Hsp90 ATPase Research Articles

Acacetin alleviates rheumatoid arthritis by targeting HSP90 ATPase domain to promote COX-2 degradation

BackgroundInflammation plays a significant role in initiating and sustaining rheumatoid arthritis (RA). Acacetin, a natural flavonoid compound, exhibits excellent anti-inflammatory effects specifically for RA. However, its relevant targets and molecular mechanisms remain to be elucidated. PurposeThis study aims to investigate the mechanism of acacetin in the therapeutic efficacy of acacetin in RA and search for new therapeutic options for RA treatment. MethodsA collagen-induced RA mouse model was established to evaluate the therapeutic effect of acacetin. Acacetin functional probes were synthesized to capture potential target proteins in RAW264.7 cells. Various small molecule-protein interaction methods were conducted to verify the binding of acacetin to target protein. Molecular docking and site directed mutagenesis tests were performed to analyze the specific binding sites. Co-immunoprecipitation, immunofluorescence assay and western blot were engineered to explore the effect of acacetin on COX-2 degradation by targeting HSP90. ResultsAcacetin specifically binds to the ATP domain of HSP90, to facilitate the dissociation between HSP90 and COX-2, inducing the ubiquitin-degradation of COX-2 in macrophages. Acacetin suppressed the production of pro-inflammatory cytokines, as well as inflammatory related pathways, exerting excellent anti-inflammatory effects in RA. ConclusionsThis research proved that acacetin, a novel HSP90 ATPase inhibitor, inhibits the functional folding of the client protein COX-2, promoting its ubiquitin degradation for anti-inflammation. Targeting HSP90 is a viable strategy to inhibit inflammation, affording a distinct way to managing joint inflammation and pains associated with RA.

Recruitment of Ahsa1 to Hsp90 is regulated by a conserved peptide that inhibits ATPase stimulation

Hsp90 is a molecular chaperone that acts on its clients through an ATP-dependent and conformationally dynamic functional cycle. The cochaperone Accelerator of Hsp90 ATPase, or Ahsa1, is the most potent stimulator of Hsp90 ATPase activity. Ahsa1 stimulates the rate of Hsp90 ATPase activity through a conserved motif, NxNNWHW. Metazoan Ahsa1, but not yeast, possesses an additional 20 amino acid peptide preceding the NxNNWHW motif that we have called the intrinsic chaperone domain (ICD). The ICD of Ahsa1 diminishes Hsp90 ATPase stimulation by interfering with the function of the NxNNWHW motif. Furthermore, the NxNNWHW modulates Hsp90’s apparent affinity to Ahsa1 and ATP. Lastly, the ICD controls the regulated recruitment of Hsp90 in cells and its deletion results in the loss of interaction with Hsp90 and the glucocorticoid receptor. This work provides clues to how Ahsa1 conserved regions modulate Hsp90 kinetics and how they may be coupled to client folding status.

Utilization of the Winkler scale of plants using big data temperature presented by the Korea Meteorological Administration.

Rice is an important food source that can provide a stable supply of calories for most people around the world. However, owing to the recent rapid temperature rise, we are facing social issues related to the increase in the Winkler scale. In this study, a strategy for screening potential candidate genes related to the yield according to the Winkler scale is presented, and the possibility of using a candidate gene identified through sequence haplotype and homology analysis as a breeding source is suggested. QTL for the Winkler scale was identified using a population of 120 double haploids derived from a cross between Cheongchoneg, Indica, and Nagdong, Japonica. A total of 79 candidate genes were detected in the identified QTL region, and OsHAq8 was finally screened. Through haplotype analysis, OsHAq8 was derived from the Indica group and orthologous to Graminae's activator of Hsp90 ATPase, suggesting that it is a candidate gene involved in yield according to temperature during the growing period. The expression level of OsHAq8 increased as the Winkler scale increased. The findings of this study can serve as a crucial indicator for predicting harvest time and grain quality while achieving a stable yield through marker selection and adaptation to climate change. Climate change occurs more frequently. In these situations, it is very important to predict harvest time and apply relevant candidate genes to breeding. The candidate genes presented in this study can be effectively applied to rice breeding in preparation for climate change.

Human Aha1's N-terminal extension confers it holdase activity in vitro.

Molecular chaperones are key components of protein quality control system, which plays an essential role in controlling protein homeostasis. Aha1 has been identified as a co-chaperone of Hsp90 known to strongly accelerate Hsp90's ATPase activity. Meanwhile, it is reported that Aha1 could also act as an autonomous chaperone and protect stressed or disordered proteins from aggregation. Here, in this article, a series of in vitro experiments were conducted to verify whether Aha1 has a non-Hsp90-dependent holdase activity and to elucidate the associated molecular mechanism for substrate recognition. According to the results of the refolding assay, the highly conserved N-terminal extension spanning M1 to R16 in Aha1 from higher eukaryotes is responsible for the holdase activity of the protein. As revealed by the NMR data, Aha1's N-terminal extension mainly adopts a disordered conformation in solution and shows no tight contacts with the core structure of Aha1's N-terminal domain. Based on the intrinsically disordered structure feature and the primary sequence of Aha1's N-terminal extension, the fuzzy-type protein-protein interactions involving this specific region and the unfolded substrate proteins are expected. The following mutation analysis data demonstrated that the Van der Waals contacts potentially involving two tryptophans including W4 and W11 do not play a dominant role in the interaction between Aha1 and unfolded maltose binding protein (MBP). Meanwhile, since the high concentration of NaCl could abolish the holdase activity of Aha1, the electrostatic interactions mediated by those charged residues in Aha1's N-terminal extension are thus indicated to play a crucial role in the substrate recognition.

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.

HSP90AB1 as the Druggable Target of Maggot Extract Reverses Cisplatin Resistance in Ovarian Cancer.

Cisplatin resistance is a crucial factor affecting ovarian cancer patient's survival rate, but the primary mechanism underlying cisplatin resistance in ovarian cancer remains unclear, and this prevents the optimal use of cisplatin therapy. Maggot extract (ME) is used in traditional Chinese medicine for patients with comas and patients with gastric cancer when combined with other drug treatments. In this study, we investigated whether ME enhances the sensitivity of ovarian cancer cells to cisplatin. Two ovarian cancer cells-A2780/CDDP and SKOV3/CDDP-were treated with cisplatin and ME in vitro. SKOV3/CDDP cells that stably expressed luciferase were subcutaneously or intraperitoneally injected into BALB/c nude mice to establish a xenograft model, and this was followed by ME/cisplatin treatment. In the presence of cisplatin, ME treatment effectively suppressed the growth and metastasis of cisplatin-resistant ovarian cancer in vivo and in vitro. RNA-sequencing data showed that HSP90AB1 and IGF1R were markedly increased in A2780/CDDP cells. ME treatment markedly decreased the expression of HSP90AB1 and IGF1R, thereby increasing the expression of the proapoptotic proteins p-p53, BAX, and p-H2AX, while the opposite effects were observed for the antiapoptotic protein BCL2. Inhibition of HSP90 ATPase was more beneficial against ovarian cancer in the presence of ME treatment. In turn, HSP90AB1 overexpression effectively inhibited the effect of ME in promoting the increased expression of apoptotic proteins and DNA damage response proteins in SKOV3/CDDP cells. Inhibition of cisplatin-induced apoptosis and DNA damage by HSP90AB1 overexpression confers chemoresistance in ovarian cancer. ME can enhance the sensitivity of ovarian cancer cells to cisplatin toxicity by inhibiting HSP90AB1/IGF1R interactions, and this might represent a novel target for overcoming cisplatin resistance in ovarian cancer chemotherapy.

A method for reducing the concentrations of Fusarium graminearum trichothecenes in durum wheat grain with the use of Debaryomyces hansenii

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is one of the most dangerous diseases of durum wheat. This hemibiotrophic pathogen transitions from the biotrophic phase, during which it penetrates host tissues and secretes trichothecenes, to the necrotrophic phase which leads to the destruction of host tissues. Yeasts applied to spikes often reduce mycotoxin concentrations, but the underlying mechanisms have not been fully elucidated. Therefore, the aim of this study was to analyze the concentrations trichothecenes in durum wheat grain and changes in the F. graminearum transcriptome under the influence the Debaryomyces hansenii antagonistic yeast strain. Debaryomyces hansenii cells adhered to and formed cell aggregates/biofilm on the surface of spikes and pathogenic hyphae. Biological control suppressed the spread of F. graminearum by 90 % and decreased the content of deoxynivalenol (DON) in spikes by 31.2 %. Yeasts significantly reduced the expression of pathogen's genes encoding the rpaI subunit of RNA polymerase I and the activator of Hsp90 ATPase, but they had no effect on mRNA transcript levels of genes encoding the enzymes involved in the biosynthesis of trichothecenes. The yeast treatment reduced the number of F. graminearum operational taxonomic units (OTUs) nearly five-fold and increased the number of D. hansenii OTUs more than six-fold in the spike mycobiome. The mechanisms that suppress infections should be explored to develop effective biological methods for reducing the concentrations mycotoxins in wheat grain.

Exploring Hsp90 as a possible pseudo-substrate receptor for E3 ligases.

Hsp90 is a chaperone protein responsible for aiding in the proper folding of its client proteins, particularly in response to cellular stresses such as heat shock. It has been demonstrated in human cell lines that inhibition of Hsp90's ATPase activity, which is required for its chaperone activity, leads to proteolytic degradation of c-Raf, which is an oncogenic kinase in the MAPK/ERK pathway and a known client protein of Hsp90. In eukaryotes, the primary mechanism of protein degradation is through the ubiquitin-proteasome system, in which E3 ligases post-translationally modify substrate proteins with ubiquitin, which marks the protein for degradation by the proteasome. It has been shown that siRNA silencing of the E3 ligases HECTD3 and Cullin5 in Hsp90-inhibited cells leads to recovery of c-Raf, suggesting that HECTD3 and Cullin5 are responsible for initiating the degradation of c-Raf. Furthermore, it has been shown that increasing concentrations of the Hsp90 inhibitor AUY922 causes HECTD3 to pull down both c-Raf and Hsp90, implying that HECTD3, and perhaps also Cullin5, directly interact with Hsp90 in order to immediately ubiquitinate misfolded Hsp90 clients and mark them for proteasomal degradation. In order to investigate the validity of this model, we are expressing these proteins in vitro and will report which proteins directly interact with which, with the goal of reconstituting the full E3 ligase complex responsible for c-Raf ubiquitination.

P23 and Aha1: Distinct Functions Promote Client Maturation.

Hsp90 is a conserved molecular chaperone regulating the folding and activation of a diverse array of several hundreds of client proteins. The function of Hsp90 in client processing is fine-tuned by a cohort of co-chaperones that modulate client activation in a client-specific manner. They affect the Hsp90 ATPase activity and the recruitment of client proteins and can in addition affect chaperoning in an Hsp90-independent way. p23 and Aha1 are central Hsp90 co-chaperones that regulate Hsp90 in opposing ways. While p23 inhibits the Hsp90 ATPase and stabilizes a client-bound Hsp90 state, Aha1 accelerates ATP hydrolysis and competes with client binding to Hsp90. Even though both proteins have been intensively studied for decades, research of the last few years has revealed intriguing new aspects of these co-chaperones that expanded our perception of how they regulate client activation. Here, we review the progress in understanding p23 and Aha1 as promoters of client processing. We highlight the structures of Aha1 and p23, their interaction with Hsp90, and how their association with Hsp90 affects the conformational cycle of Hsp90 in the context of client maturation.

Maggot Extract Inhibits Cell Migration and Tumor Growth by Targeting HSP90AB1 in Ovarian Cancer.

Ovarian cancer is one of the most lethal gynecological malignancies, because of metastatic dissemination with poor late clinical therapy. Maggots have been used in traditional Chinese medicine, where they are also known as 'Wu Gu Chong'. Previous studies have indicated that maggot extract (ME) was beneficial for the treatment of gastric cancer when combined with other drugs, but the effect on anti-ovarian cancer and the underlying mechanism remains unclear. The aim of this study was to investigate the effects of ME on suppressing the proliferation and migration of ovarian cancer cells, and to clarify the underlying mechanism. In this research, Cell Counting Kit-8 (CCK-8), colony formation assay, and luciferase-positive cell quantification assay were employed to identify the inhibitory effects of ME on cell proliferation. Then, the pro-apoptosis and anti-metastasis effects of ME were explored by Western blot, dual annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) assay, immunofluorescent staining, and wound-healing assay. We further established a xenograft model by subcutaneously or intraperitoneally injecting BALB/c nude mice with SKOV3 cells stably expressing luciferase, and the mice were treated with ME. The results showed that ME therapy effectively restrained the growth and metastasis of ovarian tumors in vivo. Furthermore, the mRNA levels of cancer factors including heat shock protein 90 alpha family class B member 1 (HSP90AB1), MYC, and insulin like growth factor 1 receptor (IGF1R) were analyzed by quantitative real-time PCR assay to explore the possible antitumor mechanisms of ME. Next, HSP90 ATPase activity was inhibited by geldanamycin in A2780, and the cell viability was shown to be dramatically reduced, decreasing further with the combination of ME and cisplatin. In turn, HSP90AB1 overexpression effectively inhibited the effect of ME in suppressing capability for cell viability and migration. In addition, HSP90AB1 overexpression limited the ability of ME to inhibit expression of MYC and IGF1R, while the opposite effect was observed for expression of pro-apoptosis protein caspase3 and BAX. Therefore, this study confirmed the potential roles and mechanisms of ME in inhibiting the growth and metastasis of ovarian tumors and promoting apoptosis of ovarian cancer cells by inhibiting overexpression of HSP90AB1.

Gene expression and functional analysis of Aha1a and Aha1b in stress response in zebrafish.

Activator of heat shock protein 90 (hsp90) ATPase (Aha1) is a Hsp90 co-chaperone required for Hsp90 ATPase activation. Aha1 is essential for yeast survival and muscle development in C. elegans under elevated temperature and hsp90-deficeiency induced stress conditions. The roles of Aha1 in vertebrates are poorly understood. Here, we characterized the expression and function of Aha1 in zebrafish. We showed that zebrafish genome contains two aha1 genes, aha1a and aha1b, that show distinct patterns of expression during development. Under the normal physiological conditions, aha1a is primarily expressed in skeletal muscle cells of zebrafish embryos, while aha1b is strongly expressed in the head region. aha1a and aha1b expression increased dramatically in response to heat shock induced stress. In addition, Aha1a-GFP fusion protein exhibited a dynamic translocation in muscle cells in response to heat shock. Moreover, upregulation of aha1 expression was also observed in hsp90a1 knockdown embryos that showed a muscle defect. Genetic studies demonstrated that knockout of aha1a, aha1b or both had no detectable effect on embryonic development, survival, and growth in zebrafish. The aha1a and aha1b mutant embryos showed normal muscle development and stress response in response to heat shock. Single or double aha1a and aha1b mutants could grow into normal reproductive adults with normal skeletal muscle structure and morphology compared with wild type control. Together, data from these studies indicate that Aha1a and Aha1b are involved in stress response. However, they are dispensable in zebrafish embryonic development, growth, and survival.

HSP90 drives the Rab11a-mediated vesicular transport of the cell surface receptors in osteoclasts.

Rab11a, which ubiquitously localizes to early and recycling endosomes, is required for regulating the vesicular transport of cellular cargos. Interestingly, our previous study revealed that Rab11a served as a negative regulator of osteoclastogenesis by facilitating the lysosomal proteolysis of (1) colony-stimulating factor-1 (c-fms) receptor and (2) receptor activator of nuclear factor-κB (RANK) receptor, thereby resulting in inhibition of osteoclast (OC) differentiation, maturation, and bone-resorbing activity. However, the molecular mechanisms of how Rab11a negatively affected osteoclastogenesis were largely unknown. Heat shock protein (HSP90), including two isoforms HSP90αand HSP90β, necessitates the stability, maturation, and activity of a broad range of its clients, and is essentially required for a vast array of signal transduction pathways in nonstressful conditions. Furthermore, cumulative evidence suggests that HSP90 is a vital element of the vesicular transport network. Indeed, our recent study revealed thatHSP90, a novel effector protein of Rab11b, modulatedRab11b-mediated osteoclastogenesis. In this study, we also found that Rab11a interacted with both HSP90α and HSP90β in OCs. Upon blockade of HSP90 ATPase activity by a specific inhibitor(17-allylamino-demethoxygeldanamycin), we showed that (1) the ATPase domain of HSP90 was a prerequisite for the interaction between HSP90 and Rab11a, and (2) the interaction of HSP90 to Rab11a sufficiently maintained the inhibitory effects of Rab11a on osteoclastogenesis. Altogether, our findings undoubtedly indicate a novel role of HSP90 in regulating Rab11a-mediated osteoclastogenesis.

Aha1 Is an Autonomous Chaperone for SULT1A1.

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.

Hsp90 S-nitrosylation at Cys521, as a conformational switch, modulates cycling of Hsp90-AHA1-CDC37 chaperone machine to aggravate atherosclerosis

Endothelial dysfunction is the initial process of atherosclerosis. Heat shock protein 90 (Hsp90), as a molecular chaperone, plays a crucial role in various cardiovascular diseases. Hsp90 function is regulated by S-nitrosylation (SNO). However, the precise role of SNO-Hsp90 in endothelial dysfunction during atherosclerosis remains unclear. We here identified Hsp90 as a highly S-nitrosylated target in endothelial cells (ECs) by biotin switch assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The elevation of SNO-Hsp90 was observed in atherosclerotic human and rodent aortas as well as in oxidized LDL (oxLDL)-treated ECs. Inhibition of inducible nitric oxide synthase (iNOS) or transfection with Hsp90 cysteine 521 (Cys521) mutation plasmid decreased the level of SNO-Hsp90 in oxLDL-cultured ECs. Coimmunoprecipitation and proximity ligation assay demonstrated that SNO-Hsp90 at Cys521 suppressed the interaction between Hsp90 and activator of Hsp90 ATPase activity 1 (AHA1), but promoted the association of Hsp90 and cell division cycle 37 (CDC37). Hsp90 Cys521 mutation increased endothelial nitric oxide synthase (eNOS) activity and inhibited nuclear factor kappa-B (NF-κB) signaling, thereby increasing nitric oxide (NO) bioavailability and alleviating endothelial adhesion, inflammation and oxidative stress in oxLDL-treated ECs. Also, administration of endothelial-specific adeno-associated viruses of Cys521-mutated Hsp90 significantly mitigated vascular oxidative stress, macrophage infiltration and atherosclerosis lesion areas in high fat diet-fed ApoE-/- mice. In conclusion, SNO-Hsp90 at Cys521, that serves as a conformational switch, disrupts Hsp90/AHA1 interaction but promotes recruitment of CDC37 to exacerbate atherosclerosis.

Geraniol and Citral as potential therapeutic agents targeting the HSP90 activity: An in silico and experimental approach

Lemongrass essential oil has antifungal and anti-cancerous properties. Heat-shock protein (HSP90), an ATP-dependent molecular chaperone found in eukaryotes, is involved in protein folding, stability, and disease, making it a promising research topic. Both in silico and in vitro approaches were used to provide a clear insight into the HSP90-ATPase 3D structures, activity, and their interaction with the essential oil constituents among various species such as fungi (S. cerevisiae), parasites (P. falciparum), and humans. For in silico studies, sequence alignment, docking (AutoDock), and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to obtain hit compounds specifically against each HSP90-ATPase. The hit compounds obtained were evaluated for their efficacy in the in vitro studies of S. cerevisiae. In vitro studies were carried out targeting HSP90-ATPases via lemongrass essential oil components individually and in combination as a function of concentration and various salt concentrations. Results suggest that sequence alignment exists of over 75% among these three species. The best docking score was possessed by Geraniol and its constituent (geldanamycin ≥ −4.93 kcal/mol) (a known antifungal and antitumor against HSP90) in all the above species. Lemongrass oil and the combination of Geraniol and Citral at concentrations of 80 μg/mL showed the maximum inhibition of ATPase and HSP90-ATPase activity compared to their individual treatment. Therefore, both in silico and in vitro studies provide clear evidence of specific inhibitory action of lemongrass oil, Geraniol, and Citral against the ATPase and HSP90–ATPase activities and might show potential as antifungal and antitumor drugs.

Reduction in Aha1 chaperone preserves hippocampal neurons and prevents tau‐related pathology in rTg4510 tau transgenic mice

Several molecular chaperones have been identified as important modulators of tau protein in aging and neurodegenerative illnesses like Alzheimer's diseases. Tau-related diseases, collectively known as tauopathies, are characterized by abnormal deposition of insoluble tau protein leading to neuronal degeneration and the formation of neurofibrillary tangles in the brain. Although the exact mechanism is largely unknown, discrete molecular chaperones are known to regulate tau stability. Our laboratory demonstrated the activator of Hsp90 ATPase Activity (Aha1) potently promotes tau fibrilization and its expression positively correlates with features of tau pathology in brains from tau overexpressing mice and individuals with Alzheimer's disease. In this study, we investigate whether reduction of Aha1 levels in tau transgenic mice is sufficient to slow the progression of tau pathology in brains of rTg4510 tau transgenic mice.To test this, we designed three AAV9 plasmids targeting Aha1 and a scramble shRNA was used as control. Aha1 knockdown was confirmed in N2A cells using Western blot analysis. Following this, 4-month-old rTg4510 male and female tau transgenic mice were injected into the hippocampus with Aha1-shRNA or scramble-shRNA (control). Two months were allowed for viral expression followed by the collection of brains for protein and tissue analysis.n N2A cells, one out of three Aha1-targeting shRNAs effectively reduced Aha1 expression after 72 hours of viral transfection. In animals, Aha1 knockdown decreased pS396/404 and AT8 phosphorylated tau as well Gallyas-silver positive tau tangles in the hippocampus. Total or oligomeric tau were not affected in mice injected with AAV9-shAha1 when compared to AAV9-scramble animals. Using unbiased stereology, we found that hippocampal CA1 neurons were preserved after reducing Aha1.Our findings demonstrate that Aha1 knockdown reduces tau phosphorylated species, prevents tau tangle formation, and rescued CA1 hippocampal neurons in rTg4510 mice. Overall, these in vivo observations suggest that Aha1 knockdown could prevent development of pathological features like tangle formation in tauopathies. Although further examinations in human brain are needed, Aha1 inhibition could be a potential therapeutic target to slow or prevent tau pathologies.

Orchardgrass ACTIVATOR OF HSP90 ATPASE possesses autonomous chaperone properties and activates Hsp90 transcription to enhance thermotolerance

High temperature stress is an environmental factor that negatively affects the growth and development of crops. Hsp90 (90 kDa heat shock protein) is a major molecular chaperone in eukaryotic cells, contributing to the maintenance of cell homeostasis through interaction with co-chaperones. Aha1 (activator of Hsp90 ATPase) is well known as a co-chaperone that activates ATPase activity of Hsp90 in mammals. However, biochemical and physiological evidence relating to Aha has not yet been identified in plants. In this study, we investigated the heat-tolerance function of orchardgrass (Dactylis glomerata L.) Aha (DgAha). Recombinant DgAha interacted with cytosolic DgHsp90s and efficiently protected substrates from thermal denaturation. Furthermore, heterologous expression of DgAha in yeast (Saccharomyces cerevisiae) cells and Arabidopsis (Arabidopsis thaliana) plants conferred thermotolerance in vivo. Enhanced expression of DgAha in Arabidopsis stimulates the transcription of Hsp90 under heat stress. Our data demonstrate that plant Aha plays a positive role in heat stress tolerance via chaperone properties and/or activation of Hsp90 to protect substrate proteins in plants from thermal injury.

AHA1 regulates cell migration and invasion via the EMT pathway in colorectal adenocarcinomas

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.

CDC37 As a Novel Target for the Treatment of NPM1-ALK Expressing Anaplastic Large Cell Lymphomas

Anaplastic large cell lymphoma (ALCL) is an aggressive CD30+ T-cell lymphoma that accounts for 2-8% and 10-15% of non-Hodgkin lymphomas in adults and children, respectively. The currently used standard therapy for anaplastic lymphoma kinase (ALK, a member of the insulin receptor superfamily) positive ALCL has limited effectiveness, resulting in a substantial percentage of cases with poor outcomes, either failing to enter remission or relapse within a few months after starting treatment. Thus, there is a clear unmet clinical need for developing novel, effective and safer therapeutic strategies for ALCL. Nucleophosmin 1 (NPM1) is a nucleolar phosphoprotein, which functions as a molecular chaperone for proteins and nucleic acids. Approximately 50% of ALCL cases are positive for the NPM1-ALK fusion chimera generated by the t(2;5) chromosomal translocation. The oligomerization domain of NPM1 in the fusion protein NPM1-ALK mediates the ligand-independent dimerization of chimeric protein, which results in constitutive activation of the chimeric tyrosine kinase activity leading to downstream signaling pathways responsible for the oncogenicity. As NPM1-ALK is an established heat shock protein 90 (HSP90) client protein, we hypothesized that disruption of the interaction between master chaperone HSP90 and co-chaperone Cdc37 would be a viable druggable target. Celastrol is a natural compound extracted from the root bark of Chinese medicinal plant Tripterygium wilfordi. Anticancer properties of celastrol are attributed to its action on the interaction of HSP90 and its co-chaperone Cdc37. Hsp90 is an ATP-dependent master molecular chaperone. Cdc37 is a Hsp90 co-chaperone and disruption of Hsp90-Cdc37 interaction will result in the degradation of Hsp90 client proteins. Celastrol disrupts the interaction of Hsp90 and Cdc37 through inhibition of Hsp90 ATPase activity without blockage of ATP binding site as observed with Hsp90 inhibitors. We used NPM1-ALK endogenously expressing Karpas299, SUDHL-1 and ectopically expressing Ba/F3-FG-NPM1-ALK cell lines in our study. Treatment with celastrol (0.1-1 µM) significantly induced apoptosis dose dependently in ALCL cells. Apoptosis was associated with activated caspase 3 and poly (ADP-ribose) polymerase cleavage. We also observed upregulation of proapoptotic protein BAD and downregulation of antiapoptotic proteins survivin and MCL1. Celastrol treatment inhibited NPM1-ALK mediated signaling and downstream signaling effectors STAT3, AKT, and ERK1/2 by degradation of the NPM1-ALK fusion protein. Cell survival was evaluated by clonogenic survival assay and results were clearly indicative that the clonogenic survival is diminished in celastrol treated samples compared to controls. Our immunoprecipitation studies clearly demonstrated that celastrol treatment disrupted the interaction between Hsp90 and Cdc37 and induced degradation of the NPM1-ALK fusion protein. Altogether, targeting Cdc37 using celastrol is a novel therapeutic approach to induce apoptosis in ALCL cells expressing NPM1-ALK and warrants developing future therapeutic intervention strategies. DisclosuresGanguly:Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau.

NudC L279P Mutation Destabilizes Filamin A by Inhibiting the Hsp90 Chaperoning Pathway and Suppresses Cell Migration.

Filamin A, the first discovered non-muscle actin filament cross-linking protein, plays a crucial role in regulating cell migration that participates in diverse cellular and developmental processes. However, the regulatory mechanism of filamin A stability remains unclear. Here, we find that nuclear distribution gene C (NudC), a cochaperone of heat shock protein 90 (Hsp90), is required to stabilize filamin A in mammalian cells. Immunoprecipitation-mass spectrometry and western blotting analyses reveal that NudC interacts with filamin A. Overexpression of human NudC-L279P (an evolutionarily conserved mutation in NudC that impairs its chaperone activity) not only decreases the protein level of filamin A but also results in actin disorganization and the suppression of cell migration. Ectopic expression of filamin A is able to reverse these defects induced by the overexpression of NudC-L279P. Furthermore, Hsp90 forms a complex with filamin A. The inhibition of Hsp90 ATPase activity by either geldanamycin or radicicol decreases the protein stability of filamin A. In addition, ectopic expression of Hsp90 efficiently restores NudC-L279P overexpression-induced protein stability and functional defects of filamin A. Taken together, these data suggest NudC L279P mutation destabilizes filamin A by inhibiting the Hsp90 chaperoning pathway and suppresses cell migration.