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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).)

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).)

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).)

Tumor-associated macrophages (TAMs) originating from monocytes are crucial for cancer progression; however, the mechanism of TAM differentiation is unclear. We investigated factors involved in the differentiation of monocytes into TAMs within the tumor microenvironment of triple-negative breast cancer (TNBC). We screened 172 compounds and found that a heat shock protein 90 (HSP90) inhibitor blocked TNBC-induced monocyte-to-TAM differentiation in human monocytes THP-1. TNBC-derived conditional medium (CM) activated cell signaling pathways, including MAP kinase, AKT and STAT3, and increased the expression of TAM-related genes and proteins. These inductions were suppressed by HSP90 inhibition or by knockdown of HSP90 in TNBC. Additionally, we confirmed that TNBC secreted HSP90 extracellularly and that HSP90 itself promoted TAM differentiation. In a mouse tumor model, treatment with an HSP90 inhibitor suppressed tumor growth and reduced TAMs in the tumor microenvironment. Our findings demonstrate the role of HSP90 in TAM differentiation, suggesting HSP90 as a potential target for TNBC immunotherapy due to its regulatory role in monocyte-to-TAM differentiation., (© 2024. The Author(s).)

PANoptosis is engaged in the program of immune response and carcinogenicity. Nonetheless, the actual impacts of PANoptosis on clinical management and oncology immunity in hepatocellular carcinoma (HCC) are not fully grasped. RNA-seq-derived computations were conducted to sort out the molecular subtypes and elucidate the disparities based on PANoptosis molecules. Single-cell sequencing (scRNA-seq) tools including Cytotrace and Addmodulescore were extracted to characterize diversification potency and quantify the PANoptosis motion. Transcriptional factors were inferred by the pySCENIC package and Cellchat program scrutinized the intercellular exchange across cell compartments. The PANoptosis score system originated by incorporating 10 machine learning algorithms and 101 compositions to project clinical results and deteriorate tendencies. Circulatory PANoptosis-associated protein HSP90AA1 was determined by enzyme-linked immunosorbent assay (ELISA). HCC individuals could be categorized into low- and high-PANoptosis groups with diverse biogenic and pharmacotherapy heterogeneity. Individuals in the elevated PANoptosis subtype were characterized as "hot tumor" conveying the increased presence of immunogenicity while reiterating an explicit negative connection with tumor stemness. Compared to immune and stromal cells, cancerous cells showcased decreased PANoptosis and heightened PANoptosis malignant cell subgroups might be tied to a substantial level of genomic expression of SREBF2, JUND, GATAD1, ZBTB20, SMAD5 and implied a more aggressive potential. The PANoptosis index, derived from machine learning, has been established to provide succinct frameworks for predicting outcomes and clarified the noteworthy utility of conventional regimens, as the differentiated power of HCC occurred together with vascular invasion and hepatocellular adenoma (HCA). The experiment confirmed that the circulating HSP90AA1 was aberrantly augmented in HCC patients, thus demonstrating its potential as a discriminatory biomarker. We systematically deciphered the molecular and immune ecosystem traits of PANoptosis in bulk and scRNA-seq degrees, which may deliver advantageous insights for customized treatment, awareness of the pathological process and prognosis scrutiny for HCC patients., (© 2024. The Author(s).)

Morgana is a ubiquitous HSP90 co-chaperone protein coded by the CHORDC1 gene. Morgana heterozygous mice develop with age a myeloid malignancy resembling human atypical myeloid leukemia (aCML), now renamed MDS/MPN with neutrophilia. Patients affected by this pathology exhibit low Morgana levels in the bone marrow (BM), suggesting that Morgana downregulation plays a causative role in the human malignancy. A decrease in Morgana expression levels is also evident in the BM of a subgroup of Philadelphia-positive (Ph+) chronic myeloid leukemia (CML) patients showing resistance or an incomplete response to imatinib. Despite the relevance of these data, the mechanism through which Morgana expression is downregulated in patients' bone marrow remains unclear. In this study, we investigated the possibility that Morgana expression is regulated by miRNAs and we demonstrated that Morgana is under the control of four miRNAs (miR-15a/b and miR-26a/b) and that miR-15a may account for Morgana downregulation in CML patients., (© 2024. The Author(s).)

The ORF9b protein, derived from the nucleocapsid's open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host-virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings., (© 2024. The Author(s).)

Opioids are the gold standard for the treatment of chronic pain but are limited by adverse side effects. In our earlier work, we showed that Heat shock protein 90 (Hsp90) has a crucial role in regulating opioid signaling in spinal cord; Hsp90 inhibition in spinal cord enhances opioid anti-nociception. Building on these findings, we injected the non-selective Hsp90 inhibitor KU-32 by the intrathecal route into male and female CD-1 mice, showing that morphine anti-nociceptive potency was boosted by 1.9-3.5-fold in acute and chronic pain models. At the same time, tolerance was reduced from 21-fold to 2.9 fold and established tolerance was rescued, while the potency of constipation and reward was unchanged. These results demonstrate that spinal Hsp90 inhibition can improve the therapeutic index of morphine. However, we also found that systemic non-selective Hsp90 inhibition blocked opioid pain relief. To avoid this effect, we used selective small molecule inhibitors and CRISPR gene editing to identify 3 Hsp90 isoforms active in spinal cord (Hsp90α, Hsp90β, and Grp94) while only Hsp90α was active in brain. We thus hypothesized that a systemically delivered selective inhibitor to Hsp90β or Grp94 could selectively inhibit spinal cord Hsp90 activity, resulting in enhanced opioid therapy. We tested this hypothesis using intravenous delivery of KUNB106 (Hsp90β) and KUNG65 (Grp94), showing that both drugs enhanced morphine anti-nociceptive potency while rescuing tolerance. Together, these results suggest that selective inhibition of spinal cord Hsp90 isoforms is a novel, translationally feasible strategy to improve the therapeutic index of opioids., (© 2024. The Author(s).)

The present study predicts the molecular targets and druglike properties of the phyto-compound piperine (PIP) by in silico studies including molecular docking simulation, druglikeness prediction and ADME analysis for prospective therapeutic benefits against diabetic complications. PIP was encapsulated in biodegradable polymer poly-lactide-co-glycolide (PLGA) to form nanopiperine (NPIP) and their physico-chemical properties were characterized by AFM and DLS. ∼ 30 nm sized NPIP showed 86.68% encapsulation efficiency and - 6 mV zeta potential, demonstrated great interactive stability and binding with CT-DNA displaying upsurge in molar ellipticity during CD spectroscopy. NPIP lowered glucose levels in peripheral circulation by > 65 mg/dL compared to disease model and improved glucose influx in alloxan-induced in vivo and in vitro diabetes models concerted with 3-folds decrease in ROS production, ROS-induced DNA damage and 27.24% decrease in nuclear condensation. The 25% increase in % cell viability and inhibition in chromosome aberration justified the initiation of p53 and PARP DNA repairing protein expression and maintenance of Hsp90. Thus, the experimental study corroborated well with in silico predictions of modulating the p53/PARP-1/Hsp90 axis, with predicted dock score value of - 8.72, - 8.57, - 8.76 kcal/mol respectively, validated docking-based preventive approaches for unravelling the intricacies of molecular signalling and nano-drug efficacy as therapeutics for diabetics., (© 2024. The Author(s).)

Mitochondrial dysfunction and cell death play important roles in diabetic cardiomyopathy, but the underlying mechanisms remain unclear. Here, we report that mitochondrial dysfunction and cell apoptosis are prominent features of primary cardiomyocytes after exposure to high glucose/palmitate conditions. The protein level of MIC60, a core component of mitochondrial cristae, is decreased via ubiquitination and degradation under these conditions. Exogenous expression of MIC60 alleviates cristae disruption, mitochondrial dysfunction and apoptosis. Moreover, we identified MARCH5 as an E3 ubiquitin ligase that specifically targets MIC60 in this process. Indeed, MARCH5 mediates K48-linked ubiquitination of MIC60 at Lys285 to promote its degradation. Mutation of the ubiquitination site in MIC60 or the MIC60-interacting motifs in MARCH5 abrogates MARCH5-mediated MIC60 ubiquitination and degradation. Silencing MARCH5 significantly alleviates high glucose/palmitate-induced mitochondrial dysfunction and apoptosis in primary cardiomyocytes. In addition to E3 ubiquitin ligases, molecular chaperones also play important roles in protein stability. We previously reported that the mitochondrial chaperone TRAP1 inhibits the ubiquitination of MIC60, but the detailed mechanism is unknown. Here, we find that TRAP1 performs this function by competing with MARCH5 for binding to MIC60. Our findings provide new insights into the mechanism underlying mitochondrial dysfunction in cardiomyocytes in diabetic cardiomyopathy. MARCH5 promotes ubiquitination of MIC60 to induce MIC60 degradation, mitochondrial dysfunction and apoptosis in cardiomyocytes under diabetic conditions. TRAP1 inhibits MARCH5-mediated ubiquitination by competitively interacting with MIC60., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Hepatitis C virus (HCV) infection is one of the major factors to trigger a sustained hepatic inflammatory response and hence hepatocellular carcinoma (HCC), but direct-acting-antiviral (DAAs) was not efficient to suppress HCC development. Heat shock protein 90 kDa (HSP90) is highly abundant in different types of cancers, and especially controls protein translation, endoplasmic reticulum stress, and viral replication. In this study we investigated the correlation between the expression levels of HSP90 isoforms and inflammatory response marker NLRP3 in different types of HCC patients as well as the effect of a natural product celastrol in suppression of HCV translation and associated inflammatory response in vivo. We identified that the expression level of HSP90β isoform was correlated with that of NLRP3 in the liver tissues of HCV positive HCC patients (R 2  = 0.3867, P < 0.0101), but not in hepatitis B virus-associated HCC or cirrhosis patients. We demonstrated that celastrol (3, 10, 30 μM) dose-dependently suppressed the ATPase activity of both HSP90α and HSP90β, while its anti-HCV activity was dependent on the Ala47 residue in the ATPase pocket of HSP90β. Celastrol (200 nM) halted HCV internal ribosomal entry site (IRES)-mediated translation at the initial step by disrupting the association between HSP90β and 4EBP1. The inhibitory activity of celastrol on HCV RNA-dependent RNA polymerase (RdRp)-triggered inflammatory response also depended on the Ala47 residue of HSP90β. Intravenous injection of adenovirus expressing HCV NS5B (pAde-NS5B) in mice induced severe hepatic inflammatory response characterized by significantly increased infiltration of immune cells and hepatic expression level of Nlrp3, which was dose-dependently ameliorated by pretreatment with celastrol (0.2, 0.5 mg/kg, i.p.). This study reveals a fundamental role of HSP90β in governing HCV IRES-mediated translation as well as hepatic inflammation, and celastrol as a novel inhibitor of HCV translation and associated inflammation by specifically targeting HSP90β, which could be developed as a lead for the treatment of HSP90β positive HCV-associated HCC., (© 2023. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.)

The 90 kDa heat shock protein, Hsp90, functions as a cancer chaperone contributing to tumor proliferation. We have encountered the mitochondrial homolog of Hsp90, the TRAP-1, regulating mitochondrial dynamics, metabolism, and tumor metastasis. Although Hsp90 is associated with a broad network of proteins regulating various cellular processes, TRAP-1-mediated cellular networks are unclear. Therefore, using TRAP-1 knockdown (KD) and overexpression (OE) systems, we compared their quantitative transcriptome (RNA Sequencing) and proteomic (LC-MS/MS) patterns to obtain molecular signatures that are altered in response to TRAP-1 KD or OE. We report TRAP-1 modulating vital metabolic pathways such as the tricarboxylic acid cycle, oxidative phosphorylation, electron transport chain, glycolysis, and gluconeogenesis. In addition, TRAP-1 facilitated the pentose phosphate pathway to shunt carbons back to glycolysis or gluconeogenesis, a much-solicited tumor response. Subsequently, we examined the TRAP-1 interactome using the tandem affinity purification system and identified 255 unique proteins. These diverse proteins appear to regulate several cellular processes, including energy metabolism, suggesting that TRAP-1, in addition to metabolic rewiring, maintains mitochondrial integrity. Our study exposes the unknown functions of TRAP-1 in cancer cells. Systematic evaluation of TRAP-1 interactors may uncover novel regulatory mechanisms in disease aggression. Since metabolic inhibitors are emerging as potential anticancer agents, our study gains importance., (© 2023. The Author(s).)

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer., (© 2022. The Author(s).)

Podocyte injury is a hallmark of glomerular diseases; however, the underlying mechanisms remain unclear. B7-1 is increased in injured podocytes, but its intrinsic role is controversial. The clinical data here revealed the intimate correlation of urinary B7-1 with severity of glomerular injury. Through transcriptomic and biological assays in B7-1 transgenic and adriamycin nephropathy models, we identified B7-1 is a key mediator in podocyte injury and glomerulosclerosis through a series of signal transmission to β-catenin. Using LC-MS/MS, Hsp90ab1, a conserved molecular chaperone, was distinguished to be an anchor for transmitting signals from B7-1 to β-catenin. Molecular docking and subsequent mutant analysis further identified the residue K69 in the N terminal domain of Hsp90ab1 was the key binding site for B7-1 to activate LRP5/β-catenin pathway. The interaction and biological functions of B7-1-Hsp90ab1-LRP5 complex were further demonstrated in vitro and in vivo. We also found B7-1 is a novel downstream target of β-catenin. Our results indicate an intercrossed network of B7-1, which collectively induces podocyte injury and glomerulosclerosis. Our study provides an important clue to improve the therapeutic strategies to target B7-1., (© 2022. The Author(s).)

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis., (© 2022. The Author(s).)

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion, decreasing both respiration and intracellular NAD + . Expression of the alternative NADH dehydrogenase NDI1 raises NAD + /NADH ratio, enhances the activity of the NAD + -dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. The antineoplastic effect of NDI1 is mimicked by administration of NAD + precursors or by rising expression of the NAD + deacetylase SIRT3 and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes. These findings shed light on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD + /NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss., (© 2022. The Author(s).)

Diffuse malignant peritoneal mesothelioma (DMPM) is a rare and rapidly lethal tumor, poorly responsive to conventional treatments. In this regards, the identification of molecular alterations underlying DMPM onset and progression might be exploited to develop novel therapeutic strategies. Here, we focused on miR-550a-3p, which we found downregulated in 45 DMPM clinical samples compared to normal tissues and whose expression levels were associated with patient outcome. Through a gain-of-function approach using miRNA mimics in 3 DMPM cell lines, we demonstrated the tumor-suppressive role of miR-550a-3p. Specifically, miRNA ectopic expression impaired cell proliferation and invasiveness, enhanced the apoptotic response, and reduced the growth of DMPM xenografts in mice. Antiproliferative and proapoptotic effects were also observed in prostate and ovarian cancer cell lines following miR-550a-3p ectopic expression. miR-550a-3p effects were mediated, at least in part, by the direct inhibition of HSP90AA1 and the consequent downregulation of its target proteins, the levels of which were rescued upon disruption of miRNA-HSP90AA1 mRNA pairing, partially abrogating miR-550a-3p-induced cellular effects. Our results show that miR-550a-3p reconstitution affects several tumor traits, thus suggesting this approach as a potential novel therapeutic strategy for DMPM., (© 2022. The Author(s).)

Heat shock proteins (HSPs) expression protect the cell from stress, this expression varies on tissue and stress level. Here, we investigated the structure and functional expression of HSPs in different chicken organs using meta-analysis. A total of 1253 studies were collected from three different electronic databases from January 1, 2015 to February 1, 2022. Of these studies, 28 were selected based on the specific criteria for this meta-analysis. The results for the expression of HSPs and the comparative expression of HSPs (HSP90, HSP70, and HSP60) in different chicken organs (brain, heart, liver, muscle, and intestine) were analyzed using the odds ratio or the random-effects model (REM) at a confidence interval (CI) of 95%. Compared to the thermoneutral groups, heat stress groups exhibited a significant (P < 0.01) change in their HSP70 expression in the chicken liver (8 trials: REM = 1.41, 95% CI: 0.41, 4.82). The expression of different HSPs in various chicken organs varied and the different organs were categorized according to their expression levels. HSP expression differed among the heart, liver, and muscle of chickens. HSPs expression level depends on the structure and molecular weight of the HSPs, as well as the type of tissue., (© 2022. The Author(s).)

Cardiac hypertrophy, as one of the major predisposing factors for chronic heart failure, lacks effective interventions. Exploring the pathogenesis of cardiac hypertrophy will reveal potential therapeutic targets. S-nitrosylation is a kind of posttranslational modification that occurs at active cysteines of proteins to mediate various cellular processes. We here identified heat shock protein 90 (Hsp90) as a highly S-nitrosylated target in the hearts of rodents with hypertrophy, and the role of Hsp90 in cardiac hypertrophy remains undefined. The S-nitrosylation of Hsp90 (SNO-Hsp90) levels were elevated in angiotensin II (Ang II)- or phenylephrine (PE)-treated neonatal rat cardiomyocytes (NRCMs) in vitro as well as in cardiomyocytes isolated from mice subjected to transverse aortic constriction (TAC) in vivo. We demonstrated that the elevated SNO-Hsp90 levels were mediated by decreased S-nitrosoglutathione reductase (GSNOR) expression during cardiac hypertrophy, and delivery of GSNOR adeno-associated virus expression vectors (AAV9-GSNOR) decreased the SNO-Hsp90 levels to attenuate cardiac hypertrophy. Mass spectrometry analysis revealed that cysteine 589 (Cys589) might be the S-nitrosylation site of Hsp90. Delivery of the mutated AAV9-Hsp90-C589A inhibited SNO-Hsp90 levels and attenuated cardiac hypertrophy. We further revealed that SNO-Hsp90 led to increased interaction of glycogen synthase kinase 3β (GSK3β) and Hsp90, leading to elevated GSK3β phosphorylation and decreased eIF2Bε phosphorylation, thereby aggravating cardiac hypertrophy. Application of GSK3β inhibitor TWS119 abolished the protective effect of Hsp90-C589A mutation in Ang II-treated NRCMs. In conclusion, this study demonstrates a critical role of SNO-Hsp90 in cardiac hypertrophy, which may be of a therapeutic target for cardiac hypertrophy treatment., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Trichophyton rubrum, a major human pathogenic dermatophyte, is responsible for the most recurrent dermatophytoses as globally important superficial fungal infections. Typical chemotherapy is used to handle such infections; however, emerging drug resistance and side effects necessitate the new remedial method development. Cold atmospheric plasma (CAP) is an emerging technology, consisted of neutral and charged particles and photons newly developed as a potent and safe antimicrobial technique to combat drug-resistant microbial pathogens. In the present study, the vast effects of CAP irradiation containing oxygen (2%) and helium (98%) on T. rubrum growth and pathogenicity were explored. After exposure of T. rubrum to CAP jet for 90, 120, 150, 180, and 210 s in 96-well microtiter plates, cell morphology and viability, ergosterol content of fungal hyphae, HSP90 gene expression, and the pattern of drug susceptibility were studied by using electron microscopy, RT-qPCR, spectrophotometry, disk diffusion and CLSI microbroth dilution methods. CAP irradiation significantly inhibited the fungal growth by 25.83 to 89.10%, reduced fungal cell viability by 11.68 to 87.71%, disrupted cellular membranous organelles and structures of the fungal hyphae, and suppressed efficiently the expression of HSP90 gene by 2 folds in 210 s exposure. Taken together, our results demonstrated that CAP is an efficient tool with potential in-vivo therapeutic applications against chronic dermatophytosis caused by T. rubrum due to its effectiveness, harmless, and ease of access., (© 2022. The Author(s).)

Despite recent advances, there remains a significant unmet need for the development of new targeted therapies for triple-negative breast cancer (TNBC). Although the heat shock protein HSP90 is a promising target, previous inhibitors have had issues during development including undesirable induction of the heat shock response (HSR) and off-target effects leading to toxicity. SL-145 is a novel, rationally-designed C-terminal HSP90 inhibitor that induces apoptosis in TNBC cells via the suppression of oncogenic AKT, MEK/ERK, and JAK2/STAT3 signaling and does not trigger the HSR, in contrast to other inhibitors. In an orthotopic allograft model incorporating breast cancer stem cell-enriched TNBC tumors, SL-145 potently suppressed tumor growth, angiogenesis, and metastases concomitant with dysregulation of the JAK2/STAT3 signaling pathway. Our findings highlight the potential of SL-145 in suppressing metastatic TNBC independent of the HSR., (© 2022. The Author(s).)

The HSP90/CDC37 chaperone system not only assists the maturation of many protein kinases but also maintains their structural integrity after folding. The interaction of mature kinases with the HSP90/CDC37 complex is governed by the conformational stability of the catalytic domain, while the initial folding of the protein kinase domain is mechanistically less well characterized. DYRK1A (Dual-specificity tyrosine (Y)-phosphorylation Regulated protein Kinase 1A) and DYRK1B are closely related protein kinases with discordant HSP90 client status. DYRK kinases stoichiometrically autophosphorylate on a tyrosine residue immediately after folding, which served us as a traceable marker of successful maturation. In the present study, we used bacterial expression systems to compare the capacity of autonomous maturation of DYRK1A and DYRK1B in the absence of eukaryotic cofactors or chaperones. Under these conditions, autophosphorylation of human DYRK1B was severely compromised when compared with DYRK1A or DYRK1B orthologs from zebrafish and Xenopus. Maturation of human DYRK1B could be restored by bacterial expression at lower temperatures, suggesting that folding was not absolutely dependent on eukaryotic chaperones. The differential folding properties of DYRK1A and DYRK1B were largely due to divergent sequences of the C-terminal lobes of the catalytic domain. Furthermore, the mature kinase domain of DYRK1B featured lower thermal stability than that of DYRK1A when exposed to heat challenge in vitro or in living cells. In summary, our study enhances the mechanistic understanding of the differential thermodynamic properties of two closely related protein kinases during initial folding and as mature kinases., (© 2022. The Author(s).)

Heat shock protein 90 (Hsp90) is the most common molecular chaperone that controls the maturation of many oncoproteins critical in tumor development. Hsp90 has been considered as a promising target for cancer treatment, but the clinical significance of Hsp90 and the mechanisms of Hsp90 regulating the tumor-promoting effects in hepatocellular carcinoma (HCC) remain obscure. Previous studies have shown that curcumin, a polyphenol derived from the plant turmeric (Curcuma longa), inhibits tumor growth, which may provide an effective alternative therapy for HCC. Compared to curcumin, a novel derivative of curcumin, 3,5-(E)-Bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride (C0818) that is more potent in Hsp90 inhibition and antitumor activity. In this study, we investigated the effect of C0818 on HCC cells in vitro and its relation to Hsp90 inhibition. We showed that C0818 concentration-dependently inhibited the proliferation, the colony formation and induced apoptosis in HepG2 and Sk-Hep-1 cells. C0818 concentration-dependently inhibited DNA synthesis and induced G 2 /M phase arrest in HepG2 and Sk-Hep-1 cells. We further demonstrated that C0818 induced ROS- and caspase-dependent apoptosis in HCC cells through the mitochondrial-mediated pathway. C0818 induced the degradation of Hsp90 client proteins as RAS, C-Raf, P-C-Raf, Erk, P-ERK, MEK, P-MEK, Akt and P-Akt, which led to subsequent inhibition of the RAS/RAF/MEK/ERK and PI3K/AKT pathways. We revealed that C0818 could inhibit the binding of Hsp90 with its clients without affecting their transcription, which subsequently induced the degradation of Hsp90 clients by the proteasome rather than the lysosome. These results are of potential importance for elucidating a novel Hsp90 inhibitor targeting HCC., (© 2021. The Author(s), under exclusive licence to CPS and SIMM.)

Maintaining a healthy proteome is fundamental for the survival of all organisms 1 . Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp90 2 . The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity 3-7 . Chaperoning GR involves a cycle of inactivation by Hsp70; formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex; conversion to an active GR-Hsp90-p23 'maturation' complex; and subsequent GR release 8 . However, to our knowledge, a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR-maturation complex structure 9 , we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Hsp90 is a conserved and essential molecular chaperone responsible for the folding and activation of hundreds of 'client' proteins 1-3 . The glucocorticoid receptor (GR) is a model client that constantly depends on Hsp90 for activity 4-9 . GR ligand binding was previously shown to nr inhibited by Hsp70 and restored by Hsp90, aided by the co-chaperone p23 10 . However, a molecular understanding of the chaperone-mediated remodelling that occurs between the inactive Hsp70-Hsp90 'client-loading complex' and an activated Hsp90-p23 'client-maturation complex' is lacking for any client, including GR. Here we present a cryo-electron microscopy (cryo-EM) structure of the human GR-maturation complex (GR-Hsp90-p23), revealing that the GR ligand-binding domain is restored to a folded, ligand-bound conformation, while being simultaneously threaded through the Hsp90 lumen. In addition, p23 directly stabilizes native GR using a C-terminal helix, resulting in enhanced ligand binding. This structure of a client bound to Hsp90 in a native conformation contrasts sharply with the unfolded kinase-Hsp90 structure 11 . Thus, aided by direct co-chaperone-client interactions, Hsp90 can directly dictate client-specific folding outcomes. Together with the GR-loading complex structure 12 , we present the molecular mechanism of chaperone-mediated GR remodelling, establishing the first, to our knowledge, complete chaperone cycle for any Hsp90 client., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R 2  = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls., (© 2021. The Author(s).)

The molecular chaperones Hsc70 and Hsp90 are required for proteostasis control and specific folding of client proteins in eukaryotic and prokaryotic organisms. Especially in eukaryotes these ATP-driven molecular chaperones are interacting with cofactors that specify the client spectrum and coordinate the ATPase cycles. Here we find that a Hsc70-cofactor of the Hsp40 family from nematodes, DNJ-13, directly interacts with the kinase-specific Hsp90-cofactor CDC-37. The interaction is specific for DNJ-13, while DNJ-12 another DnaJ-like protein of C. elegans, does not bind to CDC-37 in a similar manner. Analytical ultracentrifugation is employed to show that one CDC-37 molecule binds to a dimeric DNJ-13 protein with low micromolar affinity. We perform cross-linking studies with mass spectrometry to identify the interaction site and obtain specific cross-links connecting the N-terminal J-domain of DNJ-13 with the N-terminal domain of CDC-37. Further AUC experiments reveal that both, the N-terminal part of CDC-37 and the C-terminal domain of CDC-37, are required for efficient interaction. Furthermore, the presence of DNJ-13 strengthens the complex formation between CDC-37 and HSP-90 and modulates the nucleotide-dependent effects. These findings on the interaction between Hsp40 proteins and Hsp90-cofactors provide evidence for a more intricate interaction between the two chaperone systems during client processing., (© 2021. The Author(s).)

A prevalent characteristic of solid tumors is intra-tumoral hypoxia. Hypoxia-inducible factor 1α (HIF1α) predominantly mediates the adaptive response to O 2 oscillation and is linked to multiple malignant hallmarks. Here we describe a strategy to robustly target HIF1α by dual inhibition of CDK(s) and heat shock protein 90 (HSP90). We show that CDK1 may contribute to HSP90-mediated HIF1α stabilization. CDK1 knockdown enhances the decrease of HIF1α by HSP90 inhibition. Dual inhibition of CDK1 and HSP90 significantly increases apoptosis and synergistically inhibits cancer cell viability. Similarly, targeting CDK4/6 using FDA-approved inhibitors in combination with HSP90 inhibition shows a class effect on HIF1α inhibition and cancer cell viability suppression not only in colorectal but also in various other cancer types, including Rb-deficient cancer cells. Dual inhibition of CDK4/6 and HSP90 suppresses tumor growth in vivo. In summary, combined targeting of CDK(s) (CDK1 or CDK4/6) and HSP90 remarkably inhibits the expression level of HIF1α and shows promising anti-cancer efficacy with therapeutic potential., (© 2021. The Author(s).)

The progression of colorectal cancer (CRC) has been well studied and understood with the development of molecular and genetic techniques. However, specific marker(s) that could be used to predict lymph node (LN) involvement, which is the most important prognostic factor for CRC, have not been identified so far. Our previous study, in which network analysis of LN(+) and LN(-) CRC gene expression was carried out with data obtained from the Cancer Genome Atlas, led to the identification of AHA1. AHA1 is a co-chaperone activator of the Hsp90 ATPase activity. However, the role of AHA1 expression in cancer cells is still unclear. To investigate how AHA1 expression regulates the cancer cell progression and/or metastasis of human CRC, the expression levels of AHA1 and Hsp90 were examined in 105 CRC tissue samples and compared with those in paired normal tissue. The RNA expression levels of AHA1 and Hsp90aa1, but not Hsp90ab, were significantly higher in cancer tissues than in adjacent paired normal tissues (p = 0.032 and p = 0.0002, respectively). In particular, AHA1, but not Hsp90aa1 and Hsp90ab, was closely associated with the TNM stage, LN stage, and tumor metastasis (p = 0.035, p = 0.012, and p = 0.0003, respectively). Moreover, the expression of AHA1 was not only higher in the CRC cell lines than in the normal colon fibroblast cell line but was also associated with the progression of these CRC cell lines. Overexpression of AHA1 in SW480 cells increased, whereas suppression of AHA1 expression in HCT116 cells reduced cell migration and invasion through the regulation of Snail, E-cadherin, pSRC, and pAKT, which are associated with EMT signaling. Taken together, our study suggests that AHA1 contributes to the metastatic advantage of human CRC., (© 2021. The Author(s).)

Hypoxia-inducible factor-1 (HIF-1), a master transcriptional factor for protecting cells from hypoxia, plays a critical role in spermatogenesis and tumorigenesis. For the past two decades, numerous small molecule inhibitors that block mRNA synthesis, protein translation, or DNA binding of HIF-1α have entered clinical trials. To date, few have advanced to FDA approval for clinical applications due to limited efficacy at their toxicity-tolerable dosages. New windows for developing effective and safe therapeutics require better understanding of the specific mechanism of action. The finding that a chaperone-defective mutant heat shock protein-90-alpha (Hsp90α) blocks spermatogenesis, a known hypoxia-driven process in mouse testis prompted us to focus on the role of Hsp90α in HIF-1α. Here we demonstrate that Hsp90α gene knockout causes a dramatic reduction of the high steady-state level of HIF-1α in the testis, blocking sperm production and causing infertility of the mice. In HIF-1α-dependent tumor cells, we found that Hsp90α forms protein complexes with hypoxia-elevated HIF-1α and Hsp90α knockout prevents hypoxia-induced HIF-1α accumulation. In contrast, downregulation of Hsp90β had little effect on hypoxia-induced accumulation of HIF-1α. Instead, Hsp90β protects signaling molecules responsible for cellular homeostasis from assault by 17-AAG (17-N-allylamino-17-demethoxygeldanamycin), a general ATPase inhibitor of both Hsp90α and Hsp90β. Since targeting Hsp90β gene is lethal in both cultured cells and in mice, our new finding explains the toxicity of the previous inhibitor trials and identifies the specific binding of Hsp90α to HIF-1α as a new therapeutic window for developing safer and more effective treatment of male infertility and cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc. part of Springer Nature.)

Periodic fever is a characteristic clinical feature of human malaria, but how parasites survive febrile episodes is not known. Although the genomes of Plasmodium species encode a full set of chaperones, they lack the conserved eukaryotic transcription factor HSF1, which activates the expression of chaperones following heat shock. Here, we show that PfAP2-HS, a transcription factor in the ApiAP2 family, regulates the protective heat-shock response in Plasmodium falciparum. PfAP2-HS activates the transcription of hsp70-1 and hsp90 at elevated temperatures. The main binding site of PfAP2-HS in the entire genome coincides with a tandem G-box DNA motif in the hsp70-1 promoter. Engineered parasites lacking PfAP2-HS have reduced heat-shock survival and severe growth defects at 37 °C but not at 35 °C. Parasites lacking PfAP2-HS also have increased sensitivity to imbalances in protein homeostasis (proteostasis) produced by artemisinin, the frontline antimalarial drug, or the proteasome inhibitor epoxomicin. We propose that PfAP2-HS contributes to the maintenance of proteostasis under basal conditions and upregulates specific chaperone-encoding genes at febrile temperatures to protect the parasite against protein damage., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Nematode development is characterized by progression through several larval stages. Thousands of genes were found in large scale RNAi-experiments to block this development at certain steps, two of which target the molecular chaperone HSP-90 and its cofactor UNC-45. Aiming to define the cause of arrest, we here investigate the status of nematodes after treatment with RNAi against hsp-90 and unc-45 by employing an in-depth transcriptional analysis of the arrested larvae. To identify misregulated transcriptional units, we calculate and validate genome-wide coexpression cliques covering the entire nematode genome. We define 307 coexpression cliques and more than half of these can be related to organismal functions by GO-term enrichment, phenotype enrichment or tissue enrichment analysis. Importantly, hsp-90 and unc-45 RNAi induce or repress many of these cliques in a coordinated manner, and then several specifically regulated cliques are observed. To map the developmental state of the arrested nematodes we define the expression behaviour of each of the cliques during development from embryo to adult nematode. hsp-90 RNAi can be seen to arrest development close to the L4 larval stage with further deviations in daf-16 regulated genes. unc-45 RNAi instead leads to arrested development at young adult stage prior to the programmatic downregulation of sperm-cell specific genes. In both cases processes can be defined to be misregulated upon depletion of the respective chaperone. With most of the defined gene cliques showing concerted behaviour at some stage of development from embryo to late adult, the "clique map" together with the clique-specific GO-terms, tissue and phenotype assignments will be a valuable tool in understanding concerted responses on the genome-wide level in Caenorhabditis elegans.

Protein kinases are important regulators in cellular signal transduction. As one major type of Hsp90 client, protein kinases rely on the ATP-dependent molecular chaperone Hsp90, which maintains their structure and supports their activation. Depending on client type, Hsp90 interacts with different cofactors. Here we report that besides the kinase-specific cofactor Cdc37 large PPIases of the Fkbp-type strongly bind to kinase•Hsp90•Cdc37 complexes. We evaluate the nucleotide regulation of these assemblies and identify prominent interaction sites in this quaternary complex. The synergistic interaction between the participating proteins and the conserved nature of the interaction suggests functions of the large PPIases Fkbp51/Fkbp52 and their nematode homolog FKB-6 as contributing factors to the kinase cycle of the Hsp90 machinery.

Disease-causing sequence variants in the highly polymorphic AIPL1 gene are associated with a broad spectrum of inherited retinal diseases ranging from severe autosomal recessive Leber congenital amaurosis to later onset retinitis pigmentosa. AIPL1 is a photoreceptor-specific co-chaperone that interacts with HSP90 to facilitate the stable assembly of retinal cGMP phosphodiesterase, PDE6. In this report, we establish unequivocal correlations between patient clinical phenotypes and in vitro functional assays of uncharacterized AIPL1 variants. We confirm that missense and nonsense variants in the FKBP-like and tetratricopeptide repeat domains of AIPL1 lead to the loss of both HSP90 interaction and PDE6 activity, confirming these variants cause LCA. In contrast, we report the association of p.G122R with milder forms of retinal degeneration, and show that while p.G122R had no effect on HSP90 binding, the modulation of PDE6 cGMP levels was impaired. The clinical history of these patients together with our functional assays suggest that the p.G122R variant is a rare hypomorphic allele with a later disease onset, amenable to therapeutic intervention. Finally, we report the primate-specific proline-rich domain to be dispensable for both HSP90 interaction and PDE6 activity. We conclude that variants investigated in this domain do not cause disease, with the exception of p.A352&#95;P355del associated with autosomal dominant cone-rod dystrophy.

Prodigiosin, a secondary metabolite red pigment produced by Serratia marcescens, has an interesting apoptotic efficacy against cancer cell lines with low or no toxicity on normal cells. HSP90α is known as a crucial and multimodal target in the treatment of TNBC. Our research attempts to assess the therapeutic potential of prodigiosin/PU-H71 combination on MDA-MB-231 cell line. The transcription and protein expression levels of different signalling pathways were assessed. Treatment of TNBC cells with both drugs resulted in a decrease of the number of adherent cells with apoptotic effects. Prodigiosin/PU-H71 combination increased the levels of caspases 3,8 and 9 and decreased the levels of mTOR expression. Additionally, there was a remarkable decrease of HSP90α transcription and expression levels upon treatment with combined therapy. Also, EGFR and VEGF expression levels decreased. This is the first study to show that prodigiosin/PU-H71 combination had potent cytotoxicity on MDA-MB-231 cells; proving to play a paramount role in interfering with key signalling pathways in TNBC. Interestingly, prodigiosin might be a potential anticancer agent to increase the sensitivity of TNBC cells to apoptosis. This study provides a new basis for upcoming studies to overcome drug resistance in TNBC cells.

ErbB2 overexpression identifies a subclass of breast cancer as ErbB2-positive that is frequently associated with poor prognosis. Current ErbB2-targeted therapies have profoundly improved patient outcomes, but mutations occurring in ErbB2 have been shown to confer drug resistance. Induction of ErbB2 degradation was proposed as an intriguing strategy to battle with ErbB2-positive breast cancer and reduced mutation-incurred drug resistance. Although multiple HSP90 inhibitors have been demonstrated to effectively trigger ErbB2 degradation, none succeeded in the clinical evaluations. To develop novel ErbB2-targeting strategies, we investigated the endocytic degradation and reversible ubiquitylation of ErbB2 in breast cancer. In this study, we reveal that HSP90 inhibition leads to efficient ubiquitylation and endocytic degradation of ErbB2 through the canonical endo-lysosomal route. USP2 associates with internalized ErbB2 and prevents its lysosomal sorting and degradation via exerting deubiquitylase activity. Accordingly, the USP2 inhibitor ML364 is capable of inducing ErbB2 ubiquitylation and accelerating its turnover. ML364 potentiates the pro-degradation effects of HSP90 inhibitors on ErbB2 and hence sensitizes ErbB2-positive breast cancer cells to HSP90 inhibition. The combination of USP2 and HSP90 inhibitors effectively restrains ErbB2-positive breast cancer xenograft growth in vivo. Based on these observations, we conclude that USP2 safeguards ErbB2 surface levels by antagonizing its ubiquitylation-mediated endocytic degradation, which can be exploited to design novel therapeutic strategies against ErbB2-driven malignancies as combinatorial treatment with HSP90 inhibitors.

Yersinia ruckeri is an important bacterial pathogen of fish, in particular salmonids, it has been associated with systemic infections worldwide and, like many enteric bacteria, it is a facultative intracellular pathogen. However, the effect of Y. ruckeri's interactions with the host at the cellular level have received little investigation. In the present study, a culture of Chinook Salmon Embryo (CHSE) cell line was exposed to Y. ruckeri. Afterwards, the proteins were investigated and identified by mass spectrometry and compared to the content of unexposed cultures. The results of this comparison showed that 4.7% of the identified proteins were found at significantly altered concentrations following infection. Interestingly, infection with Y. ruckeri was associated with significant changes in the concentration of surface adhesion proteins, including a significantly decreased presence of β-integrins. These surface adhesion molecules are known to be the target for several adhesion molecules of Yersiniaceae. The concentration of several anti-apoptotic regulators (HSP90 and two DNAj molecules) appeared similarly downregulated. Taken together, these findings suggest that Y. ruckeri affects the proteome of infected cells in a notable manner and our results shed some light on the interaction between this important bacterial pathogen and its host.

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.

Oncogenic client-proteins of the chaperone Heat shock protein 90 (HSP90) insure unlimited tumor growth and are involved in resistance to chemo- and radiotherapy. The HSP90 inhibitor Onalespib initiates the degradation of oncoproteins, and might also act as a radiosensitizer. The aim of this study was therefore to evaluate the efficacy of Onalespib in combination with external beam radiotherapy in an in vitro and in vivo approach. Onalespib downregulated client proteins, lead to increased apoptosis and caused DNA-double-strands. Monotherapy and combination with radiotherapy reduced colony formation, proliferation and migration assessed in radiosensitive HCT116 and radioresistant A431 cells. In vivo, a minimal treatment regimen for 3 consecutive days of Onalespib (3 × 10 mg/kg) doubled survival, whereas Onalespib with radiotherapy (3 × 2 Gy) caused a substantial delay in tumor growth and prolonged the survival by a factor of 3 compared to the HCT116 xenografted control group. Our results demonstrate that Onalespib exerts synergistic anti-cancer effects when combined with radiotherapy, most prominent in the radiosensitive cell models. We speculate that the depletion and downregulation of client proteins involved in signalling, migration and DNA repair mechanisms is the cause. Thus, individually, or in combination with radiotherapy Onalespib inhibits tumor growth and has the potential to improve radiotherapy outcomes, prolonging the overall survival of cancer patients.

Biliary atresia (BA) is a severe pediatric liver disease resulting in necroinflammatory obliteration of the extrahepatic biliary tree. BA presents within the first few months of life as either an isolated finding or with additional syndromic features. The etiology of isolated BA is unknown, with evidence for infectious, environmental, and genetic risk factors described. However, to date, there are no definitive causal genes identified for isolated BA in humans, and the question of whether single gene defects play a major role remains open. We performed exome-sequencing in 101 North American patients of European descent with isolated BA (including 30 parent-child trios) and considered several experimental designs to identify potentially deleterious protein-altering variants that may be involved in the disease. In a case-only analysis, we did not identify genes with variants shared among more than two probands, and burden tests of rare variants using a case-case control design did not yield significant results. In the trio analysis of 30 simplex families (patient and parent trios), we identified 66 de novo variants in 66 genes including potentially deleterious variants in STIP1 and REV1. STIP1 is a co-chaperone for the heat-shock protein, HSP90, and has been shown to have diverse functions in yeast, flies and mammals, including stress-responses. REV1 is known to be a key player in DNA repair pathway and to interact with HSP90. In conclusion, our results do not support the hypothesis that a simple genetic model is responsible for the majority of cases of isolated BA. Our finding of de novo variants in genes linked to evolutionarily conserved stress responses (STIP1 and REV1) suggests that exploration of how genetic susceptibility and environmental exposure may interact to cause BA is warranted.

The Hsp90 family proteins Hsp90, Grp94, and TRAP1 are present in the cell cytoplasm, endoplasmic reticulum, and mitochondria, respectively; all play important roles in tumorigenesis by regulating protein homeostasis in response to stress. Thus, simultaneous inhibition of all Hsp90 paralogs is a reasonable strategy for cancer therapy. However, since the existing pan-Hsp90 inhibitor does not accumulate in mitochondria, the potential anticancer activity of pan-Hsp90 inhibition has not yet been fully examined in vivo. Analysis of The Cancer Genome Atlas database revealed that all Hsp90 paralogs were upregulated in prostate cancer. Inactivation of all Hsp90 paralogs induced mitochondrial dysfunction, increased cytosolic calcium, and activated calcineurin. Active calcineurin blocked prosurvival heat shock responses upon Hsp90 inhibition by preventing nuclear translocation of HSF1. The purine scaffold derivative DN401 inhibited all Hsp90 paralogs simultaneously and showed stronger anticancer activity than other Hsp90 inhibitors. Pan-Hsp90 inhibition increased cytotoxicity and suppressed mechanisms that protect cancer cells, suggesting that it is a feasible strategy for the development of potent anticancer drugs. The mitochondria-permeable drug DN401 is a newly identified in vivo pan-Hsp90 inhibitor with potent anticancer activity.

Mitragyna speciosa Korth (M. speciosa) has been widely used as a recreational product, however, there are growing concerns on the abuse potentials and toxicity of the plant. Several poisoning and fatal cases involving kratom and mitragynine have been reported but the underlying causes remain unclear. The human ether-a-go-go-related gene 1 (hERG1) encodes the pore-forming subunit underlying cardiac rapidly delayed rectifier potassium current (I Kr ). Pharmacological blockade of the I Kr can cause acquired long QT syndrome, leading to lethal cardiac arrhythmias. This study aims to elucidate the mechanisms of mitragynine-induced inhibition on hERG1a/1b current. Electrophysiology experiments were carried out using Port-a-Patch system. Quantitative RT-PCR, Western blot analysis, immunofluorescence and co-immunoprecipitation methods were used to determine the effects of mitragynine on hERG1a/1b expression and hERG1-cytosolic chaperones interaction. Mitragynine was found to inhibit the I Kr current with an IC 50 value of 332.70 nM. It causes a significant reduction of the fully-glycosylated (fg) hERG1a protein expression but upregulates both core-glycosylated (cg) expression and hERG1a-Hsp90 complexes, suggesting possible impaired hERG1a trafficking. In conclusion, mitragynine inhibits hERG1a/1b current through direct channel blockade at lower concentration, but at higher concentration, it upregulates the complexation of hERG1a-Hsp90 which may be inhibitory towards channel trafficking.

Heat shock protein 90 (Hsp90) is a widely investigated target for anticancer therapy. The experimental Hsp90 inhibitors ICPD47 and ICPD62 demonstrated anticancer activity against colorectal, osteosarcoma and cervical cancer cell lines. However, their anticancer activity has not been investigated against pancreatic cancer cell lines yet, and there are no data about synergistic activity of these compounds in combination with clinically used anticancer agents. Pancreatic cancer cell lines, MIA PaCa-2 and PANC-1 were exposed to ICPD47 and ICPD62 alone and in combinations with antimetabolites gemcitabine (GEM), 5-fluorouracil (5-FU) and topoisomerase inhibitor doxorubicin (DOX). Effects on cell viability were determined by MTT assay. The synergistic activity was evaluated using Chou-Talalay method. Also, 3D cell cultures were formed using 3D Bioprinting method and the activity of each compound and their combinations was examined by measuring the size change of spheroids. The strongest synergistic activities were determined in combinations using all ratios of ICPD47 with GEM and ICPD62 with GEM in MIA PaCa-2 cell line (combination index <0.5). The combinations of ICPD47 with 5-FU and ICPD47 with GEM in a ratio of 1:5 showed the greatest effect on tumour spheroid growth in both cell lines. The ICPD47 in combination with mild hyperthermia showed significant results, where the EC 50 value in PANC-1 cell line dropped from 4.04 ± 0.046 to 1.68 ± 0.004 µM. The ICPD47 and ICPD62 under the same conditions could act synergistically with GEM, 5-FU and DOX and is worth of further investigations, and studies of synergistic effect is a promising path for more efficient anticancer therapies.

Renal hypoxia and loss of proximal tubular cells (PTC) are relevant in diabetic nephropathy. Hypoxia inhibits hypoxia-inducible factor-1α (HIF-1α) degradation, which leads to cellular adaptive responses through HIF-1-dependent activation of gene hypoxia-responsive elements (HRE). However, the diabetic microenvironment represses the HIF-1/HRE response in PTC. Here we studied the mechanism and consequences of impaired HIF-1α regulation in human proximal tubular HK-2 cells incubated in hyperglycemia. Inhibition at different levels of the canonical pathway of HIF-1α degradation did not activate the HIF-1/HRE response under hyperglycemia, except when proteasome was inhibited. Further studies suggested that hyperglycemia disrupts the interaction of HIF-1α with Hsp90, a known cause of proteasomal degradation of HIF-1α. Impaired HIF-1α regulation in cells exposed to hyperglycemic, hypoxic diabetic-like milieu led to diminished production of vascular endothelial growth factor-A and inhibition of cell migration (responses respectively involved in tubular protection and repair). These effects, as well as impaired HIF-1α regulation, were reproduced in normoglycemia in HK-2 cells incubated with microparticles released by HK-2 cells exposed to diabetic-like milieu. In summary, these results highlight the role of proteasome-dependent mechanisms of HIF-1α degradation on diabetes-induced HK-2 cells dysfunction and suggest that cell-derived microparticles may mediate negative effects of the diabetic milieu on PTC.

Extracellular heat shock protein-90alpha (eHsp90α) plays an essential role in tumour invasion and metastasis. The plasma eHsp90α levels in patients with various cancers correlate with the stages of the diseases. Nonetheless, the mechanism of action by tumour-secreted eHsp90α remained unclear. Here we show that eHsp90α accounts for approximately 1% of the total cellular Hsp90α and is associated with tumour-secreted exosomes. CRISPR-cas9 knockout of Hsp90α did not affect the overall distribution and quantity of secreted exosomes, but it caused increased exosome-associated CD9 and decreased exosome-associated TSG101, Alix, and CD63. However, Hsp90α-knockout tumour cells have not only lost their own constitutive motility, but also the ability to recruit stromal cells via secreted exosomes. These defects are specifically due to the lack of eHsp90α on tumour cell-secreted exosomes. Anti-Hsp90α antibody nullified the pro-motility activity of tumour-secreted exosomes and human recombinant Hsp90α protein fully rescued the functional defects of eHsp90α-free exosomes. Finally, while current exosome biogenesis models exclusively implicate the luminal location of host cytosolic proteins inside secreted exosomes, we provide evidence for eHsp90α location on the external surface of tumour-secreted exosomes. Taken together, this study elucidates a new mechanism of action by exosome-associated eHsp90α.

Leishmaniasis is a neglected disease that affects millions of individuals around the world. Regardless of clinical form, treatment is based primarily on the use of pentavalent antimonials. However, such treatments are prolonged and present intense side effects, which lead to patient abandonment in many cases. The search for chemotherapeutic alternatives has become a priority. Heat Shock Protein 90 (Hsp90) inhibitors have recently come under investigation due to antiparasitic activity in Plasmodium sp., Trypanosoma sp. and Leishmania sp. Some of these inhibitors, such as geldanamycin and its analogs, 17-AAG and 17-DMAG, bind directly to Hsp90, thereby inhibiting its activity. Previous studies have demonstrated that different parasite species are more susceptible to some of these inhibitors than host cells. We hypothesized that this increased susceptibility may be due to differences in binding of Hsp90 inhibitors to Leishmania protein compared to host protein. Based on the results of the in silico approach used in the present study, we propose that geldanamycin, 17-AAG and 17-DMAG present an increased tendency to bind to the N-terminal domain of Leishmania amazonensis Hsp83 in comparison to human Hsp90. This could be partially explained by differences in intermolecular interactions between each of these inhibitors and Hsp83 or Hsp90. The present findings demonstrate potential for the use of these inhibitors in the context of anti-Leishmania therapy.

The honey bee acetylcholinesterase 1 (AmAChE1) has been suggested to be related to stress response as judged from its elevated expression level under brood rearing-suppressed conditions. To further investigate the involvement of AmAChE1 expression in the stress response and its physiological functions, we analyzed altered expression profiles of AmAChE1 induced by diverse stress factors. In addition, transcription profiles of several heat shock protein (Hsp) genes (hsps) and the vitellogenin (Vg) gene (vg) known as general stress markers were investigated as positive references. Among the tested stress conditions, AmAChE1 expression was induced under the brood rearing-suppressed, crowding and heat shock conditions. The hsps, particularly hsp70 and hsp90, responded to seven of nine stress conditions tested, confirming that hsp expression profiles can serve as a general stress marker. Taken together, AmAChE1 expression is not suitable for using as a stress marker due to its limited response. Nevertheless, AmAChE1 expression appears to be connected, at least in part, to heat shock response and other pathways. Considering that AmAChE1 likely regulates the ACh titer particularly in non-neuronal tissues, thereby modulating the signal cascades mediated by mAChR, the AmAChE1 expression profile under different conditions likely provides important information on its physiological roles in honey bees.

AIP56 (apoptosis inducing protein of 56 kDa) is a key virulence factor secreted by virulent strains of Photobacterium damselae subsp. piscicida (Phdp), a Gram-negative bacterium that causes septicemic infections in several warm water marine fish species. AIP56 is systemically disseminated during infection and induces massive apoptosis of host macrophages and neutrophils, playing a decisive role in the disease outcome. AIP56 is a single-chain AB-type toxin, being composed by a metalloprotease A domain located at the N-terminal region connected to a C-terminal B domain, required for internalization of the toxin into susceptible cells. After binding to a still unidentified surface receptor, AIP56 is internalised through clathrin-mediated endocytosis, reaches early endosomes and translocates into the cytosol through a mechanism requiring endosomal acidification and involving low pH-induced unfolding of the toxin. At the cytosol, the catalytic domain of AIP56 cleaves NF-κB p65, leading to the apoptotic death of the intoxicated cells. It has been reported that host cytosolic factors, including host cell chaperones such as heat shock protein 90 (Hsp90) and peptidyl-prolyl cis/trans isomerases (PPIases), namely cyclophilin A/D (Cyp) and FK506-binding proteins (FKBP) are involved in the uptake of several bacterial AB toxins with ADP-ribosylating activity, but are dispensable for the uptake of other AB toxins with different enzymatic activities, such as Bacillus anthracis lethal toxin (a metalloprotease) or the large glycosylating toxins A and B of Clostridium difficile. Based on these findings, it has been proposed that the requirement for Hsp90/PPIases is a common and specific characteristic of ADP-ribosylating toxins. In the present work, we demonstrate that Hsp90 and the PPIases cyclophilin A/D are required for efficient intoxication by the metalloprotease toxin AIP56. We further show that those host cell factors interact with AIP56 in vitro and that the interactions increase when AIP56 is unfolded. The interaction with Hsp90 was also demonstrated in intact cells, at 30 min post-treatment with AIP56, suggesting that it occurs during or shortly after translocation of the toxin from endosomes into the cytosol. Based on these findings, we propose that the participation of Hsp90 and Cyp in bacterial toxin entry may be more disseminated than initially expected, and may include toxins with different catalytic activities.