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The rational installation of pharmacophores targeting HSP90 and LSD1 axes has achieved significant anti-cancer capacity in prostate and colorectal cancer. Among the series of hybrids, inhibitor 6 exhibited remarkable anti-proliferative activity against prostate cancer cell lines PC-3 and DU145, with GI 50 values of 0.24 and 0.30 μM, respectively. It demonstrated notable efficacy in combinatorial attack and cell death initiation towards apoptosis. The cell death process was mediated by PARP induction and γH2AX signaling, and was also characterized as caspase-dependent and Bcl-xL/Bax-independent. Notably, no difference in eye size or morphology was observed in the zebrafish treated with compound 6 compared to the reference group (AUY922). The profound treatment response in docetaxel-resistant PC-3 cells highlighted the dual inhibitory ability in improving docetaxel sensitivity. Additionally, at a minimum concentration of 1.25 μM, compound 6 effectively inhibited the growth of patient-derived colorectal cancer (CRC) organoids for up to 10 days in vitro. Together, the designed HSP90/LSD1 inhibitors present a novel route and significant clinical value for anti-cancer drug therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jing-Ping Liou reports financial support was provided by National Science and Technology, Taiwan. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

The correction of protein folding is fundamental for cellular functionality and its failure can lead to severe diseases. In this context, molecular chaperones are crucial players involved in the tricky process of assisting in protein folding, stabilization, and degradation. Chaperones, such as heat shock proteins (HSP) 90, 70, and 60, operate within complex systems, interacting with co-chaperones both to prevent protein misfolding and direct to the correct folding. Chaperone targeting drugs could represent a challenging approach for the treatment of cystic fibrosis (CF), an autosomal recessive genetic disease caused by mutations in the CFTR gene, encoding for the CFTR chloride channel. In this review, we discuss the potential role of molecular chaperones as proteostasis modulators affecting CFTR biogenesis. In particular, we focused on HSP90 and HSP70, for their key role in CFTR folding and trafficking, as well as on HSP60 for its involvement in the inflammation process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Heat shock protein 90 (Hsp90), a molecular chaperone, contributes to the preservation of folding, structure, stability, and function proteins. In this study, novel compounds comprising isoxazole structure were designed, synthesized and their potential ability as Hsp90 inhibitors was validated through docking studies. The active site-based compounds were prepared through a multi-step synthesis process and their chemical structures were characterized employing FT-IR, NMR, and mass spectrometry analysis. Cytotoxic and Hsp90 inhibition activities of synthesized compounds were assessed by MTT assay and ELISA kit, respectively. Based on the obtained results, compound 5 exhibited the highest cytotoxicity (IC 50 ; 14 µM) against cancer cells and reduced Hsp90 expression from 5.54 ng/mL in untreated (normal cells) to 1.56 ng/mL in cancer cells. Moreover, molecular dynamics (MD) simulation results indicated its high affinity to target protein and approved its excellent stability which is essential for exerting an inhibitory effect on cancer cell proliferation., Competing Interests: Declarations Competing interests The authors declare no competing interests. Institutional review board statement The project was found to be in accordance to the ethical principle and national norms and standards for conducting medical research in Iran (Approval ID IR.MUI.RESEARCH.REC.1398.425 and Approval date 2019-11-02)., (© 2024. The Author(s).)

Pediatric burn injuries are a leading cause of morbidity with infections being the most common acute complication. Thermal injuries elicit a heightened cytokine response while suppressing immune function; however, the mechanisms leading to this dysfunction are still unknown. Our aim was to identify extracellular proteins and circulating phosphoprotein expression in the plasma after burn injury to predict the development of nosocomial infection (NI). Plasma was collected within 72 hours after injury from 64 pediatric burn subjects; of these, 18 went on to develop an NI. Extracellular damage-associated molecular proteins, FAS(APO), and protein kinase b (AKT) signaling phosphoproteins were analyzed. Subjects who went on to develop an NI had elevated high-mobility group box 1, heat shock protein 90 (HSP90), and FAS expression than those who did not develop an NI after injury (NoNI). Concurrently, phosphorylated (p-)AKT and mammalian target of rapamycin (p-mTOR) were elevated in those subjects who went on to develop an NI. Quadratic discriminant analysis revealed distinct differential profiles between NI and NoNI burn subjects using HSP90, FAS, and p-mTOR. The area under the receiver-operator characteristic curves displayed significant ability to distinguish between these 2 burn subject cohorts. These findings provide insight into predicting the signaling proteins involved in the development of NI in pediatric burn patients. Further, these proteins show promise as a diagnostic tool for pediatric burn patients at risk of developing infection while additional investigation may lead to potential therapeutics to prevent NI., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Burn Association. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCDC134, an ER luminal protein. During its translocation into the ER, an N-terminal peptide in HSP90B1 templates the assembly of a translocon complex containing CCDC134 and OST-A that protects HSP90B1 during folding, preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT and IGF1R signaling and causes the bone developmental disorder osteogenesis imperfecta. Thus, N-glycosylation can be regulated by specificity factors in the ER to control cell surface receptor signaling and tissue development.

The central role of the chaperome in maintaining cellular proteostasis has seen numerous viral families evolve to parasitically exploit host chaperones in their life cycle. The HSP90 chaperone protein and its cochaperone Hop have both individually been shown to be essential factors for Kaposi sarcoma-associated herpesvirus (KSHV) lytic replication. Given the fundamental regulatory role that protein-protein interactions (PPIs) play in cellular biology, we reasoned that disrupting the Hop-HSP90 PPI may provide a new host-based target for inhibiting KSHV lytic replication. This study expands upon a previous report of non-natural peptides, which were found to disrupt the association between the Hop TPR2A domain and its interacting HSP90 CTD . Here, in addition to providing insight into the structure-activity relationships of PPI inhibition, we show disruption of the full-length Hop-HSP90 PPI. The inhibitory peptides selectively engaged the Hop TPR2A domain in cell lysates and when tethered to a cell-penetrating peptide acted as noncytotoxic inhibitors of KSHV lytic replication by lowering the viral load, preventing the production of infectious virions, and reducing the expression of KSHV lytic genes. In addition to tentative evidence of Hop-HSP90 PPI as a much-needed target for KSHV drug discovery, this study represents an important step in understanding viral interactions with the host proteostasis machinery.

Heat shock protein 90 alpha (Hsp90α) is an abundantly expressed and evolutionarily conserved molecular chaperone. Hsp90α is the inducible Hsp90 isoform, and its expression and secretion extracellularly (eHsp90α) can be triggered in response to a variety of cellular stresses to protect/activate client proteins and to facilitate cellular adjustment to the stress. As a result, cancers often have high expression levels of intracellular and extracellular (plasma) Hsp90α, allowing them to support their oncogenesis and progression. In fact, (e)Hsp90α has been implicated in regulating processes such as cell signaling transduction, DNA repair, promotion of the Epithelial-to-Mesenchymal Transition (EMT), promotion of angiogenesis, immune response, and cell migration. Hsp90α levels have been correlated with cancer progression and severity in several cancers, indicating that it may be a useful biomarker or drug-target for cancer. To date, the development of intracellular Hsp90α-targeted therapies include standard N-terminal ATP-competitive inhibitors and allosteric regulators that bind to Hsp90α's middle or C-terminal domain. On-target toxicities and dosing complications as a result of Hsp90α inhibition has driven the development of eHsp90α-targeted therapies. Examples include anti-Hsp90α monoclonal antibodies and cell-impermeable Hsp90α small molecule inhibitors. This review aims to discuss the many roles Hsp90α plays in cancer progression with a focus on the current development of Hsp90α-targeted therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Lytic induction therapy was devised to selectively combat malignancies associated with Epstein-Barr virus (EBV) by triggering viral reactivation from latency. At present, the major challenges of lytic induction therapy are to maximize reactivating efficiencies and meanwhile minimize infectious virion production. C210, a novel curcumin derivative with potent Hsp90 inhibitory activity, was explored for EBV-reactivating and virion-producing effects in EBV-positive nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC) cell lines. And the molecular mechanisms underlying these effects were determined. Follow C210 treatment, EBV lytic RNAs and proteins were upregulated, but infectious virions were not produced. Knockdown of heat shock protein 90 (Hsp90) induced expression of lytic RNAs and proteins, and diminished C210-driven EBV lytic induction. Pretreatment with an X box binding protein 1 (XBP1) inhibitor reduced C210-induced EBV lytic RNA. Furthermore, we demonstrated that C210 inhibited the binding of Hsp90 with its clients, signal transducer and activator of transcription 3 (STAT3) and xeroderma pigmentosum group B-complementing protein (XPB), which subsequently promoted their proteasomal degradation. Degradation of STAT3 by C210 enhanced the EBV-reactivating and anticancer capacity of suberoylanilide hydroxamic acid (SAHA). Depletion of XPB blocked SAHA-induced expression of late viral genes and production of infectious virions. These results elucidate a novel Hsp90 inhibitor targeting EBV lytic phase and extend the research on lytic induction strategy, which may offer reference value in the treatment of EBV-positive malignancies., (© 2024. The Author(s).)

Retinal ganglion cells (RGCs) are among the first neurons to undergo apoptosis in diabetic retinopathy (DR), with their relationship to endoplasmic reticulum stress (ERS)-induced apoptosis still unclear. While glucocorticoid-induced transcript 1 (GLCCI1) has been shown to inhibit apoptosis, its role in ERS-induced apoptosis and its mechanisms in DR remain unclarified. Our findings indicated that GLCCI1 is predominantly localized in the ganglion cell layer and is downregulated in DR. GLCCI1 overexpression mitigated the apoptosis of RGCs and the swelling of endoplasmic reticulum and mitochondria under hyperglycemia, and downregulated ERS-induced apoptosis related markers (GRP78, CHOP and cleaved CASP3), whereas GLCCI1 knockdown has the opposite effect. In vivo, GLCCI1 overexpression not only prevents structural lesions but also protects against microvascular dysfunctions in the retinas of DR mice. We found that GLCCI1 directly interacts with HSP90AB1, which in turn interacts with GRP78. Additionally, GLCCI1 is an upstream regulator of HSP90AB1, which regulates GRP78. Thus, the impact of GLCCI1 on the ERS-induced apoptosis is mainly through the regulation of HSP90AB1, and subsequently inhibiting GRP78-initiated ERS-induced apoptosis. These findings offer a promising avenue for further treatment of DR., (© 2024. The Author(s).)

Heat shock proteins (HSPs) are induced in response to stressful stimuli and play an important role in cell repair and protection. This study, using immunohistochemistry, aimed to determine whether HSPs are induced in the cerebellum of rats subjected to hyperthermia during postnatal development (PND). The results showed that unlike HSP27 and HSP70, HSP60 and HSP90 were constitutively expressed in the cerebellum of rats. However, hyperthermia induced HSP27 in the white matter (WM) and HSP70 in the Bergmann glial cells, the internal granule layer (IGL), and the WM. In the WM, HSP27 induction was only observed on days PND20, PND25, and PND30, and HSP27 expression was higher on day PND30 compared with days PND20 and PND25 (p < 0.001). In the Bergmann glial cells, HSP70 induction was only observed on days PND5, PND10, and PND20, and HSP70 expression was greater on days PND5 and PND10 compared with day PND20 (p < 0.001). In the IGL and the WM, HSP70 expression was higher on days PND20, PND25, and PND30 compared with days PND5 and PND10 (p < 0.001). These findings indicate that unlike HSP60 and HSP90, HSP27 and HSP70 have different expression patterns in the cerebellum of rats after hyperthermia during PND., Competing Interests: Conflict of Interest: There is no conflict of interest between the authors. All authors have read and approved this article., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Microscopy Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hemipteran insects transmit viruses when infesting plants, during which vectors activate salicylic acid (SA)-regulated antiviral defenses. How vector-borne plant viruses circumvent these antiviral defenses is largely unexplored. During co-infections of begomoviruses and betasatellites in plants, betasatellite-encoded βC1 proteins interfere with SA signaling and reduce the activation of antiviral resistance. βC1 inhibits SA-induced degradation of NbNPR3 (Nicotiana benthamiana nonexpressor of pathogenesis-related genes 3), a negative regulator of SA signaling. βC1 does not bind directly to NbNPR3, but regulates NbNPR3 degradation via heat shock protein 90s (NbHSP90s). NbHSP90s bind to both NbNPR3 and βC1 and suppress SA signaling. This viral success strategy appears to be conserved as it is also documented for viral proteins encoded by two aphid-borne viruses. Our findings reveal an exquisite mechanism that facilitates the persistence of vector-borne plant viruses and provide important insights into the intricacies of the virus life cycle., (© 2024. The Author(s).)

Given the unclear, complex pathogenesis of neuropathic pain and the potential of paeoniflorin in relieving neuropathic pain, this study aimed to further clarify the therapeutic effect of paeoniflorin on neuropathic pain and to preliminarily explore the possible protective mechanisms of paeoniflorin. Chronic constrictive injury-induced Sprague Dawley rats and lipopolysaccharide-induced BV-2 cells were used for in vivo and in vitro experiments, respectively. The exosome uptake assay of mouse astrocytes (PKH-67 fluorescent labeling) and the mechanical nociceptive assay (the von Frey fibrous filaments) were performed. The effects of paeoniflorin and its downstream mechanisms on microglial and astrocyte activation, inflammation-associated proteins and exosome marker were determined. Paeoniflorin alleviated mechanical abnormal pain, decreased levels of ionized calcium binding adapter molecule-1 (Iba-1), glial fibrillary acidic protein, Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1, inflammatory factor) and High Mobility Group Box 1 (HMGB1, inflammation-related protein), and inhibited neuronal apoptosis in chronic constrictive injury rats or lipopolysaccharide-induced BV-2 cells. However, these effects were offset by HSP90AA1 overexpression in lipopolysaccharide-induced BV-2 cells. Exosomes of BV-2 cells could be absorbed by mouse astrocytes. In addition, HSP90AA1 overexpression reversed the effects of paeoniflorin on HMGB1 expression and inflammatory factors and proteins in mouse astrocytes co-cultured with exosome. Collectively, paeoniflorin alleviates neuropathic pain and inhibits inflammatory responses in chronic constrictive injury by modulating microglia-astrocyte crosstalk through HSP90AA1/HMGB1 pathways, which further evidences the potential of paeoniflorin in the treatment of neuropathic pain., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 Elsevier Ltd. All rights reserved.)

There is increasing evidence of mitochondrial dysfunction in autism spectrum disorders (ASD), but the causal relationships are unclear. In an ASD patient whose identical twin was unaffected, we identified a postzygotic mosaic mutation p.Q639* in the TRAP1 gene, which encodes a mitochondrial chaperone of the HSP90 family. Additional screening of 176 unrelated ASD probands revealed an identical TRAP1 variant in a male patient who had inherited it from a healthy mother. Notably, newly generated knock-in Trap1 p.Q641* mice display ASD-related behavioral abnormalities that are more pronounced in males than in females. Accordingly, Trap1 p.Q641* mutation also resulted in sex-specific changes in synaptic plasticity, the number of presynaptic mitochondria, and mitochondrial respiration. Thus, the TRAP1 p.Q639* mutation is the first example of a monogenic ASD caused by impaired mitochondrial protein homeostasis., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

Heat shock protein 90 (HSP90) has a recognized anti-heat stress injury effect, but its function and corresponding molecular mechanism in heat-stressed hepatocytes are not fully understood, especially in tropical animals. In the present study, we identified several key factors affecting resistance to injury liver tissues from heat-stressed Wenchang chickens (a typical tropical species), such as HSP90, cellular pyroptosis and mitophagy. Heat stress upregulated the NLRP3/Caspase-1/GSDMD-N-mediated cellular pyroptosis pathway and the Pink1/Parkin-mediated mitophagy pathway in chicken hepatocytes, accompanied by the upregulation of HSP90. We also found that HSP90 overexpression significantly reduced heat stress-induced hepatocyte pyroptosis and enhanced mitophagy in primary hepatocytes from Wenchang chickens (PHWCs). HSP90 knockdown significantly increased heat stress-induced hepatocyte pyroptosis and decreased mitophagy in PHWCs. Interestingly, we performed immunoprecipitation and immunofluorescence colocalization and found that HSP90 and Pink1 can interact and directly regulate the level of mitophagy in PHWCs. Our results suggest that HSP90, which regulates Pink1, is an important factor in mitophagy that attenuates heat stress injury by inhibiting cellular pyroptosis., Competing Interests: Declaration of competing interest The author(s) declared no potential conflicts of interest with respect to the research, author- ship, and/or publication of this article., (Copyright © 2024. Published by Elsevier B.V.)

Previously, we have shown that endothelial nitric-oxide synthase (eNOS) dimer levels directly correlate with the interaction of eNOS with hsp90 (heat shock protein 90). Further, the disruption of eNOS dimerization correlates with its redistribution to the mitochondria. However, the causal link between these events has yet to be investigated and was the focus of this study. Our data demonstrates that simvastatin, which decreases the mitochondrial redistribution of eNOS, increased eNOS-hsp90 interactions and enhanced eNOS dimerization in cultured pulmonary arterial endothelial cells (PAEC) from a lamb model of pulmonary hypertension (PH). Our data also show that the dimerization of a monomeric fraction of human recombinant eNOS was stimulated in the presence of hsp90 and ATP. The over-expression of a dominant negative mutant of hsp90 (DNHsp90) decreased eNOS dimer levels and enhanced its mitochondrial redistribution. We also found that the peroxynitrite donor3-morpholinosydnonimine (SIN-1) increased the mitochondrial redistribution of eNOS in PAEC and this was again associated with decreased eNOS dimer levels. Our data also show in COS-7 cells, the SIN-1 mediated mitochondrial redistribution of wildtype eNOS (WT-eNOS) is significantly higher than a dimer stable eNOS mutant protein (C94R/C99R-eNOS). Conversely, the mitochondrial redistribution of a monomeric eNOS mutant protein (C96A-eNOS) was enhanced. Finally, we linked the SIN-1-mediated mitochondrial redistribution of eNOS to the Akt1-mediated phosphorylation of eNOS at Serine(S) 617 and showed that the accessibility of this residue to phosphorylation is regulated by dimerization status. Thus, our data reveal a novel mechanism of pulmonary endothelial dysfunction mediated by mitochondrial redistribution of eNOS, regulated by dimerization status and the phosphorylation of S 617 ., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2024. Published by Elsevier Inc.)

Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is a principal mechanism for systemic glucose homeostasis, of which regulatory mechanisms are still unclear. Here we show that kinesin molecular motor KIF5B is essential for GSIS through maintaining the voltage-gated calcium channel Ca V 1.2 levels, by facilitating an Hsp70-to-Hsp90 chaperone exchange to pass through the quality control in the endoplasmic reticulum (ER). Phenotypic analyses of KIF5B conditional knockout (cKO) mouse beta cells revealed significant abolishment of glucose-stimulated calcium transients, which altered the behaviors of insulin granules via abnormally stabilized cortical F-actin. KIF5B and Hsp90 colocalize to microdroplets on ER sheets, where Ca V 1.2 but not K ir 6.2 is accumulated. In the absence of KIF5B, Ca V 1.2 fails to be transferred from Hsp70 to Hsp90 via STIP1, and is likely degraded via the proteasomal pathway. KIF5B and Hsc70 overexpression increased Ca V 1.2 expression via enhancing its chaperone binding. Thus, ER sheets may serve as the place of KIF5B- and Hsp90-dependent chaperone exchange, which predominantly facilitates Ca V 1.2 production in beta cells and properly enterprises GSIS against diabetes., (© 2024. The Author(s).)

This study investigated the effects of Bisphenol A (BPA), a common endocrine-disrupting chemical, on the antioxidant enzyme activities in the hepatopancreas and the expression of genes related to ovarian development in oriental river prawns (Macrobrachium nipponense). The 24hLC50 and 48hLC50 values for BPA were 80.59 mg/L and 63.90 mg/L, respectively, with a safe concentration of 12.06 mg/L. Prawns were exposed to low (4.85 mg/L), safe (12.06 mg/L), and high (30.00 mg/L) concentrations of BPA for 10 days to measure enzyme activities, and for 20 days followed by 7 days in BPA-free water to measure gene expression. Short-term exposure (12 h, 1d, 3d) to low concentration BPA did not significantly affect superoxide dismutase (SOD) activity in the hepatopancreas (P > 0.05), but long-term exposure (6d, 10d) significantly reduced SOD activity (P < 0.05). Catalase (CAT) activity showed no significant changes throughout the low concentration exposure period (P > 0.05). At safe and high concentrations, SOD and CAT activities significantly decreased after 12 h of exposure (P < 0.05). BPA affected heat shock protein 90 (HSP90) expression in the ovary, with low concentration BPA significantly upregulating HSP90 after 1 day (P < 0.05), but returning to normal levels after 10 and 20 days. At the safe concentration, HSP90 was significantly upregulated at all three sampling points (1d, 10d, 20d) (P < 0.05), while high concentration exposure led to significant upregulation only on day 10 (P < 0.05). Low concentration BPA had no significant effect on Cathepsin B (CB) and Cathepsin L (CL) gene expression in the ovaries (P > 0.05). However, safe concentration exposure promoted CB expression on days 1, 10, and 20 (P < 0.05), while high concentration exposure significantly increased CB expression on day 1 (P < 0.05), with levels returning to normal on days 10 and 20. CL expression significantly increased after 20 days of exposure to both safe and high concentrations (P < 0.05). Gene expression levels in the ovaries returned to normal after transfer to BPA-free water, with HSP90 and CB normalizing by day 1, and CL by day 7. These results indicate that even safe concentrations of BPA impose stress on the hepatopancreas and increase the expression of HSP90, CB, and CL genes in the ovaries, affecting ovarian development. And, these effects are reversible within a certain period after the removal of BPA., Competing Interests: Declaration of competing interest The authors declare that there are no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study., (Copyright © 2024. Published by Elsevier B.V.)

The heat shock protein 90 (HSP90) family members are not only widely involved in animal cellular immune response and signal transduction pathway regulation, but also play an important role in plant development and environmental stress response. Here,we identified a HSP90 family member in Ginkgo biloba, designated as GbHSP90, which performs a dual functional role to regulate telomere stability. GbHSP90 was screened by a yeast one-hybrid library using the Ginkgo biloba telomeric DNA (TTTAGGG) 5 . Fluorescence polarization, surface plasmon resonance(SPR) and EMSA technologyies revealed a specific interaction between GbHSP90 and the double-stranded telomeric DNA via its N-CR region, with no affinity for the single-stranded telomeric DNA or human double-stranded telomeric DNA. Furthermore, yeast two-hybrid system and Split-LUC assay demonstrated that GbHSP90 can interacts with two telomere end-binding proteins:the ginkgo telomerase reverse transcriptase (GbTERT) and the ginkgo Structural Maintenance of Chromosomes protein 1 (GbSMC1). Overexpression of GbHSP90 in human 293 T and HeLa cells increased cell growth rate, the content of telomerase reverse transcriptase (TERT), and promote cell division and inhibit cell apoptosis. Our results indicated GbHSP90 have dually functions: as a telomere-binding protein that binds specifically to double-stranded telomeric DNA and as a molecular chaperone that modulates cell differentiation and apoptosis by binding to telomere protein complexes in Ginkgo biloba. This study contributes to a significantly understanding of the unique telomere complex structure and regulatory mechanisms in Ginkgo biloba, a long-lived tree species., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Doxorubicin (Dox) has been limited in clinical application due to its cardiac toxicity that varies with the dose. This study aimed to explore how Rhein modulates Dox-induced myocardial toxicity. The general condition and echocardiographic changes of mice were observed to evaluate cardiac function and structure, with myocardial cell injury and apoptosis checked by TUNEL and HE staining. The ELISA assessed markers of myocardial damage and inflammation. The TCMSP and SwissTargetPrediction databases were used to retrieve Rhein's targets while GeneCards was used to find genes related to Dox-induced myocardial injury. Intersection genes were analyzed by Protein-Protein Interaction Networks. The core network genes underwent GO and KEGG enrichment analysis using R software. Western blot was used to detect protein expression. Compared to the Dox group, there was no remarkable difference in heart mass /body mass ratio in the Rhein+Dox group. However, heart mass/tibia length increased. Mice in the Rhein+Dox group had significantly increased LVEF, LVPWs, and LVFS compared to those in the Dox group. Myocardial cell damage, inflammation, and apoptosis significantly reduced in the Rhein+Dox group compared to the model group. Eleven core network genes were selected. Further, Rhein+Dox group showed significantly downregulated expression of p38/p-p38, HSP90AA1, c-Jun/p-c-Jun, c-Fos/p-c-Fos, Bax, and cleaved-caspase-3/caspase-3 while Bcl-2 expression significantly upregulated compared to the Dox group. The study suggests that Rhein mediates cardioprotection against Dox-induced myocardial injury, at least partly, by influencing multiple core genes in the MAPK signaling pathway to inhibit myocardial cell apoptosis., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Five active compounds, daidzein, luteolin, alpinumisoflavone (AI), 6,8-diprenylgenistein (DG), and warangalone (WA), were identified from the fruits of Maclura tricuspidata via LC-Q/TOF-MS. WA and DG were shown to reverse the high glucose (HG)-induced injury in human umbilical vein endothelial cells (HUVECs), indicating their potential protective effects in alleviating diabetic symptoms. Network pharmacology was conducted to reveal the potential mechanisms of action of the compounds, and Hsp90α (degree: 47), Src (degree: 49), Akt (degree: 69) and p53 (degree: 60) were shown as the core targets related to antidiabetic properties. Further experimental verification suggested that the compounds could enhance phosphorylation of Src and Akt, increase p53 expression act as Hsp90 inhibitors, and protect against HG induced endothelial dysfunction. Our findings will provide a comprehensive understanding of the active substances of M. tricuspidata , which will be helpful for their utilisation.

Despite recent advances in treatment, non-small cell lung cancer (NSCLC) continues to have a high mortality rate. Currently, NSCLC pathogenesis requires further investigation, and therapeutic drugs are still under development. Homologous recombination repair (HRR) repairs severe DNA double-strand breaks. Homologous recombination repair deficiency (HRD) occurs when HRR is impaired and causes irreparable double-strand DNA damage, leading to genomic instability and increasing the risk of cancer development. Poly(ADP-ribose) polymerase (PARP) inhibitors can effectively treat HRD-positive tumors. Extracellular heat shock protein 90α (eHSP90α) is highly expressed in hypoxic environments and inhibits apoptosis, thereby increasing cellular tolerance. Here, we investigated the relationship between eHSP90α and HRR in NSCLC. DNA damage models were established in NSCLC cell lines (A549 and H1299). The activation of DNA damage and HRR markers, apoptosis, proliferation, and migration were investigated. In vivo tumor models were established using BALB/c nude mice and A549 cells. We found that human recombinant HSP90α stimulation further activated HRR and reduced DNA damage extent; however, eHSP90α monoclonal antibody, 1G6-D7, effectively inhibited HRR. HRR inhibition and increased apoptosis were observed after LRP1 knockdown; this effect could not be reversed with hrHSP90α addition. The combined use of 1G6-D7 and olaparib caused significant apoptosis and HRR inhibition in vitro and demonstrated promising anti-tumor effects in vivo. Extracellular HSP90α may be involved in HRR in NSCLC through LRP1. The combined use of 1G6-D7 and PARP inhibitors may exert anti-tumor effects by inhibiting DNA repair and further inducing apoptosis of NSCLC cells., (© 2024 Wiley Periodicals LLC.)

Heat shock protein 90 (HSP90) is recognized for its protective effects against heat stress damage; however, the specific functions and underlying molecular mechanisms of HSP90 in heat-stressed cardiomyocytes remain largely unexplored, particularly in tropical species. In our study, Wenchang chickens (WCCs) were classified into two groups: the heat stress survival (HSS) group and the heat stress death (HSD) group, based on their survival following exposure to heat stress. Heat stress resulted in significant cardiomyocyte damage, mitochondrial dysfunction, and apoptosis in the HSD group, while the damage was less pronounced in the HSS group. We further validated these findings in primary cardiomyocytes derived from Wenchang chickens (PCWs). Additionally, heat stress was found to upregulate Pink1/Parkin-mediated mitophagy, which was accompanied by an increase in HSP90 expression in both cardiomyocytes and PCWs. Our results demonstrated that HSP90 overexpression enhances PINK1/Parkin-mediated mitophagy, ultimately inhibiting apoptosis and oxidative stress in heat-stressed PCWs. However, the application of Geldanamycin (GA) reversed these effects. Notably, we discovered that HSP90 interacts with Beclin-1 through mitochondrial translocation and directly regulates mitophagy levels in PCWs. In summary, we have elucidated a novel role for HSP90 and mitophagy in regulating heat stress-induced acute cardiomyocyte injury.

Brain damage caused by acute hypoxia is associated with the physiological activities of mitochondria. Although mitochondria being dynamically regulated, our comprehensive understanding of the response of specific brain cell types to acute hypoxia remains ambiguous. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a mitochondrial-based molecular chaperone, plays a role in controlling mitochondrial movements. Herein, we demonstrated that acute hypoxia significantly alters mitochondria morphology and functionality in both in vivo and in vitro brain injury experiments. Summary-data-based Mendelian Randomization (SMR) analyses revealed possible causative links between mitochondria-related genes and hypoxia injury. Advancing the protein-protein interaction network and molecular docking further elucidated the associations between TRAP1 and mitochondrial dynamics. Furthermore, it was shown that TRAP1 knockdown levels variably affected the expression of key mitochondrial dynamics proteins (DRP1, FIS1, and MFN1/2) in primary hippocampal neurons, astrocytes, and BV-2 cell, leading to changes in mitochondrial structure and function. Understanding the function of TRAP1 in altering mitochondrial physiological activity during hypoxia-induced acute brain injury could help serve as a potential therapeutic target to mitigate neurological damage., (© 2024. The Author(s).)

Photothermal therapy (PTT) is a prospective therapeutic method for breast cancer. However, excess inflammatory response induced by PTT may aggravate tumor metastasis. Meanwhile, the overexpressed heat shock proteins (HSPs) by cancer cells can protect them from hyperthermia during PTT. Therefore, to attenuate the PTT-induced inflammation and inhibit tumor metastasis, a folate receptor-targeted thermo-sensitive liposome (BI-FA-LP) co-loading Berberine (BBR) and Indocyanine green (ICG) was developed. BI-FA-LP utilized enhanced permeability and retention (EPR) effect and FA receptor-mediated endocytosis to selectively accumulate at tumor, reducing off-target toxicity during the treatment. After targeting to the tumor site, BBR and ICG were released from BI-FA-LP upon laser irradiation, and ICG showed good photothermal performance, while BBR inhibited HSP70 and HSP90 expression during PTT, exerting chemo-photothermal synergetic anti-tumor effect. Moreover, BBR could suppress the PTT induced inflammation, thus inhibiting tumor metastasis and ameliorating tissue injury. Thus, this versatile liposome provided a new strategy to enhance PTT and anti-inflammatory effects for breast cancer treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Geldanamycin remains a driver in the medicinal chemistry of heat shock protein 90 (Hsp90) inhibition, even half a century after its original isolation from nature. This Perspective focuses on the properties of the benzoquinone ring of the natural product that enable a range of functionalization reactions to take place. Therefore, inherent reactivity at C-17, where the methoxy group serves as a vinylogous ester, and at C-19 that demonstrates nucleophilic, enamide-type character toward electrophiles, and also as a conjugate acceptor to react with nucleophiles, has facilitated the synthesis of semisynthetic derivatives. Thus, a range of C-17-substituted amine derivatives has been investigated in oncology applications, with a number of compounds in this series reaching clinical trials. In contrast, the 19-position of geldanamycin has received less attention, although 19-substituted derivatives offer promise with markedly reduced toxicity compared to geldanamycin itself, while retaining Hsp90 inhibitory activity albeit with diminished potency in cellular studies.

Lung cancer stands out as a leading cause of death among various cancer types, highlighting the urgent need for effective anticancer drugs and the discovery of new compounds with potent therapeutic properties. Natural sources, such as the Conamomum genus, offer various bioactive compounds. Adunctin E (AE), a dihydrochalcone derived from Conamomum rubidum , exhibited several pharmacological activities, and its potential as an anticancer agent remains largely unexplored. Thus, this study aimed to elucidate its apoptotic-inducing effect and identify its molecular targets. The network pharmacology analysis led to the identification of 71 potential targets of AE against lung cancer. Subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway enrichment analyses revealed the involvement of these targets in cancer-associated signaling pathways. Notably, HSP90AA1, MAPK1, and PIK3CA emerged as key players in apoptosis. In silico molecular docking and dynamic simulations suggested a strong and stable interaction between AE and HSP90AA1. In vitro experiments further confirmed a significant apoptotic-inducing effect of AE on lung cancer cell lines A549 and H460. Furthermore, immunoblot analysis exhibited a substantial decrease in HSP90AA1 levels in response to AE treatment. These findings support the potential anticancer activity of AE through the HSP90AA1 mechanism, underscoring its promise as a novel compound worthy of further research and development for anti-lung cancer therapy.

Polo-like kinase 2 (PLK2) is a serine/threonine protein kinase associated with the regulation of synaptic plasticity and centriole duplication. We identify PLK2 as a crucial early-response gene in lipopolysaccharide (LPS)-stimulated microglial cells. Knockdown or inhibition of PLK2 remarkably attenuates LPS-induced expression of proinflammatory factors in microglial cells by suppressing the inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ)-nuclear factor (NF)-κB signaling pathway. We identify heat shock protein 90 alpha (HSP90α), a regulator of IKKβ activity, as a novel PLK2 substrate. Knockdown or pharmacological inhibition of HSP90α abolishes PLK2-mediated activation of NF-κB transcriptional activity and microglial inflammatory activation. Furthermore, phosphoproteomic analysis pinpoints Ser252 and Ser263 on HSP90α as novel phosphorylation targets of PLK2. Lastly, conditional knockout of PLK2 in microglial cells dramatically ameliorates neuroinflammation and subsequent dopaminergic neuron loss in an intracranial LPS-induced mouse Parkinson's disease (PD) model. The present study reveals that PLK2 promotes microglial activation through the phosphorylation of HSP90α and subsequent activation of the IKKβ-NF-κB signaling pathway., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Nucleotide-binding leucine-rich repeat (NLR) proteins contribute widely to plant immunity by regulating defense mechanisms through the elicitation of a hypersensitive response (HR). Here, we find that TaRACK1B (the receptor for activated C-kinase 1B) regulates wheat immune response against Chinese wheat mosaic virus (CWMV) infection. TaRACK1B recruits TaSGT1 and TaHSP90 to form the TaRACK1B-TaSGT1-TaHSP90 complex. This complex is essential for maintaining NLR proteins' stability (TaRGA5-like and TaRGH1A-like) in order to control HR activation and inhibit viral infection. However, the cysteine-rich protein encoded by CWMV can disrupt TaRACK1B-TaSGT1-TaHSP90 complex formation, leading to the reduction of NLR-protein stability and suppression of HR activation, thus promoting CWMV infection. Interestingly, the 7K protein of wheat yellow mosaic virus also interferes with this antiviral immunity. Our findings show a shared viral counter-defense strategy whereby two soil-borne viruses may disrupt the TaRACK1B-TaSGT1-TaHSP90 complex, suppressing NLR-protein-mediated broad-spectrum antiviral immunity and promoting viral infection in wheat., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Geldanamycin, an ansa -macrolide composed of a rigid benzoquinone ring and an aliphatic ansa -bridge, was isolated from Streptomyces hygroscopicus . Geldanamycin is a potent heat shock protein inhibitor with remarkable antiproliferative activity. However, it shows pronounced hepatotoxicity in animal models and unfavorable pharmacokinetic properties. Four geldanamycin analogs have progressed through various phases of clinical trials, but none have yet completed clinical evaluation or received FDA approval. To enhance the efficacy of these Hsp90 inhibitors, strategies such as prodrug approaches or nanocarrier delivery systems could be employed to minimize systemic and organ toxicity. Furthermore, exploring new drug combinations may help overcome resistance, potentially improving therapeutic outcomes. This review discusses the mechanism of action of geldanamycin, its pharmacokinetic properties, and the various approaches employed to alleviate its toxicity and maximize its clinical efficacy. The main focus is on those derivatives that have progressed to clinical trials or that have shown important in vivo activity in preclinical models.

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90's interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine., (© 2024. The Author(s).)

Acute lymphoblastic leukemia is a challenging disease to treat, especially in older adults who are most commonly diagnosed and have a high risk of relapse, even with current treatment options. MST-312, targets the RNA component of telomerase, inhibiting its activity and leading to growth arrest and telomere shortening in cancer cells. This study aimed to investigate the effects of MST-312 on apoptosis rates and the expression of telomerase target genes, CCND1, MDM2, MYC, and HSP90AA1, in Jurkat cell line. Jurkat cell line was cultured and treated with various concentrations of MST-312(0 µM, 0.5 µM, 1 µM, 2 µM, and 4 µM). The optimal concentration of MST-312 was determined using MTT assay. Flow cytometry was employed to evaluate the apoptosis induced by MST-312 treatment. The expression levels of the target genes were measured using real-time polymerase chain reaction before and after the treatment with MST-312. P-value < 0.05 was considered statistically significant. The percentages of apoptotic cells after 48 h, as determined by flow cytometry analysis, were 30.32%, 52.35%, 57.60%, and 68.82%, respectively, compared to the control group which was 4.6%. The expression levels of all genes, including CCND1, MDM2, MYC, and HSP90AA1, were decreased compared to the control group. The results showed that MST-312 induced dose- and time-dependent apoptosis and downregulated the expression of CCND1, MDM2, MYC, and HSP90AA1in Jurkat cell line., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Aims: Vascular smooth muscle cell (VSMC) senescence is crucial for the development of atherosclerosis, characterized by metabolic abnormalities. Tumour necrosis factor receptor-associated protein 1 (TRAP1), a metabolic regulator associated with ageing, might be implicated in atherosclerosis. As the role of TRAP1 in atherosclerosis remains elusive, this study aimed to examine the function of TRAP1 in VSMC senescence and atherosclerosis., Methods: TRAP1 expression was measured in the aortic tissues of patients and mice with atherosclerosis using western blot and RT-qPCR. Senescent VSMC models were established by oncogenic Ras, and cellular senescence was evaluated by measuring senescence-associated β-galactosidase expression and other senescence markers. Chromatin immunoprecipitation (ChIP) analysis was performed to explore the potential role of TRAP1 in atherosclerosis., Results: VSMC-specific TRAP1 deficiency mitigated VSMC senescence and atherosclerosis via metabolic reprogramming. Mechanistically, TRAP1 significantly increased aerobic glycolysis, leading to elevated lactate production. Accumulated lactate promoted histone H4 lysine 12 lactylation (H4K12la) by down-regulating the unique histone lysine delactylase HDAC3. H4K12la was enriched in the senescence-associated secretory phenotype (SASP) promoter, activating SASP transcription and exacerbating VSMC senescence. In VSMC-specific Trap1 knockout ApoeKO mice (ApoeKOTrap1SMCKO), the plaque area, senescence markers, H4K12la, and SASP were reduced. Additionally, pharmacological inhibition and proteolysis-targeting chimera (PROTAC)-mediated TRAP1 degradation effectively attenuated atherosclerosis in vivo., Conclusions: This study reveals a novel mechanism by which mitonuclear communication orchestrates gene expression in VSMC senescence and atherosclerosis. TRAP1-mediated metabolic reprogramming increases lactate-dependent H4K12la via HDAC3, promoting SASP expression and offering a new therapeutic direction for VSMC senescence and atherosclerosis., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Heat shock protein 90 (Hsp90) is a promising target for cancer therapy and imaging. Accurate detection of Hsp90 levels in tumors via noninvasive PET imaging might be beneficial for management. To achieve this, the precursor compound Dimer-Sansalvamide A (Dimer-San A) was PEGylated and modified by conjugating it with the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). The 18 F-labeled PEGylated Dimer-SanA decapeptide ( 18 F-PEGylated San A) was completed within 30 min using a two-step process. In vitro stability and specificity were assessed, including competition studies with the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). MicroPET imaging was performed on PL45 tumor-bearing mice to evaluate probe accumulation and tumor-to-muscle ratios. Biodistribution studies determined the route of excretion. The probe resulted in a radiochemical yield of 23.11% with a purity exceeding 95%. In vitro , 18 F-PEGylated San A exhibited high stability and selectively accumulated in Hsp90-positive PL45 cells, with binding effectively blocked by the Hsp90 inhibitor 17AAG, confirming its specificity. MicroPET imaging of PL45 tumor-bearing mice showed significant probe accumulation in tumor tissues at 1 and 2 h postinjection (4.06 ± 0.30 and 3.72 ± 0.61%ID/g, respectively), with optimal tumor-to-muscle ratios observed at 2 h postinjection (6.09 ± 1.92). While 18 F-PEGylated San A demonstrates enhanced water solubility, as indicated by increased kidney uptake relative to liver accumulation. The study successfully incorporated PEG units to create the novel probe 18 F-PEGylated San A targeting to Hsp90 without affecting its targeting capability, aimed at improving the pharmacokinetics and PET imaging of Hsp90 expression noninvasively.

A series of indazole analogs, derived from the B,C-ring-truncated scaffold of deguelin, were designed to function as C-terminal inhibitors of heat shock protein 90 (HSP90) and investigated as novel antitumor agents against HER2-positive breast cancer. Among the synthesized compounds, compound 12d exhibited substantial inhibitory effects in trastuzumab-sensitive (BT474) and trastuzumab-resistant (JIMT-1) breast cancer cells, with IC 50 values of 6.86 and 4.42 μM, respectively. Notably, compound 12d exhibited no cytotoxicity in normal cells. Compound 12d markedly downregulated the expression of the major HSP90 client proteins in both cell types, attributing its cytotoxicity to the destabilization and inactivation of HSP90 client proteins. Molecular docking studies using the homology model of an HSP90 homodimer demonstrated that inhibitor 12d fit nicely into the C-terminal domain, boasting a higher electrostatic complementary score than ATP. In vivo pharmacokinetic study indicated the high oral bioavailability of compound 12 d at F = 66.9 %, while toxicological studies indicated its negligible impact on hERG channels and CYP isozymes. Genotoxicity tests further confirmed its safety profile. The findings collectively position compound 12d as a promising candidate for further development as an antitumor agent against HER2-positive breast cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Colorectal cancer (CRC) has been becoming one of the most common causes of cancer mortality worldwide. Accumulating studies suggest that the progressive up-regulation of Wnt/β-catenin signaling is a crucial hallmark of CRC, and suppressing it is a promising strategy to treat CRC. Herein, we reported our latest efforts in the discovery of novel fused tetrahydroisoquinoline derivatives with good anti-CRC activities by screening our in-house berberine-like library and further structure-activity relationship (SAR) studies, in which we identified compound 10 is a potent lead compound with significant antiproliferation potencies. By the biotinylated probe and LC-MS/MS study, Hsp90 was identified as its molecular target, which is a fully different mechanism of action from what we reported before. Further studies showed compound 10 directly engaged the N-terminal site of Hsp90 and promoted the degradation of β-catenin, thereby suppressing the Wnt/β-catenin signaling. More importantly, compound 10 exhibits favorable pharmacokinetic parameters and significant anti-tumor efficacies in the HCT116 xenograft model. Taken together, this study furnished the discovery of candidate drug compound 10 possessing a novel fused tetrahydroisoquinoline scaffold with excellent in vitro and in vivo anti-CRC activities by targeting Hsp90 to disturb Wnt/β-catenin signaling pathway, which lay a foundation for discovering more effective CRC-targeted therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Hsp90 is a molecular chaperone of central importance for protein homeostasis in the cytosol of eukaryotic cells, with key functional and structural traits conserved from yeast to man. During evolution, Hsp90 has gained additional functional importance, leading to an increased number of interacting co-chaperones and client proteins. Here, we show that the overall conformational transitions coupled to the ATPase cycle of Hsp90 are conserved from yeast to humans, but cycle timing as well as the dynamics are significantly altered. In contrast to yeast Hsp90, the human Hsp90 is characterized by broad ensembles of conformational states, irrespective of the absence or presence of ATP. The differences in the ATPase rate and conformational transitions between yeast and human Hsp90 are based on two residues in otherwise conserved structural elements that are involved in triggering structural changes in response to ATP binding. The exchange of these two mutations allows swapping of the ATPase rate and of the conformational transitions between human and yeast Hsp90. Our combined results show that Hsp90 evolved to a protein with increased conformational dynamics that populates ensembles of different states with strong preferences for the N-terminally open, client-accepting states., (© 2024. The Author(s).)

The calculation of absolute binding free energies (ABFEs) for protein-ligand systems has long been a challenge. Recently, refined force fields and algorithms have improved the quality of the ABFE calculations. However, achieving the level of accuracy required to inform drug discovery efforts remains difficult. Here, we present a transferable enhanced sampling strategy to accurately calculate absolute binding free energies using OneOPES with simple geometric collective variables. We tested the strategy on two protein targets, BRD4 and Hsp90, complexed with a total of 17 chemically diverse ligands, including both molecular fragments and drug-like molecules. Our results show that OneOPES accurately predicts protein-ligand binding affinities with a mean unsigned error within 1 kcal mol -1 of experimentally determined free energies, without the need to tailor the collective variables to each system. Furthermore, our strategy effectively samples different ligand binding modes and consistently matches the experimentally determined structures regardless of the initial protein-ligand configuration. Our results suggest that the proposed OneOPES strategy can be used to inform lead optimization campaigns in drug discovery and to study protein-ligand binding and unbinding mechanisms.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

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.

Metabolic rewiring promotes cancer cell adaptation to a hostile microenvironment, representing a hallmark of cancer. This process involves mitochondrial function and is mechanistically linked to the balance between mitochondrial biogenesis (MB) and mitophagy. The molecular chaperone TRAP1 is overexpressed in 60-70% of human colorectal cancers (CRC) and its over-expression correlates with poor clinical outcome, being associated with many cancer cell functions (i.e. adaptation to stress, protection from apoptosis and drug resistance, protein synthesis quality control, metabolic rewiring from glycolysis to mitochondrial respiration and vice versa). Here, the potential new role of TRAP1 in regulating mitochondrial dynamics was investigated in CRC cell lines and human CRCs. Our results revealed an inverse correlation between TRAP1 and mitochondrial-encoded respiratory chain proteins both at transcriptional and translational levels. Furthermore, TRAP1 silencing is associated with increased mitochondrial mass and mitochondrial DNA copy number (mtDNA-CN) as well as enhanced MB through PGC-1α/TFAM signalling pathway, promoting the formation of new functioning mitochondria and, likely, underlying the metabolic shift towards oxidative phosphorylation. These results suggest an involvement of TRAP1 in regulating MB process in human CRC cells. KEY MESSAGES: TRAP1 inversely correlates with protein-coding mitochondrial gene expression in CRC cells and tumours. TRAP1 silencing correlates with increased mitochondrial mass and mtDNA copy number in CRC cells. TRAP1 silencing favours mitochondrial biogenesis in CRC cells., (© 2024. The Author(s).)

Accurate prediction of protein-ligand binding affinities is an essential challenge in structure-based drug design. Despite recent advances in data-driven methods for affinity prediction, their accuracy is still limited, partially because they only take advantage of static crystal structures while the actual binding affinities are generally determined by the thermodynamic ensembles between proteins and ligands. One effective way to approximate such a thermodynamic ensemble is to use molecular dynamics (MD) simulation. Here, an MD dataset containing 3,218 different protein-ligand complexes is curated, and Dynaformer, a graph-based deep learning model is further developed to predict the binding affinities by learning the geometric characteristics of the protein-ligand interactions from the MD trajectories. In silico experiments demonstrated that the model exhibits state-of-the-art scoring and ranking power on the CASF-2016 benchmark dataset, outperforming the methods hitherto reported. Moreover, in a virtual screening on heat shock protein 90 (HSP90) using Dynaformer, 20 candidates are identified and their binding affinities are further experimentally validated. Dynaformer displayed promising results in virtual drug screening, revealing 12 hit compounds (two are in the submicromolar range), including several novel scaffolds. Overall, these results demonstrated that the approach offer a promising avenue for accelerating the early drug discovery process., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Endothelial cAMP-specific phosphodiesterase PDE3A is one of the major negative regulators of the endothelial barrier function in acute lung injury models. However, the mechanisms underlying its regulation still need to be fully resolved. We show here that the PDE3A is a newly described client of the molecular chaperone heat shock protein 90 (hsp90). In endothelial cells (ECs), hsp90 inhibition by geldanamycin (GA) led to a disruption of the hsp90/PDE3A complex, followed by a significant decrease in PDE3A protein levels. The decrease in PDE3A protein levels was ubiquitin-proteasome-dependent and required the activity of the E3 ubiquitin ligase C terminus of Hsc70-interacting protein. GA treatment also enhanced the association of PDE3A with hsp70, which partially prevented PDE3A degradation. GA-induced decreases in PDE3A protein levels correlated with decreased PDE3 activity and increased cAMP levels in EC. We also demonstrated that protein kinase G-dependent phosphorylation of PDE3A at Ser 654 can signal the dissociation of PDE3A from hsp90 and PDE3A degradation. This was confirmed by endogenous PDE3A phosphorylation and degradation in 8-Br-cGMP- or 8-CPT-cGMP- and Bay 41-8543-stimulated EC and comparisons of WT- and phospho-mimic S 654 D mutant PDE3A protein stability in transiently transfected HEK293 cells. In conclusion, we have identified a new mechanism of PDE3A regulation mediated by the ubiquitin-proteasome system. Further, the degradation of PDE3A is controlled by the phosphorylation of S 654 and the interaction with hsp90. We speculate that targeting the PDE3A/hsp90 complex could be a therapeutic approach for acute lung injury., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Early-onset epilepsy following ischemic stroke is a severe neurological condition, the pathogenesis of which remains incompletely understood. Recent studies suggest that Neural stem/progenitor cells (NSPCs) play a crucial role in the disease process, yet the precise molecular mechanisms regulating NSPCs have not been thoroughly investigated. This study utilized single-cell transcriptome sequencing and bioinformatics analysis to identify disease-related genes, which were subsequently validated in both in vitro and in vivo experiments. The findings revealed that Hsp90aa1 (heat shock protein 90 kDa alpha, class A member 1), Jun proto-oncogene (JUN), and CC Motif Ligation 2 (Ccl2) constitute an important regulatory axis influencing the migration and differentiation of NSPCs, potentially impacting the onset and progression of early-onset epilepsy post-ischemic stroke. Additionally, the expression of Hsp90aa1 was found to influence the likelihood of seizure occurrence and the severity of brain ischemia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytokinesis is required for faithful division of cytoplasmic components and duplicated nuclei into two daughter cells. Midbody, a protein-dense organelle that forms at the intercellular bridge, is indispensable for successful cytokinesis. However, the regulatory mechanism of cytokinesis at the midbody still remains elusive. Here, we unveil a critical role for NudC-like protein 2 (NudCL2), a co-chaperone of heat shock protein 90 (Hsp90), in cytokinesis regulation by stabilizing regulator of chromosome condensation 2 (RCC2) at the midbody in mammalian cells. NudCL2 localizes at the midbody, and its downregulation results in cytokinesis failure, multinucleation, and midbody disorganization. Using iTRAQ-based quantitative proteomic analysis, we find that RCC2 levels are decreased in NudCL2 knockout (KO) cells. Moreover, Hsp90 forms a complex with NudCL2 to stabilize RCC2, which is essential for cytokinesis. RCC2 depletion mirrors phenotypes observed in NudCL2-downregulated cells. Importantly, ectopic expression of RCC2 rescues the cytokinesis defects induced by NudCL2 deletion, but not vice versa. Together, our data reveal the significance of the NudCL2/Hsp90/RCC2 pathway in cytokinesis at the midbody., (© The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.)

Background and Purpose: Ulcerative colitis (UC) is a refractory inflammatory disease associated with immune dysregulation. Elevated levels of heat shock protein (HSP) 90 in the β but not α subtype were positively associated with disease status in UC patients. This study validated the possibility that pharmacological inhibition or reduction of HSP90β would alleviate colitis, induced by dextran sulfate sodium, in mice and elucidated its mechanisms., Experimental Approach: Histopathological and biochemical analysis assessed disease severity, and bioinformatics and correlation analysis explained the association between the many immune cells and HSP90β. Flow cytometry was used to analyse the homeostasis and transdifferentiation of Th17 and Treg cells. In vitro inhibition and adoptive transfer assays were used to investigate functions of the phenotypically transformed Th17 cells. Metabolomic analysis, DNA methylation detection and chromatin immunoprecipitation were used to explore these mechanisms., Key Results: The selective pharmacological inhibitor (HSP90βi) and shHSP90β significantly mitigated UC in mice and promoted transformation of Th17 to Treg cell phenotype, via Foxp3 transcription. The phenotypically-transformed Th17 cells by HSP90βi or shHSP90β were able to inhibit lymphocyte proliferation and colitis in mice. HSP90βi and shHSP90β selectively weakened glycolysis by stopping the direct association of HSP90β and GLUT1, the key glucose transporter, to accelerate ubiquitination degradation of GLUT1, and enhance the methylation of Foxp3 CNS2 region. Then, the mediator path was identified as the "lactate-STAT5-TET2" cascade., Conclusion and Implications: HSP90β shapes the fate of Th17 cells via glycolysis-controlled methylation modification to affect UC progression, which provides a new therapeutic target for UC., (© 2024 British Pharmacological Society.)

Molecular chaperone heat shock protein 90 (Hsp90) is a ubiquitous regulator that fine-tunes and remodels diverse client proteins, exerting profound effects on normal biology and diseases. Unraveling the mechanistic details of Hsp90's function requires atomic-level insights into its client interactions throughout the adenosine triphosphate-coupled functional cycle. However, the structural details of the initial encounter complex in the chaperone cycle, wherein Hsp90 adopts an open conformation while engaging with the client, remain elusive. Here, using nuclear magnetic resonance spectroscopy, we determined the solution structure of Hsp90 in its open state, bound to a disordered client. Our findings reveal that Hsp90 uses two distinct binding sites, collaborating synergistically to capture discrete hydrophobic segments within client proteins. This bipartite interaction generates a versatile complex that facilitates rapid conformational sampling. Moreover, our investigations spanning various clients and Hsp90 orthologs demonstrate a pervasive mechanism used by Hsp90 orthologs to accommodate the vast array of client proteins. Collectively, our work contributes to establish a unified conceptual and mechanistic framework, elucidating the intricate interplay between Hsp90 and its clients., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Aberrant activation of the NLRP3 inflammasome is recognized to induce a chronic inflammatory response in the liver, ultimately leading to hepatic fibrosis. HSP90 is suggested to regulate NLRP3 activation and its downstream signaling. This study is the first to explore the potential therapeutic role of pimitespib in mitigating liver fibrosis in rats. The results of the study revealed that pimitespib effectively suppressed hepatic inflammation and fibrogenesis by modulating HSP90's control over the NFκB/NLRP3/caspase-1 axis. In vitro experiments demonstrated that pimitespib reduced LDH levels and increased hepatocyte survival, whereas in vivo, it prolonged the survival of rats with hepatic fibrosis. Additionally, pimitespib exhibited improvements in the function and microscopic characteristics of rat livers. Pimitespib effectively inhibited NFκB, which serves as the priming signal for NLRP3 activation. Pimitespib's inhibitory effect on NLRP3, identified as an HSP90 client protein, plays a central role in the observed anti-fibrotic effect. The simultaneous inhibition of both priming and activation signals of NLRP3 by pimitespib led to a reduction in caspase-1 activity and subsequent suppression of the N-terminal fragment of gasdermin D, ultimately constraining hepatocyte pyroptotic cell death. These diverse effects were associated with a decrease in the transcription of inflammatory mediators IL-1β, IL-18, and TNF-α, as well as the fibrogenic mediators TGF-β, TIMP-1, PDGF-BB, and Col1a1. Moreover, pimitespib induced the expression of HSP70, which could further contribute to the repression of fibrosis development. In summary, our findings provide an evolutionary perspective on managing liver fibrosis, positioning pimitespib as a promising candidate for anti-inflammatory and antifibrotic therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Targeting the heat shock protein-90 (Hsp90) chaperone machinery in various cancers with 200 monotherapy or combined-therapy clinical trials since 1999 has not yielded any success of food and drug administration approval. Blames for the failures were unanimously directed at the Hsp90 inhibitors or tumors or both. However, analyses of recent cellular and genetic studies together with the Hsp90 data from the Human Protein Atlas database suggest that the vast variations in Hsp90 expression among different organs in patients might have been the actual cause. It is evident now that Hsp90β is the root of dose-limiting toxicity (DLT), whereas Hsp90α is a buffer of penetrated Hsp90 inhibitors. The more Hsp90α, the safer Hsp90β, and the lower DLT are for the host. Unfortunately, the dramatic variations of Hsp90, from total absence in the eye, muscle, pancreas, and heart to abundance in reproduction organs, lung, liver, and gastrointestinal track, would cause the selection of any fair toxicity biomarker and an effective maximum tolerable dose (MTD) of Hsp90 inhibitor extremely challenging. In theory, a safe MTD for the organs with high Hsp90 could harm the organs with low Hsp90. In reverse, a safe MTD for organs with low or undetectable Hsp90 would have little impact on the tumors, whose cells exhibit average 3-7% Hsp90 over the average 2-3% Hsp90 in normal cells. Moreover, not all tumor cell lines tested follow the "inhibitor binding-client protein degradation" paradigm. It is likely why the oral Hsp90 inhibitor TAS-116 (Pimitespib), which bypasses blood circulation and other organs, showed some beneficiary efficacy by conveniently hitting tumors along the gastrointestinal track. The critical question is what the next step will be for the Hsp90 chaperone as a cancer therapeutic target., Competing Interests: Declarations of interest The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests. Wei Li reports financial support was provided by the National Institutes of Health. Mei Chen reports financial support was provided by the National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glucose-regulated protein 94 (Grp94) is an isoform of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Inhibiting Grp94 has been implicated for many diseases. Co-crystal structures of two generations of Grp94 inhibitors revealed the importance of investigating the ester group, which is projected into the site 2 pocket unique to Grp94. Therefore, a series of KUNG65 benzamide analogs was designed and synthesized to evaluate their impact on the affinity and selectivity for Grp94. The data demonstrated that substituents with small and saturated ring systems that contain hydrogen bond acceptors exhibited increased affinity for Grp94, whereas larger saturated ring system manifested increased selectivity for Grp94 over Hsp90α., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)