Co-chaperone P23 Research Articles

Molecular chaperones: Guardians of tumor suppressor stability and function.

The term 'tumor suppressor' describes a widely diverse set of genes that are generally involved in the suppression of metastasis, but lead to tumorigenesis upon loss-of-function mutations. Despite the protein products of tumor suppressors exhibiting drastically different structures and functions, many share a common regulatory mechanism-they are molecular chaperone 'clients'. Clients of molecular chaperones depend on an intracellular network of chaperones and co-chaperones to maintain stability. Mutations of tumor suppressors that disrupt proper chaperoning prevent the cell from maintaining sufficient protein levels for physiological function. This review discusses the role of the molecular chaperones Hsp70 and Hsp90 in maintaining the stability and functional integrity of tumor suppressors. The contribution of cochaperones prefoldin, HOP, Aha1, p23, FNIP1/2 and Tsc1 as well as the chaperonin TRiC to tumor suppressor stability is also discussed. Genes implicated in renal cell carcinoma development-VHL, TSC1/2, and FLCN-will be used as examples to explore this concept, as well as how pathogenic mutations of tumor suppressors cause disease by disrupting protein chaperoning, maturation, and function.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1−/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Molecular mechanisms of chaperone-directed protein folding: Insights from atomistic simulations.

Molecular chaperones, a family of proteins of which Hsp90 and Hsp70 are integral members, form an essential machinery to maintain healthy proteomes by controlling the folding and activation of a plethora of substrate client proteins. This is achieved through cycles in which Hsp90 and Hsp70, regulated by task-specific co-chaperones, process ATP and become part of a complex network that undergoes extensive compositional and conformational variations. Despite impressive advances in structural knowledge, the mechanisms that regulate the dynamics of functional assemblies, their response to nucleotides, and their relevance for client remodeling are still elusive. Here, we focus on the glucocorticoid receptor (GR):Hsp90:Hsp70:co-chaperone Hop client-loading and the GR:Hsp90:co-chaperone p23 client-maturation complexes, key assemblies in the folding cycle of glucocorticoid receptor (GR), a client strictly dependent upon Hsp90/Hsp70 for activity. Using a combination of molecular dynamics simulation approaches, we unveil with unprecedented detail the mechanisms that underpin function in these chaperone machineries. Specifically, we dissect the processes by which the nucleotide-encoded message is relayed to the client and how the distinct partners of the assemblies cooperate to (pre)organize partially folded GR during Loading and Maturation. We show how different ligand states determine distinct dynamic profiles for the functional interfaces defining the interactions in the complexes and modulate their overall flexibility to facilitate progress along the chaperone cycle. Finally, we also show that the GR regions engaged by the chaperone machinery display peculiar energetic signatures in the folded state, which enhance the probability of partial unfolding fluctuations. From these results, we propose a model where a dynamic cross-talk emerges between the chaperone dynamics states and remodeling of client-interacting regions. This factor, coupled to the highly dynamic nature of the assemblies and the conformational heterogeneity of their interactions, provides the basis for regulating the functions of distinct assemblies during the chaperoning cycle.

The transportosome system as a model for the retrotransport of soluble proteins

The classic model of action of the glucocorticoid receptor (GR) sustains that its associated heat-shock protein of 90-kDa (HSP90) favours the cytoplasmic retention of the unliganded GR, whereas the binding of steroid triggers the dissociation of HSP90 allowing the passive nuclear accumulation of GR. In recent years, it was described a molecular machinery called transportosome that is responsible for the active retrograde transport of GR. The transportosome heterocomplex includes a dimer of HSP90, the stabilizer co-chaperone p23, and FKBP52 (FK506-binding protein of 52-kDa), an immunophilin that binds dynein/dynactin motor proteins. The model shows that upon steroid binding, FKBP52 is recruited to the GR allowing its active retrograde transport on cytoskeletal tracks. Then, the entire GR heterocomplex translocates through the nuclear pore complex. The HSP90-based heterocomplex is released in the nucleoplasm followed by receptor dimerization. Subsequent findings demonstrated that the transportosome is also responsible for the retrotransport of other soluble proteins. Importantly, the disruption of this molecular oligomer leads to several diseases. In this article, we discuss the relevance of this transport machinery in health and disease.

The p23 co-chaperone is a succinate-activated COX-2 transcription factor in lung adenocarcinoma tumorigenesis.

P23, historically known as a heat shock protein 90 (HSP90) co-chaperone, exerts some of its critical functions in an HSP90-independent manner, particularly when it translocates into the nucleus. The molecular nature underlying how this HSP90-independent p23 function is achieved remains as a biological mystery. Here, we found that p23 is a previously unidentified transcription factor of COX-2, and its nuclear localization predicts the poor clinical outcomes. Intratumor succinate promotes p23 succinylation at K7, K33, and K79, which drives its nuclear translocation for COX-2 transcription and consequently fascinates tumor growth. We then identified M16 as a potent p23 succinylation inhibitor from 1.6 million compounds through a combined virtual and biological screening. M16 inhibited p23 succinylation and nuclear translocation, attenuated COX-2 transcription in a p23-dependent manner, and markedly suppressed tumor growth. Therefore, our study defines p23 as a succinate-activated transcription factor in tumor progression and provides a rationale for inhibiting p23 succinylation as an anticancer chemotherapy.

A snapshot of the aryl hydrocarbon receptor complex

In this issue of Structure, Wen etal. present the cryo-EM structure of the aryl hydrocarbon receptor (AhR) and show how it is recruited and stabilized by the HSP90 molecular chaperone and its co-chaperones XAP2 and p23.

Hypoviral-regulated HSP90 co-chaperone p23 (CpCop23) determines the colony morphology, virulence, and viral response of chestnut blight fungus Cryphonectria parasitica.

We previously identified a protein spot that showed down-regulation in the presence of Cryphonectria hypovirus 1 (CHV1) and tannic acid supplementation as a Hsp90 co-chaperone p23 gene (CpCop23). The CpCop23-null mutant strain showed retarded growth with less aerial mycelia and intense pigmentation. Conidia of the CpCop23-null mutant were significantly decreased and their viability was dramatically diminished. The CpCop23-null mutant showed hypersensitivity to Hsp90 inhibitors. However, no differences in responsiveness were observed after exposure to other stressors such as temperature, reactive oxygen species, and high osmosis, the exception being cell wall-disturbing agents. A severe reduction in virulence was observed in the CpCop23-null mutant. Interestingly, viral transfer to the CpCop23-null mutant from CHV1-infected strain via anastomosis was more inefficient than a comparable transfer with the wild type as a result of decreased hyphal branching of the CpCop23-null mutant around the peripheral region, which resulted in less fusion of the hyphae. The CHV1-infected CpCop23-null mutant exhibited recovered mycelial growth with less pigmentation and sporulation. The CHV1-transfected CpCop23-null mutant demonstrated almost no virulence, that is, even less than that of the CHV1-infected wild type (UEP1), a further indication that reduced virulence of the mutant is not attributable exclusively to the retarded growth but rather is a function of the CpCop23 gene. Thus, this study indicates that CpCop23 plays a role in ensuring appropriate mycelial growth and development, spore viability, responses to antifungal drugs, and fungal virulence. Moreover, the CpCop23 gene acts as a host factor that affects CHV1-infected fungal growth and maintains viral symptom development.

OR26-3 USP19 Deubiquitinating Enzyme Functions as a Novel Co-Chaperone of Hsp90 to Regulate Glucocorticoid Receptor Levels

Abstract Ubiquitin specific protease 19 (USP19) is a deubiquitinating enzyme involved in metabolism. Its expression is upregulated in skeletal muscle in many conditions of muscle wasting. Genetic inactivation of USP19 (USP19KO) in mice protects from muscle atrophy and diet induced obesity and insulin resistance. We previously reported that the protection against muscle atrophy in USP19KO is due at least in part to reduction in muscle glucocorticoid receptor (GR) levels and signaling. Here, we report that USP19KO mice show > 50% decrease in GR protein level in multiple tissues with no observed changes in GR mRNA levels. Overexpression of the endoplasmic reticulum or cytosol-localized isoforms of USP19 in cells resulted in increased GR protein levels, while knockdown by siRNA or knockout by CRISPR-Cas9 resulted in decreased GR protein levels. Intriguingly, GR was not more ubiquitinated in the absence of USP19. Furthermore, expression of a catalytically inactive form of USP19 also increased GR protein levels and exposure of cells to a small molecule inhibitor of USP19 enzymatic activity had no effects on levels of GR. Deletion analysis revealed that the N-terminal CS domains of USP19, which are structurally similar to the p23 co-chaperone of HSP90, were required for increasing levels of GR. Since HSP90 is known to regulate GR folding and stability, this suggested that USP19 may function as a p23 like co-chaperone. Indeed, overexpression of USP19 not only increased GR levels, but translocation to the nucleus and transcriptional activity of a reporter gene. Using a novel BRET assay, we observed that USP19 interacts indirectly with GR through binding with HSP90. This interaction with HSP90 is critically dependent on the CS2 domain of USP19 and can be competed by expression of the p23 co-chaperone of HSP90. We have therefore identified a novel co-chaperone-like mechanism through which USP19 regulates GR protein stability independent of its catalytic activity. Targeting the interaction of USP19 with the GR-HSP90 complex could be a strategy to decrease GR levels, reduce glucocorticoid signalling and prevent muscle atrophy. Presentation: Monday, June 13, 2022 11:30 a.m. - 11:45 a.m.

Mutual antagonism between aryl hydrocarbon receptor and hypoxia-inducible factor-1α (AhR/HIF-1α) signaling: Impact on the aging process

The ambient oxygen level, many environmental toxins, and the rays of ultraviolet light (UV) provide a significant risk for the maintenance of organismal homeostasis. The aryl hydrocarbon receptors (AhR) represent a complex sensor system not only for environmental toxins and UV radiation but also for many endogenous ligands, e.g., L-tryptophan metabolites. The AhR signaling system is evolutionarily conserved and AhR homologs existed as many as 600 million years ago. The ancient atmosphere demanded the evolution of an oxygen-sensing system, i.e., hypoxia-inducible transcription factors (HIF) and their prolyl hydroxylase regulators (PHD). Given that both signaling systems have important roles in embryogenesis, it seems that they have been involved in the evolution of multicellular organisms. The evolutionary origin of the aging process is unknown although it is most likely associated with the evolution of multicellularity. Intriguingly, there is compelling evidence that while HIF-1α signaling extends the lifespan, that of AhR promotes many age-related degenerative processes, e.g., it increases oxidative stress, inhibits autophagy, promotes cellular senescence, and aggravates extracellular matrix degeneration. In contrast, HIF-1α signaling stimulates autophagy, inhibits cellular senescence, and enhances cell proliferation. Interestingly, there is a clear antagonism between the AhR and HIF-1α signaling pathways. For instance, (i) AhR and HIF-1α factors heterodimerize with the same factor, ARNT/HIF-1β, leading to their competition for DNA-binding, (ii) AhR and HIF-1α signaling exert antagonistic effects on autophagy, and (iii) co-chaperone p23 exhibits specific functions in the signaling of AhR and HIF-1α factors. One might speculate that it is the competition between the AhR and HIF-1α signaling pathways that is a driving force in the aging process.

Hsp multichaperone complex buffers pathologically modified Tau

Alzheimer’s disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau’s aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.

The switch from client holding to folding in the Hsp70/Hsp90 chaperone machineries is regulated by a direct interplay between co-chaperones

Folding of stringent clients requires transfer from Hsp70 to Hsp90. The co-chaperone Hop physically connects the chaperone machineries. Here, we define its role from the remodeling of Hsp70/40-client complexes to the mechanism of client transfer and the conformational switching from stalled to active client-processing states of Hsp90. We show that Hsp70 together with Hsp40 completely unfold a stringent client, the glucocorticoid receptor ligand-binding domain (GR-LBD) in large assemblies. Hop remodels these for efficient transfer onto Hsp90. As p23 enters, Hsp70 leaves the complex via switching between binding sites in Hop. Current concepts assume that to proceed to client folding, Hop dissociates and the co-chaperone p23 stabilizes the Hsp90 closed state. In contrast, we show that p23 functionally interacts with Hop, relieves the stalling Hsp90-Hop interaction, and closes Hsp90. This reaction allows folding of the client and is thus the key regulatory step for the progression of the chaperone cycle.

Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.

Cyclophilin A is a mitochondrial factor that forms complexes with p23 - correlative evidence for an anti-apoptotic action.

Cyclophilin A (CyPA, also known as PPIA) is an abundant and ubiquitously expressed protein belonging to the immunophilin family, which has intrinsic peptidyl-prolyl-(cis/trans)-isomerase enzymatic activity. CyPA mediates immunosuppressive action of the cyclic undecapeptide cyclosporine A and is also involved in multiple cellular processes, such as protein folding, intracellular trafficking, signal transduction and transcriptional regulation. CyPA is abundantly expressed in cancer cells, and, owing to its chaperone nature, its expression is induced upon the onset of stress. In this study, we demonstrated that a significant pool of this immunophilin is primarily an intramitochondrial factor that migrates to the nucleus when cells are stimulated with stressors. CyPA shows anti-apoptotic action per se and the capability of forming ternary complexes with cytochrome c and the small acidic co-chaperone p23, the latter interaction being independent of the usual association of p23 with the heat-shock protein of 90 kDa, Hsp90. These CyPA•p23 complexes enhance the anti-apoptotic response of the cell, suggesting that both proteins form a functional unit, the high level of expression of which plays a significant role in cell survival.

Simulation Study of the Stability of Heat Shock Protein 90 (HSP90) and Co-chaperone p23 Complex

Simulation Study of the Stability of Heat Shock Protein 90 (HSP90) and Co-chaperone p23 Complex

Casein kinase 1.2 over expression restores stress resistance to Leishmania donovani HSP23 null mutants

Leishmania donovani is a trypanosomatidic parasite and causes the lethal kala-azar fever, a neglected tropical disease. The Trypanosomatida are devoid of transcriptional gene regulation and rely on gene copy number variations and translational control for their adaption to changing conditions. To survive at mammalian tissue temperatures, L. donovani relies on the small heat shock protein HSP23, the loss of which renders the parasites stress sensitive and impairs their proliferation. Here, we analysed a spontaneous escape mutant with wild type-like in vitro growth. Further selection of this escape strains resulted in a complete reversion of the phenotype. Whole genome sequencing revealed a correlation between stress tolerance and the massive amplification of a six-gene cluster on chromosome 35, with further analysis showing over expression of the casein kinase 1.2 gene as responsible. In vitro phosphorylation experiments established both HSP23 and the related P23 co-chaperone as substrates and modulators of casein kinase 1.2, providing evidence for another crucial link between chaperones and signal transduction protein kinases in this early branching eukaryote.

Glucocorticoid receptor complexes form cooperatively with the Hsp90 co-chaperones Pp5 and FKBPs

The function of steroid receptors in the cell depends on the chaperone machinery of Hsp90, as Hsp90 primes steroid receptors for hormone binding and transcriptional activation. Several conserved proteins are known to additionally participate in receptor chaperone assemblies, but the regulation of the process is not understood in detail. Also, it is unknown to what extent the contribution of these cofactors is conserved in other eukaryotes. We here examine the reconstituted C. elegans and human chaperone assemblies. We find that the nematode phosphatase PPH-5 and the prolyl isomerase FKB-6 facilitate the formation of glucocorticoid receptor (GR) complexes with Hsp90. Within these complexes, Hsp90 can perform its closing reaction more efficiently. By combining chemical crosslinking and mass spectrometry, we define contact sites within these assemblies. Compared to the nematode Hsp90 system, the human system shows less cooperative client interaction and a stricter requirement for the co-chaperone p23 to complete the closing reaction of GR·Hsp90·Pp5/Fkbp51/Fkbp52 complexes. In both systems, hormone binding to GR is accelerated by Hsp90 alone and in the presence of its cofactors. Our results show that cooperative complex formation and hormone binding patterns are, in many aspects, conserved between the nematode and human systems.

Identification of the Hsp90 Isoforms and Co‐Chaperones that Repress Opioid Anti‐Nociception in the Spinal Cord

The various isoforms of the chaperone protein heat shock protein 90 (Hsp90) in conjunction with specific co‐chaperones are key regulators of signal transduction. We have recently identified a novel role for Hsp90 within downstream mu opioid receptor (MOR) signaling both within the brain and spinal cord. Within the brain, we found that Hsp90α and the co‐chaperones Cdc37 and p23 promote MOR‐induced ERK phosphorylation and subsequent anti‐nociception. However, in the spinal cord Hsp90 represses MOR‐induced ERK phosphorylation and subsequent anti‐nociception, so that Hsp90 inhibitor treatment in the spinal cord results in enhanced opioid pain relief. This study aimed to identify the Hsp90 isoforms and co‐chaperones necessary for this mechanism within the spinal cord, and whether they differ from the brain. We used novel isoform and co‐chaperone selective small molecule inhibitors and in vivo gene knockdown using CRISPR/Cas9 to selectively block each of the Hsp90 isoforms and co‐chaperones in the spinal cords of male and female CD‐1 mice. We then used opioid‐induced anti‐nociception in the tail flick assay as a readout, combined with immunohistochemistry to validate CRISPR/Cas9 knockdown in the spinal cord. In contrast to our earlier brain studies, we found that the isoforms Hsp90α, Hsp90β, and Grp94 all acted to repress opioid anti‐nociception in the spinal cord, in that inhibiting these isoforms resulted in increased opioid anti‐nociception. We similarly found that the co‐chaperones Cdc37, p23, and Aha1 all similarly repressed opioid anti‐nociception in the spinal cord. These findings suggest that Hsp90β, Grp94, and Aha1 all act within the spinal cord but not the brain to regulate opioid anti‐nociception. These findings also suggest, in line with our earlier studies, that blocking these isoforms with systemic selective inhibitors could represent a novel therapeutic approach to enhance opioid therapy, increasing analgesic efficacy while reducing side effects like tolerance and reward/addiction.Support or Funding InformationThese studies were supported by an Arizona Biomedical Research Commission New Investigator Award (#ADHS18‐198875) and institutional funds from the University of Arizona to JMS, and NIH R01CA213566 to BB. BB is a founder with an equity stake in Grannus Therapeutics, a virtual startup for developing novel Hsp90 inhibitors. No other author has any relevant conflicts to declare.

Anticancer properties and mechanism of action of the quassinoid ailanthone.

Ailanthone (AIT) is a quassinoid natural product isolated from the worldwide-distributed plant Ailanthus altissima. The drug displays multiple pharmacological properties, in particular significant antitumor effects against a variety of cancer cell lines in vitro. Potent in vivo activities have been evidenced in mice bearing hepatocellular carcinoma, nonsmall cell lung cancer and castration-resistant prostate cancer. This review focusses on the mechanism of action of AIT, notably to highlight the capacity of the drug to activate DNA damage responses, to inhibit the Hsp90 co-chaperone p23 and to modulate the expression of several microRNA. The interconnexion between these effects is discussed. The unique capacity of AIT to downregulate oncogenic miR-21 and to upregulate the tumor suppressor miRNAs miR-126, miR-148a, miR-195, and miR-449a is presented. AIT exploits several microRNAs to exert its anticancer effects in distinct tumor types. AIT is one of the rare antitumor natural products that binds to and strongly inhibits cochaperone p23, opening interesting perspectives to treat cancers. However, the toxicity profile of the molecule may limit its development as an anticancer drug, unless it can be properly formulated to prevent AIT-induced gastro-intestinal damages in particular. The antitumor properties of AIT and analogs are underlined, with the aim to encourage further pharmacological studies with this underexplored natural product and related quassinoids. HIGHLIGHTS: Ailanthone (AIT) is an anticancer quassinoid isolated from Ailanthus altissima It inhibits proliferation and induces cell death of many cancer cell types The drug activates DNA damage response and targets p23 cochaperone Up or downregulation of several microRNA by AIT contributes to the anticancer activity Analogs or specific formulations must be developed to prevent the toxicity of AIT.

A cytosolic heat shock protein 90 and co-chaperone p23 complex activates RIPK3/MLKL during necroptosis of endothelial cells in acute respiratory distress syndrome

Necrosis with inflammation plays a crucial role in acute respiratory distress syndrome (ARDS). Receptor-interacting protein 3 (RIPK3) regulates a newly discovered programmed form of necrosis called necroptosis. However, the underlying mechanism of necroptosis in ARDS remains unknown. Thus, the purpose of this study was to examine the possible involvement of RIPK3 in ARDS-associated necroptosis. RIPK3 protein levels were found to be significantly elevated in the plasma and bronchoalveolar lavage fluid of ARDS patients. Next, we utilised a mouse model of severe ARDS induced with high-dose lipopolysaccharide and found that lung injury was mainly due to RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL)-mediated necroptosis and endothelial dysfunction. The activation of RIPK3-MLKL by tumour necrosis factor receptor 1 (TNFR1) and TNFR1-associated death domain protein (TRADD) required catalytically active RIPK1 and the inhibition of Fas-associated protein with death domain (FADD)/caspase-8 catalytic activity. We further showed that the molecular chaperone heat shock protein 90 (Hsp90)/p23, as a novel RIPK3- and MLKL-interacting complex, played an important role in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction in the pulmonary vasculature, which resulted in ARDS. Collectively, the results of our study indicate that necroptosis is an important mechanism of cell death in ARDS and the inhibition of necroptosis may be a therapeutic intervention for ARDS. KEY MESSAGES: Lung injury in high-dose LPS-induced severe ARDS is mainly due to RIP3-MLKL-mediated necroptosis and endothelial dysfunction. Chaperone HSP90/p23 is a novel RIP3- and MLKL-interacting complex in HPAECs. HSP90/p23 is a novel RIP3- and MLKL-interacting complex in RIP-MLKL-mediated necroptosis, inflammation and endothelial dysfunction.

The Alpha Isoform of Heat Shock Protein 90 and the Co-chaperones p23 and Cdc37 Promote Opioid Anti-nociception in the Brain.

Opioid activation of the mu opioid receptor (MOR) promotes signaling cascades that evoke both analgesic responses to pain and side effects like addiction and dependence. Manipulation of these cascades, such as by biased agonism, has great promise to improve opioid therapy. However, the signaling cascades of the MOR are in general poorly understood, providing few targets for drug development. In our earlier work, we identified Heat shock protein 90 (Hsp90) as a novel and crucial regulator of opioid anti-nociception in the brain by promoting ERK MAPK activation. In this study, we sought to identify the molecular isoforms and co-chaperones by which Hsp90 carried out this role, which could provide specific targets for future clinical intervention. We used novel selective small molecule inhibitors as well as CRISPR/Cas9 gene editing constructs delivered by the intracerebroventricular (icv) route to the brains of adult CD-1 mice to target Hsp90 isoforms (Hsp90α/β, Grp94) and co-chaperones (p23, Cdc37, Aha1). We found that inhibition of the isoform Hsp90α fully blocked morphine anti-nociception in a model of post-surgical paw incision pain, while blocking ERK and JNK MAPK activation, suggesting Hsp90α as the main regulator of opioid response in the brain. We further found that inhibition of the co-chaperones p23 and Cdc37 blocked morphine anti-nociception, suggesting that these co-chaperones assist Hsp90α in promoting opioid anti-nociception. Lastly, we used cycloheximide treatment in the brain to demonstrate that rapid protein translation within 30 min of opioid treatment is required for Hsp90 regulation of opioid response. Together these studies provide insight into the molecular mechanisms by which Hsp90 promotes opioid anti-nociception. These findings thus both improve our basic science knowledge of MOR signal transduction and could provide future targets for clinical intervention to improve opioid therapy.