development of an in vitro system for the study of this chaperone-mediated heme regulation would be extremely useful. Here, we show that use of an antibody-immobilized apo-nNOS led not only to successful assembly of chaperone complexes but the ability to show a clear dependence on Hsp90 and GAPDH for heme-mediated activation of apo-nNOS. The kinetics of binding for Hsp70 and Hsp90, the ATP and K+ dependence, and the absolute requirement for Hsp70 in assembly of Hsp90•apo-nNOS heterocomplexes all point to a similar chaperone machinery to the well-established canonical machine regulating steroid hormone receptors. However, unlike steroid receptors, the use of a purified protein system containing Hsp90, Hsp70, Hsp40, Hop, and p23 is unable to activate apo-nNOS. Thus, heme insertion requires a unique Hsp90-chaperone complex. With this newly developed in vitro system, which recapitulates the cellular process requiring GAPDH as well as Hsp90, further mechanistic studies are now possible to better understand the components of the Hsp90-based chaperone system as well as how this heterocomplex works with GAPDH to regulate nNOS and possibly other hemeproteins.

Low-Dose Hsp90 Inhibitor Selectively Radiosensitizes HNSCC and Pancreatic Xenografts.

PURPOSE: Treatment approaches using Hsp90 inhibitors at their maximum tolerated doses (MTDs) have not produced selective tumor toxicity. Inhibition of Hsp90 activity causes degradation of client proteins including those involved in recognizing and repairing DNA lesions. We hypothesized that if DNA repair proteins were degraded by concentrations of an Hsp90 inhibitor below those required to cause nonspecific cytotoxicity, significant tumor-selective radiosensitization might be achieved. EXPERIMENTAL DESIGN: Tandem mass tagged-mass spectrometry was performed to determine the effect of a subcytotoxic concentration of the Hsp90 inhibitor, AT13387 (onalespib), on global protein abundance. The effect of AT13387 on in vitro radiosensitization was assessed using a clonogenic assay. Pharmacokinetics profiling was performed in mice bearing xenografts. Finally, the effect of low-dose AT13387 on the radiosensitization of three tumor models was assessed. RESULTS: A subcytotoxic concentration of AT13387 reduced levels of DNA repair proteins, without affecting the majority of Hsp90 clients. The pharmacokinetics study using one-third of the MTD showed 40-fold higher levels of AT13387 in tumors compared with plasma. This low dose enhanced Hsp70 expression in peripheral blood mononuclear cells (PBMCs), which is a biomarker of Hsp90 inhibition. Low dose monotherapy was ineffective, but when combined with radiotherapy, produced significant tumor growth inhibition. CONCLUSIONS: This study shows that a significant therapeutic ratio can be achieved by a low dose of Hsp90 inhibitor in combination with radiotherapy. Hsp90 inhibition, even at a low dose, can be monitored by measuring Hsp70 expression in PBMCs in human studies.

Targeting Hsp70 facilitated protein quality control for treatment of polyglutamine diseases.

The polyglutamine (polyQ) diseases are a group of nine fatal, adult-onset neurodegenerative disorders characterized by the misfolding and aggregation of mutant proteins containing toxic expansions of CAG/polyQ tracts. The heat shock protein 90 and 70 (Hsp90/Hsp70) chaperone machinery is a key component of cellular protein quality control, playing a role in the regulation of folding, aggregation, and degradation of polyQ proteins. The ability of Hsp70 to facilitate disaggregation and degradation of misfolded proteins makes it an attractive therapeutic target in polyQ diseases. Genetic studies have demonstrated that manipulation of Hsp70 and related co-chaperones can enhance the disaggregation and/or degradation of misfolded proteins in models of polyQ disease. Therefore, the development of small molecules that enhance Hsp70 activity is of great interest. However, it is still unclear if currently available Hsp70 modulators can selectively enhance disaggregation or degradation of misfolded proteins without perturbing other Hsp70 functions essential for cellular homeostasis. This review discusses the multifaceted role of Hsp70 in protein quality control and the opportunities and challenges Hsp70 poses as a potential therapeutic target in polyQ disease.

Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90ß in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery.

Several hundred proteins cycle into heterocomplexes with a dimer of the chaperone heat shock protein 90 (Hsp90), regulating their activity and turnover. There are two isoforms of Hsp90, Hsp90α and Hsp90ß, and their relative chaperone activities and composition in these client protein•Hsp90 heterocomplexes has not been determined. Here, we examined the activity of human Hsp90α and Hsp90ß in a purified five-protein chaperone machinery that assembles glucocorticoid receptor (GR)•Hsp90 heterocomplexes to generate high-affinity steroid-binding activity. We found that human Hsp90α and Hsp90ß have equivalent chaperone activities, and when mixed together in this assay, they formed only GR•Hsp90αα and GR•Hsp90ßß homodimers and no GR•Hsp90αß heterodimers. In contrast, GR•Hsp90 heterocomplexes formed in human embryonic kidney (HEK) cells also contain GR•Hsp90αß heterodimers. The formation of GR•Hsp90αß heterodimers in HEK cells probably reflects the longer time permitted for exchange to form Hsp90αß heterodimers in the cell versus in the cell-free assembly conditions. This purified GR-activating chaperone machinery can be used to determine how modifications of Hsp90 affect its chaperone activity. To that effect, we have tested whether the unique phosphorylation of Hsp90α at threonines 5 and 7 that occurs during DNA damage repair affects its chaperone activity. We showed that the phosphomimetic mutant Hsp90α T5/7D has the same intrinsic chaperone activity as wild-type human Hsp90α in activation of GR steroid-binding activity by the five-protein machinery, supporting the conclusion that T5/7 phosphorylation does not affect Hsp90α chaperone activity.

ATM is the primary kinase responsible for phosphorylation of Hsp90α after ionizing radiation.

Heat shock protein 90 is a chaperone that plays an essential role in the stabilization of a large number of signal transduction molecules, many of which are associated with oncogenesis. An Hsp90 isoform (Hsp90α) has been shown to be selectively phosphorylated on two N-terminal threonine residues (threonine 5 and 7) and is involved in the DNA damage response and apoptosis. However, the kinase that phosphorylates Hsp90α after ionizing radiation (IR) and its role in post-radiation DNA repair remains unclear. Inasmuch as several proteins of the DNA damage response machinery are Hsp90 clients, the functional consequences of Hsp90α phosphorylation following IR have implications for the design of novel radiosensitizing agents that specifically target the Hsp90α isoform. Here we show that ATM phosphorylates Hsp90α at the T5/7 residues immediately after IR. The kinetics of Hsp90α T5/7 phosphorylation correlate with the kinetics of H2AX S139 phosphorylation (γH2AX). Although Hsp90α is located in both the cytoplasm and nucleus, only nuclear Hsp90α is phosphorylated by ATM after IR. The siRNA mediated knockdown of Hsp90α sensitizes head and neck squamous cell carcinoma cells, lung cancer cells and lung fibroblasts to IR. Furthermore, MEF cells that are Hsp90α null have reduced levels of γH2AX indicating that Hsp90α is important for the formation of γH2AX. Thus, this study provides evidence that Hsp90α is a component of the signal transduction events mediated by ATM following IR, and that Hsp90α loss decreases γH2AX levels. This work supports additional investigation into Hsp90α T5/7 phosphorylation with the goal of developing targeted radiosensitizing therapies.

Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases.

Currently available therapies for adult onset neurodegenerative diseases provide symptomatic relief but do not modify disease progression. Here we explore a new neuroprotective approach based on drugs targeting chaperone-directed protein quality control. Critical target proteins that unfold and aggregate in these diseases, such as the polyglutamine androgen receptor in spinal and bulbar muscular atrophy, huntingtin in Huntington's disease, α-synuclein in Parkinson's disease, and tau in Alzheimer's disease, are client proteins of heat shock protein 90 (Hsp90), and their turnover is regulated by the protein quality control function of the Hsp90/Hsp70-based chaperone machinery. Hsp90 and Hsp70 have opposing effects on client protein stability in protein quality control; Hsp90 stabilizes the clients and inhibits their ubiquitination, whereas Hsp70 promotes ubiquitination dependent on CHIP (C terminus of Hsc70-interacting protein) and proteasomal degradation. We discuss how drugs that modulate proteostasis by inhibiting Hsp90 function or promoting Hsp70 function enhance the degradation of the critical aggregating proteins and ameliorate toxic symptoms in cell and animal disease models.

A model in which heat shock protein 90 targets protein-folding clefts: rationale for a new approach to neuroprotective treatment of protein folding diseases.

In an EBM Minireview published in 2010, we proposed that the heat shock protein (Hsp)90/Hsp70-based chaperone machinery played a major role in determining the selection of proteins that have undergone oxidative or other toxic damage for ubiquitination and proteasomal degradation. The proposal was based on a model in which the Hsp90 chaperone machinery regulates signaling by modulating ligand-binding clefts. The model provides a framework for thinking about the development of neuroprotective therapies for protein-folding diseases like Alzheimer's disease (AD), Parkinson's disease (PD), and the polyglutamine expansion disorders, such as Huntington's disease (HD) and spinal and bulbar muscular atrophy (SBMA). Major aberrant proteins that misfold and accumulate in these diseases are "client" proteins of the abundant and ubiquitous stress chaperone Hsp90. These Hsp90 client proteins include tau (AD), α-synuclein (PD), huntingtin (HD), and the expanded glutamine androgen receptor (polyQ AR) (SBMA). In this Minireview, we update our model in which Hsp90 acts on protein-folding clefts and show how it forms a rational basis for developing drugs that promote the targeted elimination of these aberrant proteins.

Efficacy of an EGFR-specific peptide against EGFR-dependent cancer cell lines and tumor xenografts.

We have recently synthesized a peptide called Disruptin, which comprised the SVDNPHVC segment of the epidermal growth factor receptor (EGFR) that inhibits binding of heat shock protein 90 (Hsp90) to the EGFR and EGF-dependent EGFR dimerization to cause EGFR degradation. The effect is specific for EGFR versus other Hsp90 client proteins [Ahsan et al.: (2013). Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization. J Biol Chem288, 26879-26886]. Here, we show that Disruptin decreases the clonogenicity of a variety of EGFR-dependent cancer cells in culture but not of EGFR-independent cancer or noncancerous cells. The selectivity of Disruptin toward EGFR-driven cancer cells is due to the high level of EGF stimulation of EGFR in EGFR-dependent tumor cells relative to normal cells. When administered by intraperitoneal injection into nude mice bearing EGFR-driven human tumor xenografts, Disruptin causes extensive degradation of EGFR in the tumor but not in adjacent host tissue. Disruptin markedly inhibits the growth of EGFR-driven tumors without producing the major toxicities caused by the Hsp90 inhibitor geldanamycin or by cisplatin. These findings provide proof of concept for development of a new Disruptin-like class of antitumor drugs that are directed specifically against EGFR-driven tumors.

Destabilization of the epidermal growth factor receptor (EGFR) by a peptide that inhibits EGFR binding to heat shock protein 90 and receptor dimerization.

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.

Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation.

We sought new strategies to reduce amounts of the polyglutamine androgen receptor (polyQ AR) and achieve benefits in models of spinobulbar muscular atrophy, a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the heat shock protein 90 (Hsp90)- and Hsp70-based chaperone machinery, but mechanisms regulating the protein's turnover are incompletely understood. We demonstrate that overexpression of Hsp70 interacting protein (Hip), a co-chaperone that enhances binding of Hsp70 to its substrates, promotes client protein ubiquitination and polyQ AR clearance. Furthermore, we identify a small molecule that acts similarly to Hip by allosterically promoting Hsp70 binding to unfolded substrates. Like Hip, this synthetic co-chaperone enhances client protein ubiquitination and polyQ AR degradation. Both genetic and pharmacologic approaches targeting Hsp70 alleviate toxicity in a Drosophila model of spinobulbar muscular atrophy. These findings highlight the therapeutic potential of allosteric regulators of Hsp70 and provide new insights into the role of the chaperone machinery in protein quality control.

Wild-type EGFR is stabilized by direct interaction with HSP90 in cancer cells and tumors.

The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.

Modulation of heme/substrate binding cleft of neuronal nitric-oxide synthase (nNOS) regulates binding of Hsp90 and Hsp70 proteins and nNOS ubiquitination.

Like other nitric-oxide synthase (NOS) enzymes, neuronal NOS (nNOS) turnover and activity are regulated by the Hsp90/Hsp70-based chaperone machinery, which regulates signaling proteins by modulating ligand binding clefts (Pratt, W. B., Morishima, Y., and Osawa, Y. (2008) J. Biol. Chem. 283, 22885-22889). We have previously shown that nNOS turnover is due to Hsp70/CHIP-dependent ubiquitination and proteasomal degradation. In this work, we use an intracellular cross-linking approach to study both chaperone binding and nNOS ubiquitination in intact HEK293 cells. Treatment of cells with N(G)-nitro-L-arginine, a slowly reversible competitive inhibitor that stabilizes nNOS, decreases both nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP. Treatment with the calcium ionophore A23187, which increases Ca(2+)-calmodulin binding to nNOS, increases nNOS ubiquitination and binding of Hsp90, Hsp70, and CHIP in a manner that is specific for changes in the heme/substrate binding cleft. Both Hsp90 and Hsp70 are bound to the expressed nNOS oxygenase domain, which contains the heme/substrate binding cleft, but not to the reductase domain, and binding is increased to an expressed fragment containing both the oxygenase domain and the calmodulin binding site. Overexpression of Hsp70 promotes nNOS ubiquitination and decreases nNOS protein, and overexpression of Hsp90 inhibits nNOS ubiquitination and increases nNOS protein, showing the opposing effects of the two chaperones as they participate in nNOS quality control in the cell. These observations support the notion that changes in the state of the heme/substrate binding cleft affect chaperone binding and thus nNOS ubiquitination.

Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft.

We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-µ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.

C331A mutant of neuronal nitric-oxide synthase is labilized for Hsp70/CHIP (C terminus of HSC70-interacting protein)-dependent ubiquitination.

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind L-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N(G)-nitro-L-arginine, but not its inactive D-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.

Proposal for a role of the Hsp90/Hsp70-based chaperone machinery in making triage decisions when proteins undergo oxidative and toxic damage.

The Hsp90/Hsp70-based chaperone machinery plays a well-established role in signaling protein function, trafficking and turnover. A number of recent observations also support the notion that Hsp90 and Hsp70 play key roles in the triage of damaged and aberrant proteins for degradation via the ubiquitin-proteasome pathway. In the mid-1990s, it was discovered that Hsp70 is required for ubiquitin-dependent degradation of short-lived and abnormal proteins, and it became clear that inhibition of Hsp90 uniformly leads to the proteasomal degradation of Hsp90 client proteins. Subsequently, CHIP and parkin were shown to be Hsp70-binding ubiquitin E3 ligases that direct ubiquitin-charged E2 enzymes to the Hsp70-bound client protein. Stabilization by Hsp90 reflects the interaction of the chaperone with the ligand binding cleft of the client protein. These hydrophobic clefts must be open to allow passage of ligands to binding sites in the protein interior, and they are inherent sites of conformational instability. Hsp90 stabilizes the open state of the cleft and prevents Hsp70-dependent ubiquitination. In the model we propose here, progressive oxidative events result in cleft opening as the initial step in protein unfolding, and as long as Hsp90 can interact to stabilize the cleft, it will buffer the effect of oxidative damage. When cleft opening is such that Hsp90 can no longer interact, Hsp70-dependent ubiquitination occurs. We summarize evidence that Hsp90 interacts very dynamically with a variety of proteins that are not classic Hsp90 clients, and we show that this dynamic cycling of Hsp90 with nitric oxide synthase protects against CHIP-mediated ubiquitination. Scientific interest to date has focused on stringent regulation of the classic client proteins, which have metastable clefts and are inherently short lived. But, the recognition that Hsp90 cycles dynamically with longer lived proteins with more stable clefts may permit extension of the triage model to the quality control of damaged proteins in general.

Inhibition of hsp70 by methylene blue affects signaling protein function and ubiquitination and modulates polyglutamine protein degradation.

The Hsp90/Hsp70-based chaperone machinery regulates the activity and degradation of many signaling proteins. Cycling with Hsp90 stabilizes client proteins, whereas Hsp70 interacts with chaperone-dependent E3 ubiquitin ligases to promote protein degradation. To probe these actions, small molecule inhibitors of Hsp70 would be extremely useful; however, few have been identified. Here we test the effects of methylene blue, a recently described inhibitor of Hsp70 ATPase activity, in three well established systems of increasing complexity. First, we demonstrate that methylene blue inhibits the ability of the purified Hsp90/Hsp70-based chaperone machinery to enable ligand binding by the glucocorticoid receptor and show that this effect is due to specific inhibition of Hsp70. Next, we establish that ubiquitination of neuronal nitric-oxide synthase by the native ubiquitinating system of reticulocyte lysate is dependent upon both Hsp70 and the E3 ubiquitin ligase CHIP and is blocked by methylene blue. Finally, we demonstrate that methylene blue impairs degradation of the polyglutamine expanded androgen receptor, an Hsp90 client mutated in spinal and bulbar muscular atrophy. In contrast, degradation of an amino-terminal fragment of the receptor, which lacks the ligand binding domain and, therefore, is not a client of the Hsp90/Hsp70-based chaperone machinery, is enhanced through homeostatic induction of autophagy that occurs when Hsp70-dependent proteasomal degradation is inhibited by methylene blue. Our data demonstrate the utility of methylene blue in defining Hsp70-dependent functions and reveal divergent effects on polyglutamine protein degradation depending on whether the substrate is an Hsp90 client.

Dynamic cycling with Hsp90 stabilizes neuronal nitric oxide synthase through calmodulin-dependent inhibition of ubiquitination.

NO production by neuronal nitric oxide synthase (nNOS) requires calmodulin and is enhanced by the chaperone Hsp90, which cycles dynamically with the enzyme. The proteasomal degradation of nNOS is enhanced by suicide inactivation and by treatment with Hsp90 inhibitors, the latter suggesting that dynamic cycling with Hsp90 stabilizes nNOS. Here, we use a purified ubiquitinating system containing CHIP (carboxyl terminus of Hsp70-interacting protein) as the E3 ligase to show that Hsp90 inhibits CHIP-dependent nNOS ubiquitination. Like the established Hsp90 enhancement of NO synthesis, Hsp90 inhibition of nNOS ubiquitination is Ca2+/calmodulin-dependent, suggesting that the same interaction of Hsp90 with the enzyme is responsible for both enhancement of nNOS activity and inhibition of ubiquitination. It is established that CHIP binds to Hsp90 as well as to Hsp70, but we show here the two chaperones have opposing actions on nNOS ubiquitination, with Hsp70 stimulating and Hsp90 inhibiting. We have used two mechanism-based inactivators, guanabenz and NG-amino-L-arginine, to alter the heme/substrate binding cleft and promote nNOS ubiquitination that can be inhibited by Hsp90. We envision that, as nNOS undergoes toxic damage, the heme/substrate binding cleft opens exposing hydrophobic residues as the initial step in unfolding. As long as Hsp90 can form even transient complexes with the opening cleft, ubiquitination by Hsp70-dependent ubiquitin E3 ligases, like CHIP, is inhibited. When unfolding of the cleft progresses to a state that cannot cycle with Hsp90, Hsp70-dependent ubiquitination is unopposed. In this way, the Hsp70/Hsp90 machinery makes the quality control decision for stabilization versus degradation of nNOS.

CHIP deletion reveals functional redundancy of E3 ligases in promoting degradation of both signaling proteins and expanded glutamine proteins.

CHIP (carboxy terminus of Hsc70-interacting protein) an E3 ubiquitin ligase that binds to Hsp70 and Hsp90, promotes degradation of several Hsp90-regulated signaling proteins and disease-causing proteins containing expanded glutamine tracts. In polyglutamine disease models, CHIP has been considered a primary protection factor by promoting degradation of these misfolded proteins. Here, we show that two CHIP substrates, the glucocorticoid receptor (GR), a classic Hsp90-regulated signaling protein, and the expanded glutamine androgen receptor (AR112Q), are degraded at the same rate in CHIP(-/-) and CHIP(+/+) mouse embryonic fibroblasts after treatment with the Hsp90 inhibitor geldanamycin. CHIP(-/-) cytosol has the same ability as CHIP(+/+) cytosol to ubiquitinate purified neuronal nitric oxide synthase (nNOS), another established CHIP substrate. To determine whether other E3 ubiquitin ligases that bind to Hsp70 (Parkin) or Hsp90 (Mdm2) act on CHIP substrates, each E3 ligase was co-expressed with the GR, nNOS, AR112Q or Q78 ataxin-3. CHIP lowered the levels of all four proteins, Parkin acted on nNOS and Q78 ataxin-3 but not on the steroid receptors, and Mdm2 did not affect any of the co-expressed proteins. Moreover, both CHIP and Parkin co-localized to aggregates of the expanded glutamine AR formed in cell culture and in a knock-in mouse model of spinal and bulbar muscular atrophy. These observations establish that CHIP does not play an exclusive role in regulating the turnover of Hsp90 client signaling proteins or expanded glutamine tract proteins, and show that the Hsp70-dependent E3 ligase Parkin acts redundantly to CHIP on some substrates.

Pharmacologic and genetic inhibition of hsp90-dependent trafficking reduces aggregation and promotes degradation of the expanded glutamine androgen receptor without stress protein induction.

The molecular chaperone hsp90 has emerged as an important therapeutic target in cancer and neurodegenerative diseases, including the polyglutamine expansion disorders, because of its ability to regulate the activity, turnover and trafficking of many proteins. For neurodegenerative disorders associated with protein aggregation, the rationale has been that inhibition of hsp90 by geldanamycin and related compounds activates heat shock factor 1 (HSF1) to induce the production of the chaperones hsp70 and hsp40 that promote disaggregation and protein degradation. However, we show here that geldanamycin blocks the development of aggregates of the expanded glutamine androgen receptor (AR112Q) of Kennedy disease in Hsf1(-/-) mouse embryonic fibroblasts where these chaperones are not induced. Geldanamycin is additionally known to inhibit hsp90-dependent protein trafficking and to promote proteasomal degradation of client proteins. Overexpression of the hsp90 cochaperone p23 also promotes AR112Q degradation, and inhibits both AR trafficking and AR112Q aggregation without altering levels of hsp70 or hsp40. The hsp90-dependent trafficking mechanism has been defined, and it is shown that key immunophilin (IMM) components of the trafficking machinery are present in polyglutamine aggregates in cell and mouse models of Kennedy disease. Our results indicate that inhibition of the hsp90-dependent trafficking mechanism prevents aggregation of the expanded glutamine androgen receptor, thereby opening a variety of novel therapeutic approaches to these neurodegenerative disorders.