Yoshihiro Morishima

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.

Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 “client” protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601–607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR·hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR·hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR·hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR·hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR·hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a ∼100-fold shift to the right in the steroid dose response for gene activation.

The Hsp90 Chaperone Machinery Regulates Signaling by Modulating Ligand Binding Clefts

In the two decades that have elapsed since the molecular chaperone Hsp902 was shown to regulate the function of steroid receptors (1), >200 signaling proteins have been found to be regulated by Hsp90 (2). These Hsp90 “client” proteins form complexes containing Hsp90 and Hsp70 that are assembled by a multichaperone machinery (3), with Hsp90 regulating both signaling protein function and turnover. Degradation of these Hsp90-regulated signaling proteins occurs via the ubiquitin-proteasome pathway (4), which in this case is initiated by Hsp70-dependent E3 ligases, such as CHIP and parkin (5). Formation of a complex with Hsp90 stabilizes the client signaling protein, and treatment with a specific inhibitor of Hsp90, such as geldanamycin, triggers its rapid degradation (6). Because many of the Hsp90-regulated signaling proteins are involved in cancer cell growth, Hsp90 inhibitors have emerged as a promising new class of anticancer drugs (7). In this Minireview, we provide a mechanistic basis for understanding how the abundant and ubiquitous chaperones Hsp90 and Hsp70 function together as essential components of the Hsp90 chaperone machinery to regulate signaling protein function and turnover. Like other chaperones, Hsp90 alone has been shown in vitro to assist the refolding of partially unfolded proteins to a properly folded, active conformation. However, Hsp90 is not required for de novo protein folding (8), and it is likely that in cells Hsp90 acts only in concert with Hsp70 in the multichaperone machinery. In contrast to the in vitro experiments on unfolded substrates, this Hsp90 machinery acts on proteins that are in their native conformations to assist the opening of ligand binding clefts. These clefts are hydrophobic clefts that must open to allow access of ligands, such as steroids, ATP, and heme, to their binding sites within the proteins interior. In the absence of the chaperone machinery, ligand binding clefts are dynamic, shifting to varying extents between closed and open states. When clefts open, hydrophobic residues of the proteins interior are exposed to solvent, and continued opening may progress to protein unfolding. Therefore, the extent to which the ligand binding cleft is open determines ligand access and thus protein function, but clefts are inherent sites of conformational instability. The chaperone machinery assists cleft opening, and Hsp90 binding stabilizes the open state of the cleft, preventing further unfolding and Hsp70-dependent ubiquitination. The Hsp90 client proteins are assembled into complexes with the chaperone that are stable enough to be isolated and analyzed biochemically. Although we will refer to these as “stable” Hsp90 complexes, they are constantly undergoing cycles of assembly and disassembly in the cytoplasm and nucleoplasm (3). We will refer to this client protein cycling with Hsp90 as stable cycling. As we will show, a variety of manipulations, including mutations of the LBD or ligand binding itself, result in heterocomplexes that very rapidly disassemble such that no (or only trace amounts of) Hsp90 heterocomplexes can be observed in cell lysates. This rapid complex disassembly we define as “dynamic” Hsp90 cycling, and some signaling proteins naturally interact with Hsp90 in this dynamic cycling mode. Because the function and turnover of these proteins are not as affected by Hsp90 inhibitors as proteins undergoing stable Hsp90 complex assembly, they have not been considered as Hsp90-regulated client proteins, but they are nevertheless Hsp90 substrates. There are several examples where the LBDs of signaling proteins with this dynamic “kiss-and-run” interaction with Hsp90 have been converted by mutation to metastable clefts that undergo stable Hsp90 complex assembly. This conversion of signaling protein-Hsp90 interaction is associated with the acquisition of stringently Hsp90-regulated behavior, typical of client proteins. As Neckers and colleagues have noted (9), many “nodes” in overlapping signaling pathways involved in cancer cell growth are subject to stringent Hsp90 regulation. These Hsp90 client proteins may have evolved from a wide variety of signaling proteins that undergo a more common dynamic cycling of Hsp90 with ligand binding clefts. However, there is no motif for Hsp90 binding, and the basis for its interaction with proteins to form stable or dynamic complexes has not been defined. Here we will present selected examples of Hsp90 effects on signaling protein function and turnover to develop a model in which ligand binding clefts are the common feature determining the interaction with the chaperone. Additional examples in support of the model are cited elsewhere (10).

Activation of Hsp70 reduces neurotoxicity by promoting polyglutamine protein degradation

We sought novel strategies to reduce levels of the polyglutamine androgen receptor (polyQ AR) and achieve therapeutic benefits in models of spinobulbar muscular atrophy (SBMA), a protein aggregation neurodegenerative disorder. Proteostasis of the polyQ AR is controlled by the Hsp90/Hsp70-based chaperone machinery, but mechanisms regulating the protein’s turnover are incompletely understood. We demonstrate that overexpression of 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 SBMA. 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.

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.

The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)·Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90·Hop·Hsp70·Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR·Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358–7366). Hop has been considered essential for the formation of receptor·Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR·Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

Major SNP (Q141K) Variant of Human ABC Transporter ABCG2 Undergoes Lysosomal and Proteasomal Degradations

PurposeSingle nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCG2 gene have been suggested to be a significant factor in patients’ responses to medication and/or the risk of diseases. We aimed to evaluate the impact of the major non-synonymous SNP Q141K on lysosomal and proteasomal degradations.MethodsABCG2 WT and the Q141K variant were expressed in Flp-In-293 cells by using the Flp recombinase system. Their expression levels and cellular localization was measured by immunoblotting and immunofluorescence microscopy, respectively.ResultsThe protein level of the Q141K variant expressed in Flp-In-293 cells was about half that of ABCG2 WT, while their mRNA levels were equal. The protein expression level of the Q141K variant increased about two-fold when Flp-In-293 cells were treated with MG132. In contrast, the protein level of ABCG2 WT was little affected by the same treatment. After treatment with bafilomycin A1, the protein levels of ABCG2 WT and Q141K increased 5- and 2-fold in Flp-In-293 cells, respectively.ConclusionsThe results strongly suggest that the major non-synonymous SNP Q141K affects the stability of the ABCG2 protein in the endoplasmic reticulum and enhances its susceptibility to ubiquitin-mediated proteasomal degradation.

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.

Differential Effects of the hsp70-binding Protein BAG-1 on Glucocorticoid Receptor Folding by the hsp90-based Chaperone Machinery

The heat shock protein hsp70/hsc70 is a required component of a five-protein (hsp90, hsp70, Hop, hsp40, and p23) minimal chaperone system reconstituted from reticulocyte lysate that forms glucocorticoid receptor (GR)·hsp90 heterocomplexes. BAG-1 is a cofactor that binds to the ATPase domain of hsp70/hsc70 and that modulates its chaperone activity. Inasmuch as BAG-1 has been found in association with several members of the steroid receptor family, we have examined the effect of BAG-1 on GR folding and GR·hsp90 heterocomplex assembly. BAG-1 was present in reticulocyte lysate at a BAG-1:hsp70/hsc70 molar ratio of ∼0.03, and its elimination by immunoadsorption did not affect GR folding and GR·hsp90 heterocomplex assembly. At low BAG-1:hsp70/hsc70 ratios, BAG-1 promoted the release of Hop from the hsp90-based chaperone system without inhibiting GR·hsp90 heterocomplex assembly. However, at molar ratios approaching stoichiometry with hsp70, BAG-1 produced a concentration-dependent inhibition of GR folding to the steroid-binding form with corresponding inhibition of GR·hsp90 heterocomplex assembly by the minimal five-protein chaperone system. Also, there was decreased steroid-binding activity in cells that were transiently or stably transfected with BAG-1. These observations suggest that, at physiological concentrations, BAG-1 modulates assembly by promoting Hop release from the assembly complex; but, at concentrations closer to those in transfected cells and some transformed cell lines, hsp70 is continuously bound by BAG-1, and heterocomplex assembly is blocked.

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.