Regulation Of Hsp70 Research Articles

Potential synergistic regulation of hsp70 and antioxidant enzyme genes in Pyropia yezoensis under high temperature stress

High temperatures have been regarded as one of the main factors limiting Pyropia yezoensis cultivation in recent years. Both the heat shock proteins and antioxidant enzymes genes were activated timely before heat damage to the cell. Here, the activities of different antioxidant enzymes under different conditions, including HSF inhibitor, CaM inhibitor, and HSP70 protein inhibitor were determined. Then, RNA-seq was performed using samples that had been pretreated with corresponding chemicals under high temperature conditions. The principal component analysis inferred that the shift of gene transcription patterns under high temperature conditions was closely related to HSF since the addition of QR suppressed the changes in gene transcription pattern. The hsp70 genes and antioxidant enzyme genes were categorized into three groups by the method of WGCNA. And the HSE sites were identified using plantPAN and FIMO in most genes of the categories showed relationship to HSF. Quantitative RT-PCR analysis of designated genes (FMPK >10) indicated that the expression of hsp70 and antioxidant enzyme genes in Py. yezoensis showed various transcriptional patterns, including upregulation, downregulation, and no significant changes in response to high temperature stress. The classification between qRT-PCR and WGCNA results showed a certain consistency. Considered the results above together, it was speculated that there were different HSFs regulated the expression of different genes of hsp70 and antioxidant enzymes, and HSP70 might also regulate the expression of anti-oxidase genes under the conditions of long-term stress. Additionally, transcription factors other than HSF might be involved in the high temperature stress response. The results indicated the complexity of heat stress response mechanisms in Py. yezoensis.

Protective effects of L-theanine and dihydromyricetin on reproductive function in male mice under heat stress.

Heat stress can impair the male reproductive function. L-Theanine and dihydromyricetin have biological activities against heat stress; however, their effects on reproductive function in heat-stressed males are unclear. In this study, male mice were given L-theanine, dihydromyricetin, or a combination of both for 28 days, followed by 2 h of heat stress daily for 7 days. All interventions alleviated heat stress-induced testicular damage, improving the testicular organ index, sperm density, acrosome integrity, sperm deformity rate, and hormone levels. Treatment increased the antioxidant enzyme activity and decreased the markers of oxidative and inflammatory stress in the testes. A combination dose of 200 + 200 mg kg-1 d-1 showed the best protective effect. The potential mechanism involves the regulation of HSP27 and HSP70, which regulate the levels of reproductive hormones through the StAR/Cyp11a1/Hsd3b1/Cyp17a1/Hsd17b3 pathway, alleviate inflammation and oxidative stress through the P38/NF-κB/Nrf2/HO-1 pathway, and regulate the Bcl-2/Fas/Caspase3 apoptotic pathway. Overall, L-theanine and dihydromyricetin may play a protective role against heat stress-induced reproductive dysfunction, suggesting their potential use in heat stress-resistant foods.

Effects of Inhibition of IKK Kinase Phosphorylation On the Cellular Defence System and HSP90 Activity

The present study was conceived to examine the effects of inhibition of BMS-345541 mediated IKK kinase phosphorylation on the cellular defence system as well as on anti-inflammatory response and HSP90 activity. The analysis was conducted in A549 cell line, since such cells carry a homozygous Keap1 mutation (G333C) that alters its interaction with Nrf2. Recent data have highlighted that Keap1, HSP90 protein and IKK kinase interact reciprocally and particularly Keap1 protein is involved in HSP90 and anti-oxidative pathway regulation. The activities of COX2 and HO1 were investigated by real time and immunoblot analysis along with the synthesis and activity of inducible forms of heat shock protein HSP90. Pre-treatment with IKK kinase inhibitor proved to be a protective means to lower the activity of inflammatory cascade, so preventing the formation of excessive amounts of pro-inflammatory molecules. The inhibitor of IKK kinase BMS-345541 was added to cultured A549 cells before the Escherichia coli lipopolysaccharide (LPS) addition. The viability of the cells was determined after 1–24 h incubation with BMS-345541 at concentrations ranging from 1,25–5 µM. It was found that 1 µM concentration does not significantly affected cell viability (data not shown). As a result, the treatment with 1 μM of BMS-345541 induces the inhibition of IKK phosphorylation. In the A549 cells treated with BMS-345541 and LPS, COX2 activity is not induced: mRNA and protein levels have not increased, while there is an increase in the level of HSP90, HO1 proteins and mRNA. The results suggest that the IKK inhibition is effective in the reduction of the inflammatory response thanks to mechanisms involving both the heat shock cellular defense system and the antioxidative pathway.Graphical

The implications of exercise in Drosophila melanogaster: insights into Akt/p38 MAPK/Nrf2 pathway associated with Hsp70 regulation in redox balance maintenance.

This study investigated the potential effects of exercise on the responses of energy metabolism, redox balance maintenance, and apoptosis regulation in Drosophila melanogaster to shed more light on the mechanisms underlying the increased performance that this emerging exercise model provides. Three groups were evaluated for seven days: the control (no exercise or locomotor limitations), movement-limited flies (MLF) (no exercise, with locomotor limitations), and EXE (with exercise, no locomotor limitations). The EXE flies demonstrated greater endurance-like tolerance in the swimming test, associated with increased citrate synthase activity, lactate dehydrogenase activity and lactate levels, and metabolic markers in exercise. Notably, the EXE protocol regulated the Akt/p38 MAPK/Nrf2 pathway, which was associated with decreased Hsp70 activation, culminating in glutathione turnover regulation. Moreover, reducing the locomotion environment in the MLF group decreased endurance-like tolerance and did not alter citrate synthase activity, lactate dehydrogenase activity, or lactate levels. The MLF treatment promoted a pro-oxidant effect, altering the Akt/p38 MAPK/Nrf2 pathway and increasing Hsp70 levels, leading to a poorly-regulated glutathione system. Lastly, we demonstrated that exercise could modulate major metabolic responses in Drosophila melanogaster aerobic and anaerobic metabolism, associated with apoptosis and cellular redox balance maintenance in an emergent exercise model.

Aha1 regulates Hsp90’s conformation and function in a stoichiometry-dependent way

The heat shock protein 90 (Hsp90) is a molecular chaperone, which plays a key role in eukaryotic protein homeostasis. Co-chaperones assist Hsp90 in client maturation and in regulating essential cellular processes such as cell survival, signal transduction, gene regulation, hormone signaling, and neurodegeneration. Aha1 (activator of Hsp90 ATPase) is a unique co-chaperone known to stimulate the ATP hydrolysis of Hsp90, but the mechanism of their interaction is still unclear. In this report, we show that one or two Aha1 molecules can bind to one Hsp90 dimer and that the binding stoichiometry affects Hsp90’s conformation, kinetics, ATPase activity, and stability. In particular, a coordination of two Aha1 molecules can be seen in stimulating the ATPase activity of Hsp90 and the unfolding of the middle domain, whereas the conformational equilibrium and kinetics are hardly affected by the stoichiometry of bound Aha1. Altogether, we show a regulation mechanism through the stoichiometry of Aha1 going far beyond a regulation of Hsp90’s conformation.

Tanespimycin and rapamycin exhibit antifungal activity against Colletotrichum gloeosporioides and enhance mango resistance to anthracnose via differentially modulating heat shock response

Postharvest mango (Mangifera indica L.) fruit suffer tremendous loss due to anthracnose inflicted by Colletotrichum spp. Fungicides and heat treatment are often used to control the disease, while biocontrol agents are eco-friendly alternatives. Streptomyces are antagonistic organisms against plant pathogens; however, the mechanisms and active components are unclear. Here, we evaluated the antifungal actions of S. hygroscopicus metabolites tanespimycin and rapamycin against anthracnose. Within the range of 0.01–0.1 μM, tanespimycin and rapamycin reduced fungal growth by at least 60.4% and 40.4%, respectively in vitro. In mango fruit, tanespimycin upregulated MiHSP101, MiHSP90, MiHSP70, and MiHSP18.2 by 5.8-, 1.9-, 4.8-, and 3.3-fold, respectively, and increased HSP101 and HSP70 protein by 2.2-fold. On the other hand, rapamycin did not affect HSP expression. Pathogen-challenge assays revealed tanespimycin and rapamycin exerted antifungal and defense-eliciting dual actions, where pre-challenge treatment of tanespimycin and rapamycin inhibited pathogen growth by 73.7% and 78.4%, respectively in mango 5 d post infection. Finally, in a 20 °C storage experiment, tanespimycin and rapamycin extended the fruit shelf life by 6 d and exerted comparable antifungal effects as hot water treatment without deteriorating fruit quality. In conclusion, we elucidated the antifungal and defense-eliciting actions of S. hygroscopicus metabolites and revealed mechanistic discrepancies regarding HSP regulation. These findings support the development of new disease management strategies and advance the knowledge regarding the roles of HSP in crop defense.

Differential expression profile of genes involved in the immune response associated to progression of chronic Chagas disease.

Patients with chronic Chagas disease present marked clinical and immunological heterogeneity. During the disease, multiple immune mechanisms are activated to fight the parasite. The purpose of this study was to investigate the expression patterns of genes involved in relevant immunological processes throughout the disease in patients with chronic Chagas disease. High-throughput RT-qPCR with QuantStudio 12K Flex real-time PCR system was used to evaluate the expression of 106 immune-related genes in PBMC from a cohort of cardiac Chagas disease patients (CCC I), asymptomatic patients (IND) and healthy donors (HD) after being stimulated with T. cruzi soluble antigens. Principal component analysis (PCA), cluster analysis and volcano plots were used to identify differentially expressed genes. In addition, gene set enrichment analysis (GSEA) was employed to identify the enriched immunological pathways in which these genes are involved. PCA revealed the existence of a statistically divergent expression profile of the 36 genes correlated with PC1 between CCC I patients and HD (p < 0.0001). Differential gene expression analysis revealed upregulation of 41 genes (expression fold-change > 1.5) and downregulation of 14 genes (expression fold-change < 0.66) (p = 8.4x10-13 to p = 0.007) in CCC I patients versus HD. Furthermore, significant differences in the expression level of specific genes have been identified between CCC I and IND patients (8 up and 1 downregulated). GSEA showed that several upregulated genes in CCC I patients participate in immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, cytokines-inflammatory response and IL-10 anti-inflammatory signaling. Cardiac Chagas disease patients show an antigen-specific differential gene expression profile in which several relevant immunological pathways seem to be activated. Assessment of gene expression profiles reveal unique insights into the immune response that occurs along chronic Chagas disease.

Cadmium exposure promotes ferroptosis by upregulating Heat Shock Protein 70 in vascular endothelial damage of zebrafish

Cadmium (Cd) exposure is a risk factor for endothelial dysfunction and cardiovascular disease. Ferroptosis is a type of cell death that relies on lipid peroxidation. Whether ferroptosis acts in Cd-induced vascular endothelial damage and the underlying mechanisms remain unclear. Herein, we found that Cd resulted in ferroptosis of vascular endothelial cells (ECs) in vivo and in vitro. In the visualized zebrafish embryos, Cd accumulated in vascular ECs, ROS and lipid peroxidation levels were increased, and the oxidoreductase system was disturbed after exposure. Moreover, Cd decreased Gpx4 in ECs and caused smaller mitochondria with increased membrane density. Accompanied by ferroptosis, the number of ECs and the area of the caudal venous plexus in zebrafish embryos were reduced, and the survival rate of HUVECs decreased. These effects were partially reversed by ferrostatin-1 and aggravated by erastin. Mechanistically, an excessive increase in Heat Shock Protein 70 (Hsp70) was identified by transcriptomics after Cd exposure. Inhibition of Hsp70 by VER-155008 or siRNA ameliorated Cd-induced ferroptosis, thereby alleviating endothelial injury. Furthermore, Hsp70 regulated Cd-induced ferroptosis by targeting multiple targets, including Gpx4, Fth1, Nrf2 and Acsl4. Our findings provide a new approach to investigating the endothelial damage of Cd and indicate that regulation of Hsp70 is an important target for alleviating this process.

The effects of Pandanus tectorius leaf extract on the resistance of White-leg shrimp Penaeus vannamei towards pathogenic Vibrio parahaemolyticus

Pandanus tectorius leaf extract effect on the White-leg shrimp Penaeus vannamei tolerance against Vibrio parahaemolyticus were investigated in this study. Thirty shrimp post-larvae measured at approximately 1 cm were exposed for 24 h to 0.5, 1, 2, 3, 4, 5 and 6 g/L leaf extract and subsequently observed for survival and immune-related genes expression (Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase), followed by determination of their tolerance and histological tissue profiles upon Vibrio challenge. Survival of shrimps treated with 6 g/L of leaf extract improved by up to 95% to controls. Hsp70, crustin, and prophenoloxidase mRNA levels were observed to be 8.5, 10.4, and 1.5-fold higher, respectively. Histopathological analysis of the hepatopancreas and the muscle tissues revealed major tissue degeneration in Vibrio-challenged shrimps but not in shrimps primed with P. tectorius leaf extract. Of all the dose examined, the best pathogen resistance results were obtained with a 24 h incubation of shrimp in 6 g/L P. tectorius methanolic leaf extract. The tolerance towards V. parahaemolyticus might be associated with the increased regulation of Hsp70, prophenoloxidase and crustin upon exposure to the extract, all immune-related proteins essential for pathogen elimination in Penaeid shrimp. The present study primarily demonstrated that P. tectorius leaf extract is a viable alternative for enhancing P. vannamei post-larvae resistance against V. parahaemolyticus, a major bacterial pathogen in aquaculture.

Analysis of signaling cascades from myeloma cells treated with pristimerin.

Multiple myeloma (MM) is the 2nd most frequently diagnosed blood cancer after non-Hodgkin's lymphoma. The present study aimed to identify the differentially expressed genes (DEGs) between the control and pristimerin-treated MM cell lines. We examined the GSE14011 microarray dataset and screened DEGs with GEO2R statistical tool using the inbuilt limma package. We used a bioinformatics pipeline to identify the differential networks, signaling cascades, and the survival of the hub genes. We implemented two different enrichment analysis including ClueGO and Metacore™, to get accurate annotation for most significant DEGs. We screened the most significant 408 DEGs from the dataset based on p-values and logFC values. Using protein network analysis, we found the genes UBC, HSP90AB1, HSPH1, HSPA1B, HSPA1L, HSPA6, HSPD1, DNAJB1, HSPE1, DNAJC10, BAG3, and DNAJC7 had higher node degree distribution. In contrast, the functional annotation provided that the DEGs were predominantly enriched in B-cell receptor signaling, unfolded protein response, positive regulation of phagocytosis, HSP70, and HSP40-dependent folding, and ubiquitin-proteasomal proteolysis. Using network algorithms, and comparing enrichment analysis, we found the hub genes enriched were INHBE, UBC, HSPA1A, HSP90AB1, IKBKB, and BAG3. These DEGs were further validated with overall survival and gene expression analysis between the tumor and control groups. Finally, pristimerin effects were validated independently in a cell line model consisting of IM9 and U266 MM cells. Pristimerin induced in vitro cytotoxicity in MM cells in a dose-dependent manner. Pristimerin inhibited NF-κB, induced accumulation of ubiquitinated proteins and inhibited HSP60 in the validation of bioinformatics findings, while pristimerin-induced caspase-3 and PARP cleavage confirmed cell death. Taken together, we found that the identified DEGs were strongly associated with the apoptosis induced in MM cell lines due to pristimerin treatment, and combinatorial therapy derived from pristimerin could act as novel anti-myeloma multifunctional agents.

Detecting Posttranslational Modifications of Hsp90 Isoforms.

The molecular chaperone heat shock protein 90 (Hsp90) is essential in eukaryotes. Hsp90 chaperones proteins that are important determinants of multistep carcinogenesis. There are multiple Hsp90 isoforms including the cytosolic Hsp90α and Hsp90β as well as GRP94 located in the endoplasmic reticulum and TRAP1 in the mitochondria. The chaperone function of Hsp90 is linked to its ability to bind and hydrolyze ATP. Co-chaperones and posttranslational modifications (such as phosphorylation, SUMOylation, and ubiquitination) are important for Hsp90 stability and regulation of its ATPase activity. Both mammalian and yeast cells can be used to express and purify Hsp90 and TRAP1 and also detect post-translational modifications by immunoblotting.

Impact of Co-chaperones and Posttranslational Modifications Toward Hsp90 Drug Sensitivity.

Posttranslational modifications (PTMs) regulate myriad cellular processes by modulating protein function and protein-protein interaction. Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone whose activity is responsible for the stabilization and maturation of more than 300 client proteins. Hsp90 is a substrate for numerous PTMs, which have diverse effects on Hsp90 function. Interestingly, many Hsp90 clients are enzymes that catalyze PTM, demonstrating one of the several modes of regulation of Hsp90 activity. Approximately 25 co-chaperone regulatory proteins of Hsp90 impact structural rearrangements, ATP hydrolysis, and client interaction, representing a second layer of influence on Hsp90 activity. A growing body of literature has also established that PTM of these co-chaperones fine-tune their activity toward Hsp90; however, many of the identified PTMs remain uncharacterized. Given the critical role of Hsp90 in supporting signaling in cancer, clinical evaluation of Hsp90 inhibitors is an area of great interest. Interestingly, differential PTM and co-chaperone interaction have been shown to impact Hsp90 binding to its inhibitors. Therefore, understanding these layers of Hsp90 regulation will provide a more complete understanding of the chaperone code, facilitating the development of new biomarkers and combination therapies.

Ydj1 interaction at nucleotide-binding-domain of yeast Ssa1 impacts Hsp90 collaboration and client maturation.

Hsp90 constitutes one of the major chaperone machinery in the cell. The Hsp70 assists Hsp90 in its client maturation though the underlying basis of the Hsp70 role remains to be explored. In the present study, using S. cerevisiae strain expressing Ssa1 as sole Ssa Hsp70, we identified novel mutations in the nucleotide-binding domain of yeast Ssa1 Hsp70 (Ssa1-T175N and Ssa1-D158N) that adversely affect the maturation of Hsp90 clients v-Src and Ste11. The identified Ssa1 amino acids critical for Hsp90 function were also found to be conserved across species such as in E.coli DnaK and the constitutive Hsp70 isoform (HspA8) in humans. These mutations are distal to the C-terminus of Hsp70, that primarily mediates Hsp90 interaction through the bridge protein Sti1, and proximal to Ydj1 (Hsp40 co-chaperone of Hsp70 family) binding region. Intriguingly, we found that the bridge protein Sti1 is critical for cellular viability in cells expressing Ssa1-T175N (A1-T175N) or Ssa1-D158N (A1-D158N) as sole Ssa Hsp70. The growth defect was specific for sti1Δ, as deletion of none of the other Hsp90 co-chaperones showed lethality in A1-T175N or A1-D158N. Mass-spectrometry based whole proteome analysis of A1-T175N cells lacking Sti1 showed an altered abundance of various kinases and transcription factors suggesting compromised Hsp90 activity. Further proteomic analysis showed that pathways involved in signaling, signal transduction, and protein phosphorylation are markedly downregulated in the A1-T175N upon repressing Sti1 expression using doxycycline regulatable promoter. In contrast to Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N), the stress inducible Hsp70 isoform, supported cell growth even in the absence of Sti1. Overall, our data suggest that Ydj1 competes with Hsp90 for binding to Hsp70, and thus regulates Hsp90 interaction with the nucleotide-binding domain of Hsp70. The study thus provides new insight into the Hsp70-mediated regulation of Hsp90 and broadens our understanding of the intricate complexities of the Hsp70-Hsp90 network.

Hsp90 and Associated Co-Chaperones of the Malaria Parasite.

Heat shock protein 90 (Hsp90) is one of the major guardians of cellular protein homeostasis, through its specialized molecular chaperone properties. While Hsp90 has been extensively studied in many prokaryotic and higher eukaryotic model organisms, its structural, functional, and biological properties in parasitic protozoans are less well defined. Hsp90 collaborates with a wide range of co-chaperones that fine-tune its protein folding pathway. Co-chaperones play many roles in the regulation of Hsp90, including selective targeting of client proteins, and the modulation of its ATPase activity, conformational changes, and post-translational modifications. Plasmodium falciparum is responsible for the most lethal form of human malaria. The survival of the malaria parasite inside the host and the vector depends on the action of molecular chaperones. The major cytosolic P. falciparum Hsp90 (PfHsp90) is known to play an essential role in the development of the parasite, particularly during the intra-erythrocytic stage in the human host. Although PfHsp90 shares significant sequence and structural similarity with human Hsp90, it has several major structural and functional differences. Furthermore, its co-chaperone network appears to be substantially different to that of the human host, with the potential absence of a key homolog. Indeed, PfHsp90 and its interface with co-chaperones represent potential drug targets for antimalarial drug discovery. In this review, we critically summarize the current understanding of the properties of Hsp90, and the associated co-chaperones of the malaria parasite.

Characterization of thermo-physiological, hematological, and molecular changes in response to seasonal variations in two tropically adapted native cattle breeds of Bos indicus lineage in hot arid ambience of Thar Desert.

The selection of climate resilient animal is necessary to secure the future of sustainable animal production. The present investigation therefore was an effort to unravel answers to the adaptation at physiological, hematological, and molecular levels in cows of hot arid region that helps them to survive harsh environment, to continue production and reproduction. This investigation was carried out in indicine cows over a period of one year, encompassing four seasons, wherein physiological data of 50 animals, hematological data of 15 animals, and gene expression profile of 5 animals from each of Sahiwal and Kankrej breeds per season was generated. In total, 5600 physiological observations, 1344 hematological observations, and 480 molecular samples were processed. The meteorological data revealed a high diurnal variation of temperature across seasons, with THI exceeding 80 during the months of summer and hot-humid seasons, indicating significant heat stress (HS). The physiological parameters showed an increasing trend with the incremental THI, with significantly (p < 0.05) higher values of rectal temperature (RT), respiration rate (RR), pulse rate (PR), and body surface temperature (BST) at ventral (VT), lateral (LT), dorsal (DT), and frontal (FT), in both breeds recorded during HS. The hematological pictures also revealed significant (p < 0.05) seasonal perturbations in erythrocytic and leucocytic parameters. Moreover, the molecular response was driven by a significant (p < 0.05) upregulation of all the key HSPs, HSP70, HSP90, HSP60, and HSP40, except HSP27 during the hotter months of summer and hot-humid seasons. The expression of HSF1, an important transcriptional regulator ofHSP70 was also significantly (p < 0.05) upregulated during summer season in both breeds. All the molecular chaperones revealed a significant upregulation during the summer season, followed by a decreasing trend by hot-humid season. The study indicated a well-developed thermotolerance mechanism in animals of both breeds, with Kankrej cows exhibiting better thermotolerance compared to Sahiwal cows.

The Keap1 signaling in the regulation of HSP90 pathway

The Keap1 protein is the master modulator of Nrf2 pathway; moreover, it is the hub of such important processes as cancer, cell stress, inflammation, and chemio- and radio-resistance. That is why Keap1 has become an intriguing pharmacological target. Many recent data show that Keap1 interacts with HSP90 protein. In this study, we use ferulic acid (FA) as antioxidant and anti-inflammatory agent, able to relieve inflammatory response. It is known that treatment with 100 μg of FA can significantly decrease the oxidative stress, so it turns to be useful to study the antioxidant regulation. The RAW 264.7 cells transfected with si-Keap1 and LPS treated are the in vitro model used to study the effects of Keap1 silencing on HSP90 activities and the FA antioxidant modulation. Immunoblot data and qPCR analysis show that Keap1 is involved in HSP90 modulation and on anti-oxidative response. Keap1 silencing affects negatively COX2 activation; in fact western blot and qPCR analysis conducted on RAW 264.7 cells Keap1silenced highlight that LPS treatment does not induce COX2 activation. In addition, the FA anti-oxidative and modulatory effect is abolished in COX2 pathway. The same results are point out using human A549 cell line with an allelic mutation on Keap1 gene, and the protein results are partially inactive. This preliminary study points out that Keap1protein is involved in HSP90 and anti-oxidative pathway regulation.

A novel C-terminal heat shock protein 90 inhibitor that overcomes STAT3-Wnt-β-catenin signaling-mediated drug resistance and adverse effects.

Rationale: The heat shock protein (Hsp) system plays important roles in cancer stem cell (CSC) and non-CSC populations. However, limited efficacy due to drug resistance and toxicity are obstacles to clinical use of Hsp90 inhibitors, suggesting the necessity to develop novel Hsp90 inhibitors overcoming these limitations.Methods: The underlying mechanism of resistance to Hsp90 inhibitors was investigated by colony formation assay, sphere formation assay, western blot analysis, and real-time PCR. To develop anticancer Hsp90 inhibitors that overcome the signal transducer and activator of transcription 3 (STAT3)-mediated resistance, we synthesized and screened a series of synthetic deguelin-based compounds in terms of inhibition of colony formation, migration, and viability of non-small cell lung cancer (NSCLC) cells and toxicity to normal cells. Regulation of Hsp90 by the selected compound NCT-80 [5-methoxy-N-(3-methoxy-4-(2-(pyridin-3-yl)ethoxy)phenyl)-2,2-dimethyl-2H-chromene-6-carboxamide] was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. The antitumor, antimetastatic, and anti-CSC effects of NCT-80 were examined in vitro and in vivo using various assays such as MTT, colony formation, and migration assays and flow cytometric analysis and tumor xenograft models.Results: We demonstrated a distinct mechanism in which Hsp90 inhibitors that block N-terminal ATP-binding pocket causes transcriptional upregulation of Wnt ligands through Akt- and ERK-mediated activation of STAT3, resulting in NSCLC cell survival in an autocrine or paracrine manner. In addition, NCT-80 effectively reduced viability, colony formation, migration, and CSC-like phenotypes of NSCLC cells and their sublines with acquired resistance to anticancer drugs by inducing apoptosis and inhibiting epithelial-mesenchymal transition and the growth of NSCLC patient-derived xenograft tumors without overt toxicity. With regards to mechanism, NCT-80 directly bound to the C-terminal ATP-binding pocket of Hsp90, disrupting the interaction between Hsp90 and STAT3 and degrading STAT3 protein. Moreover, NCT-80 inhibited chemotherapy- and EGFR TKI-induced programmed cell death ligand 1 expression and potentiated the antitumor effect of chemotherapy in the LLC-Luc allograft model.Conclusions: These data indicate the potential of STAT3/Wnt signaling pathway as a target to overcome resistance to Hsp90 inhibitors and NCT-80 as a novel Hsp90 inhibitor that targets both CSCs and non-CSCs in NSCLC.

Structural basis for recognition of the Hsp90 closed, ATP state by the TPR‐containing co‐chaperone FKBP51

The Hsp90 molecular chaperone drives activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct co-chaperone regulators. The FKBP51 immunophilin co-chaperone binds Hsp90 through its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. However, for many TPR cochaperones, the protein:protein interactions and mechanisms which enable Hsp90 specificity and regulation in the folding pathway remain unclear. High resolution structure of the Hsp90:FKBP51:p23 complex was determined using cryo-electron microscopy (EM). Native gel electrophoresis, gel filtration chromatography coupled with multiangle light scattering, and negative stain-EM were used to test interaction of FKBP51/p23 with Hsp90 in different nucleotide binding states. The structure of the Hsp90:FKBP51:p23 complex was verified with mutational analysis and site-specific photocrosslinking experiments. We developed in vitro conditions that promote and stabilize the closed, ATP conformation of Hsp90, enabling characterization of binding interactions between defined open (apo) and closed (ATP) states. We find that FKBP51 preferentially binds the closed, ATP state, forming a complex with a 2:1 stoichiometry that is further stabilized by p23 binding. Using cryo-EM we determined a 3.3 Å structure the Hsp90:FKBP51:p23 complex that reveals distinct contacts by the TPR domain of FKBP51 that define its asymmetric interaction and recognition of C-terminal Hsp90 dimer. We propose a model in which helix 7 of the TPR domain of FKBP51 functions as a critical specificity element for engaging Hsp90 in the p23-stabilized, ATP state to promote client maturation. This arrangement flexibly positions the FK1 domain of FKBP51 adjacent Hsp90 client binding sites in a manner we predict facilitates site-specific PPIase activity towards clients in coordination with the Hsp90 chaperone cycle.

Induction of apoptosis in the gonads of Mytilus edulis by metformin and increased temperature, via regulation of HSP70, CASP8, BCL2 and FAS

Pharmaceutically active compounds have been considered contaminants of emerging concern, in response to evidence that these substances may adversely affect aquatic organisms. Here we expose mussels for 7 days to metformin, the most commonly prescribed anti-diabetes treatment, at a concentration of 40 μg/L and a high temperature of 20 °C. The apoptosis-related genes HSP70, CASP8, BCL2 and FAS showed variation in expression in gonadal tissue. The results suggest that complex interactions between these genes are modulating the onset of apoptotic changes such as atresia and follicle degeneration. The temperature induced apoptosis may be initiated by overexpression of CASP8. Conversely, metformin may induce apoptosis by suppressing the anti-apoptotic gene BCL2, thus promoting the process. Interestingly, apoptosis and follicle degeneration are likely FAS-mediated, following the synergistic effect of metformin and temperature. The potential of metformin to act as a non-traditional EDC, due to its impact on the reproductive system in mussels is discussed.

Differential Expression of Immune Response Genes in Asymptomatic Chronic Chagas Disease Patients Versus Healthy Subjects.

Infection by the Trypanosoma cruzi parasite causes Chagas disease and triggers multiple immune mechanisms in the host to combat the pathogen. Chagas disease has a variable clinical presentation and progression, producing in the chronic phase a fragile balance between the host immune response and parasite replication that keeps patients in a clinically silent asymptomatic stage for years. Since the parasite is intracellular and replicates within cells, the cell-mediated response of the host adaptive immunity plays a critical role. This function is mainly orchestrated by T lymphocytes, which recognize parasite antigens and promote specific functions to control the infection. However, little is known about the immunological markers associated with this asymptomatic stage of the disease. In this large-scale analysis, the differential expression of 106 immune system-related genes has been analyzed using high-throughput qPCR in T. cruzi antigen-stimulated PBMC from chronic Chagas disease patients with indeterminate form (IND) and healthy donors (HD) from endemic and non-endemic areas of Chagas disease. This analysis revealed that there were no differences in the expression level of most genes under study between healthy donors from endemic and non-endemic areas determined by PCA and differential gene expression analysis. Instead, PCA revealed the existence of different expression profiles between IND patients and HD (p < 0.0001), dependent on the 32 genes included in PC1. Differential gene expression analysis also revealed 23 upregulated genes (expression fold change > 2) and 11 downregulated genes (expression fold change < 0.5) in IND patients versus HD. Enrichment analysis showed that several upregulated genes in IND patients participate in relevant immunological pathways such as antigen-dependent B cell activation, stress induction of HSP regulation, NO2-dependent IL12 pathway in NK cells, and cytokine-inflammatory response. The antigen-specific differential gene expression profile detected in these patients and the relevant immunological pathways that seem to be activated could represent potential biomarkers of the asymptomatic form of Chagas disease, helpful to diagnosis and infection control.