Exploring the Roles of Key Mediators IKBKE and HSPA1A in Alzheimer's Disease and Hepatocellular Carcinoma through Bioinformatics Analysis.

Recent studies have hinted at a potential link between Alzheimer's Disease (AD) and cancer. Thus, our study focused on finding genes common to AD and Liver Hepatocellular Carcinoma (LIHC), assessing their promise as diagnostic indicators and guiding future treatment approaches for both conditions. Our research utilized a broad methodology, including differential gene expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), gene enrichment analysis, Receiver Operating Characteristic (ROC) curves, and Kaplan-Meier plots, supplemented with immunohistochemistry data from the Human Protein Atlas (HPA) and machine learning techniques, to identify critical genes and significant pathways shared between AD and LIHC. Through differential gene expression analysis, WGCNA, and machine learning methods, we identified nine key genes associated with AD, which served as entry points for LIHC analysis. Subsequent analyses revealed IKBKE and HSPA1A as shared pivotal genes in patients with AD and LIHC, suggesting these genes as potential targets for intervention in both conditions. Our study indicates that IKBKE and HSPA1A could influence the onset and progression of AD and LIHC by modulating the infiltration levels of immune cells. This lays a foundation for future research into targeted therapies based on their shared mechanisms.

Integrating single-nucleus sequence profiling to reveal the transcriptional dynamics of Alzheimer's disease, Parkinson's disease, and multiple sclerosis.

BACKGROUND: Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS) are three nervous system diseases that partially overlap clinically and genetically. However, bulk RNA-sequencing did not accurately detect the core pathogenic molecules in them. The availability of high-quality single cell RNA-sequencing data of post-mortem brain collections permits the generation of a large-scale gene expression in different cells in human brain, focusing on the molecular features and relationships between diseases and genes. We integrated single-nucleus RNA-sequencing (snRNA-seq) datasets of human brains with AD, PD, and MS to identify transcriptomic commonalities and distinctions among them. METHODS: The snRNA-seq datasets were downloaded from Gene Expression Omnibus (GEO) database. The Seurat package was used for snRNA-seq data processing. The uniform manifold approximation and projection (UMAP) were utilized for cluster identification. The FindMarker function in Seurat was used to identify the differently expressed genes. Functional enrichment analysis was carried out using the Gene Set Enrichment Analysis (GSEA) and Gene ontology (GO). The protein-protein interaction (PPI) analysis of differentially expressed genes (DEGs) was analyzed using STRING database ( http://string-db.org ). SCENIC analysis was performed using utilizing pySCENIC (v0.10.0) based on the hg19-tss-centered-10 kb-10species databases. The analysis of potential therapeutic drugs was analyzed on Connectivity Map ( https://clue.io ). RESULTS: The gene regulatory network analysis identified several hub genes regulated in AD, PD, and MS, in which HSPB1 and HSPA1A were key molecules. These upregulated HSP family genes interact with ribosome genes in AD and MS, and with immunomodulatory genes in PD. We further identified several transcriptional regulators (SPI1, CEBPA, TFE3, GRHPR, and TP53) of the hub genes, which has important implications for uncovering the molecular crosstalk among AD, PD, and MS. Arctigenin was identified as a potential therapeutic drug for AD, PD, and MS. CONCLUSIONS: Together, the integrated snRNA-seq data and findings have significant implications for unraveling the shared and unique molecular crosstalk among AD, PD, and MS. HSPB1 and HSPA1A as promising targets involved in the pathological mechanisms of neurodegenerative diseases. Additionally, the identification of arctigenin as a potential therapeutic drug for AD, PD, and MS further highlights its potential in treating these neurological disorders. These discoveries lay the groundwork for future research and interventions to enhance our understanding and treatment of AD, PD, and MS.

The use of CO2 laser in vulvar lichen sclerosus treatment - molecular evidence.

Vulvar lichen sclerosus is chronic and difficult to treat disorder, which offer is recurrent and leads to multiple complications. The limited efficacy of pharmacologic treatment directed the search for new therapies including use of CO2 laser. In our study we focused on collagen and elastin gene expression as well as heat shock proteins and p53 expression in two patients with vulvar lichen sclerosus who underwent CO2 laser therapy. In both patients we observed decreased clinical symptoms observed by an experienced gynecologist as well as significant changes in gene expression before and after laser treatment.

The profile of HSPA1A gene expression and its association with heat tolerance in crossbred cattle and the tropically adapted dwarf Vechur and Kasaragod.

Certain livestock breeds are adapted to hot and humid environments, and these breeds have genetics that could be useful in a changing climate. The expression of several genes has been identified as a useful biomarker for heat stress. In this study, the responses to heat exposure of heat-tolerant Vechur and Kasaragod cattle found in Kerala state in India (also known as dwarf Bos taurus indicus) were compared to crossbred cattle (crosses of Bos t. taurus with Bos t. indicus). At various time points during heat exposure, rectal temperature and the expression of HSPA1A were determined, and the relationship between them was characterized. We characterized HSPA1A mRNA in Vechur cattle and performed molecular clock analysis. The expression of HSPA1A between the lineages and at different temperature humidity index (THI) was significant. There were significant differences between the expression profiles of HSPA1A in Kasaragod and crossbred (p < 0.01) and Vechur and crossbred (p < 0.01) cattle, but no significant difference in expression was observed between Vechur and Kasaragod cattle. The genetic distance between Vechur, B. grunniens, B. t. taurus, and B. t. indicus was 0.0233, 0.0059, and 0.007, respectively. The genetic distance between Vechur and the Indian dwarf breed Malnad Gidda was 0.0081. A molecular clock analysis revealed divergent adaptive evolution of Vechur cattle to B. t. taurus, with adaptations to the high temperatures and humidity that are prevalent in their breeding tract in Kerala, India. These results could also prove useful in selecting heat-tolerant animals using HSPA1A as a marker.

Heat-Induced Proteotoxic Stress Response in Placenta-Derived Stem Cells (PDSCs) Is Mediated through HSPA1A and HSPA1B with a Potential Higher Role for HSPA1B.

Placenta-derived stem cells (PDSCs), due to unique traits such as mesenchymal and embryonic characteristics and the absence of ethical constraints, are in a clinically and therapeutically advantageous position. To aid in stemness maintenance, counter pathophysiological stresses, and withstand post-differentiation challenges, stem cells require elevated protein synthesis and consequently augmented proteostasis. Stem cells exhibit source-specific proteostasis traits, making it imperative to study them individually from different sources. These studies have implications for understanding stem cell biology and exploitation in the augmentation of therapeutic applications. Here, we aim to identify the primary determinants of proteotoxic stress response in PDSCs. We generated heat-induced dose-responsive proteotoxic stress models of three stem cell types: placental origin cells, the placenta-derived mesenchymal stem cells (pMSCs), maternal origin cells, the decidua parietalis mesenchymal stem cells (DPMSCs), and the maternal-fetal interface cells, decidua basalis mesenchymal stem cells (DBMSCs), and measured stress induction through biochemical and cell proliferation assays. RT-PCR array analysis of 84 genes involved in protein folding and protein quality control led to the identification of Hsp70 members HSPA1A and HSPA1B as the prominent ones among 17 significantly expressed genes and with further analysis at the protein level through Western blotting. A kinetic analysis of HSPA1A and HSPA1B gene and protein expression allowed a time series evaluation of stress response. As identified by protein expression, an active stress response is in play even at 24 h. More prominent differences in expression between the two homologs are detected at the translational level, alluding to a potential higher requirement for HSPA1B during proteotoxic stress response in PDSCs.

Transcriptomic and Proteomic Analysis of CRISPR/Cas9-Mediated ARC-Knockout HEK293 Cells.

Arc/Arg3.1 (activity-regulated cytoskeletal-associated protein (ARC)) is a critical regulator of long-term synaptic plasticity and is involved in the pathophysiology of schizophrenia. The functions and mechanisms of human ARC action are poorly understood and worthy of further investigation. To investigate the function of the ARC gene in vitro, we generated an ARC-knockout (KO) HEK293 cell line via CRISPR/Cas9-mediated gene editing and conducted RNA sequencing and label-free LC-MS/MS analysis to identify the differentially expressed genes and proteins in isogenic ARC-KO HEK293 cells. Furthermore, we used bioluminescence resonance energy transfer (BRET) assays to detect interactions between the ARC protein and differentially expressed proteins. Genetic deletion of ARC disturbed multiple genes involved in the extracellular matrix and synaptic membrane. Seven proteins (HSPA1A, ENO1, VCP, HMGCS1, ALDH1B1, FSCN1, and HINT2) were found to be differentially expressed between ARC-KO cells and ARC wild-type cells. BRET assay results showed that ARC interacted with PSD95 and HSPA1A. Overall, we found that ARC regulates the differential expression of genes involved in the extracellular matrix, synaptic membrane, and heat shock protein family. The transcriptomic and proteomic profiles of ARC-KO HEK293 cells presented here provide new evidence for the mechanisms underlying the effects of ARC and molecular pathways involved in schizophrenia pathophysiology.

Corrigendum: Long Noncoding RNA NONHSAT079852.2 Contributes to GBM Recurrence by Functioning as a ceRNA for has-mir-10401-3p to Facilitate HSPA1A Upregulation.

[This corrects the article DOI: 10.3389/fonc.2021.636632.].

Targeting HSPA1A in ARID2-deficient lung adenocarcinoma.

Somatic mutations of the chromatin remodeling gene ARID2 are observed in ∼7% of human lung adenocarcinomas (LUADs). However, the role of ARID2 in the pathogenesis of LUADs remains largely unknown. Here we find that ARID2 expression is decreased during the malignant progression of both human and mice LUADs. Using two KrasG12D -based genetically engineered murine models, we demonstrate that ARID2 knockout significantly promotes lung cancer malignant progression and shortens overall survival. Consistently, ARID2 knockdown significantly promotes cell proliferation in human and mice lung cancer cells. Through integrative analyses of ChIP-Seq and RNA-Seq data, we find that Hspa1a is up-regulated by Arid2 loss. Knockdown of Hspa1a specifically inhibits malignant progression of Arid2-deficient but not Arid2-wt lung cancers in both cell lines as well as animal models. Treatment with an HSPA1A inhibitor could significantly inhibit the malignant progression of lung cancer with ARID2 deficiency. Together, our findings establish ARID2 as an important tumor suppressor in LUADs with novel mechanistic insights, and further identify HSPA1A as a potential therapeutic target in ARID2-deficient LUADs.

Upregulation of HSPA1A/HSPA1B/HSPA7 and Downregulation of HSPA9 Were Related to Poor Survival in Colon Cancer.

The human HSP70 family is a type of heat shock protein (HSP), consisting of 13 members encoded by the HSPA genes. HSPs play important roles in regulating cellular responses and functions during carcinogenesis, but their relationship with colon cancer is unclear. In our study, we found that the expressions of HSPA1B, HSPA4, HSPA5, HSPA6, HSPA8, HSPA9, HSPA13, and HSPA14 were significantly increased, while those of HSPA1A, HSPA2, HSPA7, and HSPA12B were significantly decreased in colon cancer tissues. The expression of HSPA gene family members was associated with some clinicopathological characteristics, including age, gender, TNM stage, pathological stage, and CEA level. Furthermore, the Kaplan-Meier method and Cox regression analysis showed that high HSPA1A, HSPA1B, and HSPA7 expressions were related to unfavorable survival, and high HSPA9 was associated with favorable survival. The relationships between HSPA1A and HSPA9 expression and survival were validated in the GEO dataset, and the HSPA1A and HSPA9 protein expression differences between colon cancer tissues and normal tissues were validated in the UALCAN database. Methylation of HSPA1A and HSPA9 was also analyzed, and it was found that the methylation of the HSPA1A promoter was significantly increased, and the methylation of the HSPA9 promoter was significantly decreased in colon cancer tissues. Increasing the methylation level of the HSPA1A gene and decreasing the methylation level of HSPA9 were related to favorable prognosis. The expression difference of HSPA1A/HSPA1B/HSPA7/HSPA9 was verified in colon cancer cell lines and colonic epithelial cells. Gene ontology analysis was used to screen signal pathways related to HSPA1A-, HSPA1B-, HSPA7-, and HSPA9- high phenotype. In summary, the increased expressions of HSPA1A1, HSPA1B, and HSPA7 were associated with poor prognosis, while that of HSPA9 was related to favorable prognosis for colon cancer patients.

Relationship between HSPA1A-regulated gene expression and alternative splicing in mouse cardiomyocytes and cardiac hypertrophy.

BACKGROUND: Cardiac hypertrophy may be classified as either physiological or pathological. Pathological hypertrophy has a complex etiology and is genetically regulated. In this study, we used a mouse model of cardiac hypertrophy to explore the mechanisms of gene regulation, in particular, modulation of the expression of target genes through transcription factor activity, regulation of immune and inflammation-associated genes and regulation of the alternative splicing of transcription factors. METHODS: Mouse models of pathological cardiac hypertrophy were established by transverse aortic constriction (TAC). We overexpressed HSPA1A in mouse cardiac HL-1 cells. GO and KEGG pathway annotation database was used to analyze all DEGs. RESULTS: The expression of HSPA1A differed significantly between TAC + dantrolene vs. sham + dantrolene (Sham was the non-TAC group, and DMSO was the contrast agent), and TAC + DMSO vs. sham + DMSO. The RNA-binding protein Zfp36 was found to be differentially expressed between both TAC + dantrolene vs. sham + dantrolene and TAC + DMSO vs. sham + DMSO. The expression of mki67 and gm5619 was significantly different between TAC + dantrolene and TAC + DMSO. HSPA1A was found to selectively regulate the expression of non-coding RNAs related to cardiac hypertrophy, including Rn7sk and RMRP. The downregulated genes were mainly related to inflammation and the immune response. HSPA1A negatively regulated alternative splicing of Asxl2 and positively regulated alternative splicing of Runx1. CONCLUSIONS: HSPA1A was closely related to cardiac hypertrophy. Zfp36 was also related to cardiac hypertrophy. Dantrolene may delay cardiac hypertrophy and ventricular remodeling by regulating the expression of the RNA-binding protein genes mki67 and gm5619. HSPA1A positively regulated the expression of the non-coding RNAs RN7SK and RMRP while negatively regulating the expression of inflammation- and immune response-related genes. HSPA1A can play a role in cardiac hypertrophy by regulating the alternative splicing of asxl2 and runx1.

Long Noncoding RNA NONHSAT079852.2 Contributes to GBM Recurrence by Functioning as a ceRNA for has-mir-10401-3p to Facilitate HSPA1A Upregulation.

Glioblastoma multiforme (GBM) is the most common brain malignancy and major cause of high mortality in patients with GBM, and its high recurrence rate is its most prominent feature. However, the pathobiological mechanisms involved in recurrent GBM remain largely unknown. Here, whole-transcriptome sequencing (RNA-sequencing, RNA-Seq) was used in characterizing the expression profile of recurrent GBM, and the aim was to identify crucial biomarkers that contribute to GBM relapse. Differentially expressed RNAs in three recurrent GBM tissues compared with three primary GBM tissues were identified through RNA-Seq. The function and mechanism of a candidate long noncoding RNA (lncRNA) in the progression and recurrence of GBM were elucidated by performing comprehensive bioinformatics analyses, such as functional enrichment analysis, protein-protein interaction prediction, and lncRNA-miRNA-mRNA regulatory network construction, and a series of in vitro assays. As the most significantly upregulated gene identified in recurrent GBM, HSPA1A is mainly related to antigen presentation and the MAPK signaling pathway, as indicated by functional enrichment analysis. HSPA1A was predicted as the target gene of the lncRNA NONHSAT079852.2. qRT-PCR revealed that NONHSAT079852.2 was significantly elevated in recurrent GBM relative to that in primary GBM, and high NONHSAT079852.2 expression was associated with the poor overall survival rates of patients with GBM. The knockdown of NONHSAT079852.2 successfully induced tumor cell apoptosis, inhibited the proliferation, migration, invasion and the expression level of HSPA1A in glioma cells. NONHSAT079852.2 was identified to be a sponge for hsa-miR-10401-3p through luciferase reporter assay. Moreover, HSPA1A was targeted and regulated by hsa-miR-10401-3p. Collectively, the results suggested that NONHSAT079852.2 acts as a sponge of hsa-mir-10401-3p and thereby enhances HSPA1A expression, promotes tumor cell proliferation and invasion, and leads to the progression and recurrence of GBM. This study will provide new insight into the regulatory mechanisms of NONHSAT079852.2-mediated competing endogenous RNA in the pathogenesis of recurrent GBM and evidence of the potential of lncRNAs as diagnostic biomarkers or potential therapeutic targets.

Human heat shock cognate protein (HSC70/HSPA8) interacts with negatively charged phospholipids by a different mechanism than other HSP70s and brings HSP90 into membranes.

Heat shock proteins (HSP) are critical elements for the preservation of cellular homeostasis by participating in an array of biological processes. In addition, HSP play an important role in cellular protection from various environmental stresses. HSP are part of a large family of different molecular mass polypeptides, displaying various expression patterns, subcellular localizations, and diversity functions. An unexpected observation was the detection of HSP on the cell surface. Subsequent studies have demonstrated that HSP have the ability to interact and penetrate lipid bilayers by a process initiated by the recognition of phospholipid heads, followed by conformational changes, membrane insertion, and oligomerization. In the present study, we described the interaction of HSPA8 (HSC70), the constitutive cytosolic member of the HSP70 family, with lipid membranes. HSPA8 showed high selectivity for negatively charged phospholipids, such as phosphatidylserine and cardiolipin, and low affinity for phosphatidylcholine. Membrane insertion was mediated by a spontaneous process driven by increases in entropy and diminished by the presence of ADP or ATP. Finally, HSPA8 was capable of driving into the lipid bilayer HSP90 that does not display any lipid biding capacity by itself. This observation suggests that HSPA8 may act as a membrane chaperone.

New insights on human Hsp70-escort protein 1: Chaperone activity, interaction with liposomes, cellular localizations and HSPA's self-assemblies remodeling.

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.

ACOT4 accumulation via AKT-mediated phosphorylation promotes pancreatic tumourigenesis.

The acyl-CoA thioesterase (ACOT) family catalyses the hydrolysis of acyl-CoA thioesters to their corresponding non-esterified fatty acid and coenzyme A (CoA). Increasing evidence suggests that cancer cells generally have altered lipid metabolism in different aspects. However, the roles of the ACOT family in cancer, especially in pancreatic ductal carcinoma (PDAC), are largely unknown. In the present study, we mined data to determine the clinical significance of all eleven ACOT genes among nine major solid tumour types from TCGA database and found that the expression of ACOT4 in PDAC was negatively correlated with patient survival, establishing ACOT4 as a potential biomarker of PDAC. Depletion of ACOT4 attenuated the proliferation and tumour formation of PDAC cells. Using mass spectrometry, HSPA1A was found to associate with ACOT4. Furthermore, we found that phosphorylation of ACOT4 at S392 by AKT decreased the binding of ACOT4 to HSPA1A, resulting in ACOT4 accumulation. The ACOT4 elevation promotes pancreatic tumourigenesis by producing excessive CoA to support tumour cell metabolism. Thus, our study expands the relationship between AKT signalling and lipid metabolism and establishes a functional role of ACOT4 in PDAC.

Andrographolide and Its 14-Aryloxy Analogues Inhibit Zika and Dengue Virus Infection.

Andrographolide is a labdene diterpenoid with potential applications against a number of viruses, including the mosquito-transmitted dengue virus (DENV). In this study, we evaluated the anti-viral activity of three 14-aryloxy analogues (ZAD-1 to ZAD-3) of andrographolide against Zika virus (ZIKV) and DENV. Interestingly, one analogue, ZAD-1, showed better activity against both ZIKV and DENV than the parental andrographolide. A two-dimension (2D) proteomic analysis of human A549 cells treated with ZAD-1 compared to cells treated with andrographolide identified four differentially expressed proteins (heat shock 70 kDa protein 1 (HSPA1A), phosphoglycerate kinase 1 (PGK1), transketolase (TKT) and GTP-binding nuclear protein Ran (Ran)). Western blot analysis confirmed that ZAD-1 treatment downregulated expression of HSPA1A and upregulated expression of PGK1 as compared to andrographolide treatment. These results suggest that 14-aryloxy analogues of andrographolide have the potential for further development as anti-DENV and anti-ZIKV agents.

HSPA1A gene polymorphism rs1008438 is associated with susceptibility to acute mountain sickness in Han Chinese individuals.

BACKGROUND: Acute mountain sickness (AMS) usually occurs among non-acclimated individuals after rapid ascending to high-altitude environments (generally ≥2,500 m). However, the precise molecular mechanism of AMS remains unclear. Our study aimed to investigate the relationship between several single nucleotide polymorphisms (SNPs) and AMS susceptibility. METHODS: In this work, sequencing data were obtained from 69 AMS patients and 95 matched acclimated Han Chinese individuals from southwest China. Five SNPs (rs1008438, rs150877473, rs1799983, rs2153364, and rs3025039) were systematically investigated in all the participants. RESULTS: In our study, we found that allele frequencies of "A" (AMS 69.57% vs. non-AMS 54.74%) and "C" (AMS 30.43% vs. non-AMS 45.26%) in the HSPA1A gene rs1008438 were significantly different between the AMS and non-AMS groups (p = .01). Genotypes "CC" and "CA" of the HSPA1A gene (rs1008438) were associated with lower risk of developing AMS than the genotype "AA." Comparing the genotypes "CC + CA" and "AA," we also observed that the "CC + CA" genotype of rs1008438 was associated with lower AMS risk. CONCLUSIONS: In our case-control study, there was a significant association between the rs1008348 polymorphism and AMS susceptibility, suggesting that this particular SNP might be a Han-specific risk factor for AMS. We believe that this study establishes a foundation for further elucidation of the genetic mechanisms underlying AMS.

MicroRNAs and Xenobiotic Toxicity: An Overview.

The advent of new technologies has paved the rise of various chemicals that are being employed in industrial as well as consumer products. This leads to the accumulation of these xenobiotic compounds in the environment where they pose a serious threat to both target and non-target species. miRNAs are one of the key epigenetic mechanisms that have been associated with toxicity by modulating the gene expression post-transcriptionally. Here, we provide a comprehensive view on miRNA biogenesis, their mechanism of action and, their possible role in xenobiotic toxicity. Further, we review the recent in vitro and in vivo studies involved in xenobiotic exposure induced miRNA alterations and the mRNA-miRNA interactions. Finally, we address the challenges associated with the miRNAs in toxicological studies.

LncRNA HOTAIR enhances breast cancer radioresistance through facilitating HSPA1A expression via sequestering miR-449b-5p.

BACKGROUND: Breast cancer (BRCA) is the leading cause of cancer-related death in women worldwide. Pre- and postoperative radiotherapy play a pivotal role in BRCA treatment but its efficacy remains limited and plagued by the emergence of radiation resistance, which aggravates patient prognosis. The long noncoding RNA (lncRNA)-implicated mechanisms underlying radiation resistance are rarely reported. The aim of this study was to determine whether lncRNA HOX transcript antisense RNA (HOTAIR) modulated the radiosensitivity of breast cancer through HSPA1A. METHODS: A Gammacell 40 Exactor was used for irradiation treatment. Bioinformatic tools and luciferase reporter assay were adopted to explore gene expression profile and demonstrate the interactions between lncRNA, miRNA and target mRNA 3'-untranslated region (3'-UTR). The expression levels of certain genes were determined by real-time PCR and western-blot analyses. in vitro and in vivo functional assays were conducted by cell viability and tumorigenicity assays. RESULTS: The levels of oncogenic lncRNA HOTAIR were positively correlated with the malignancy of BRCA but reversely correlated with the radiosensitivity of breast cancer cells. Moreover, the expression levels of HOTAIR were positively associated with those of heat shock protein family A (Hsp70) member 1A (HSPA1A) in clinical BRCA tissues and HOTAIR upregulated HSPA1A at the mRNA and protein levels in irradiated BRCA cells. Mechanistically, miR-449b-5p restrained HSPA1A expression through targeting the 3'-UTR of HSPA1A mRNA, whereas HOTAIR acted as a competing sponge to sequester miR-449b-5p and thereby relieved the miR-449b-5p-mediated HSPA1A repression. Functionally, HOTAIR conferred decreased radiosensitivity on BRCA cells, while miR-449b-5p overexpression or HSPA1A knockdown abrogated the HOTAIR-enhanced BRCA growth under the irradiation exposure both in vitro and in vivo. CONCLUSIONS: LncRNA HOTAIR facilitates the expression of HSPA1A by sequestering miR-449b-5p post-transcriptionally and thereby endows BRCA with radiation resistance. KEY POINTS: Therapeutically, HOTAIR and HSPA1A may be employed as potential targets for BRCA radiotherapy. Our findings shed new light into the mechanism by which lncRNAs modulate the radiosensitivity of tumors.

S-Glutathionylation of human inducible Hsp70 reveals a regulatory mechanism involving the C-terminal α-helical lid.

Heat shock protein 70 (Hsp70) proteins are a family of ancient and conserved chaperones. Cysteine modifications have been widely detected among different Hsp70 family members in vivo, but their effects on Hsp70 structure and function are unclear. Here, we treated HeLa cells with diamide, which typically induces disulfide bond formation except in the presence of excess GSH, when glutathionylated cysteines predominate. We show that in these cells, HspA1A (hHsp70) undergoes reversible cysteine modifications, including glutathionylation, potentially at all five cysteine residues. In vitro experiments revealed that modification of cysteines in the nucleotide-binding domain of hHsp70 is prevented by nucleotide binding but that Cys-574 and Cys-603, located in the C-terminal α-helical lid of the substrate-binding domain, can undergo glutathionylation in both the presence and absence of nucleotide. We found that glutathionylation of these cysteine residues results in unfolding of the α-helical lid structure. The unfolded region mimics substrate by binding to and blocking the substrate-binding site, thereby promoting intrinsic ATPase activity and competing with binding of external substrates, including heat shock transcription factor 1 (Hsf1). Thus, post-translational modification can alter the structure and regulate the function of hHsp70.

Inhibition of HSP90 and Activation of HSF1 Diminish Macrophage NLRP3 Inflammasome Activity in Alcohol-Associated Liver Injury.

BACKGROUND: Activation of NLRP3 in liver macrophages contributes to alcohol-associated liver disease (ALD). Molecular chaperone heat shock protein (HSP) 90 facilitates NLRP3 inflammasome activity during infections and inflammatory diseases. We previously reported that HSP90 is induced in ALD and regulates proinflammatory cytokines, tumor necrosis factor alpha, and IL-6. Whether HSP90 affects IL-1ß and IL-18 regulated by NLRP3 inflammasome in ALD is unknown. Here, we hypothesize that HSP90 modulated NLRP3 inflammasome activity and affects IL-1ß and IL-18 secretion in ALD. METHODS: The expression of HSP90AA1 and NLRP3 inflammasome genes was evaluated in human alcoholic livers and in mouse model of ALD. The importance of HSP90 on NLRP3 inflammasome activation in ALD was evaluated by administering HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) to mice subjected to ALD, and in vitro to bone marrow-derived macrophages (BMDM) stimulated with LPS and ATP. The effect of activation of HSF1/HSPA1A axis during HSP90 inhibition or direct activation during heat shock of BMDMs on NLRP3 activity and secretion of downstream cytokines was evaluated. RESULTS: We found positive correlation between induction of HSP90 and NLRP3 inflammasome genes in human alcoholic cirrhotic livers. Administration of 17-DMAG in mouse model of ALD significantly down-regulated NLRP3 inflammasome-mediated caspase-1 (CASP-1) activity and cytokine secretion, with reduction in ALD. 17-DMAG-mediated decrease in NLRP3 was restricted to liver macrophages. Using BMDMs, we show that inhibition of HSP90 prevented CASP-1 activity, and Gasdermin D (GSDMD) cleavage, important in release of active IL-1ß and IL-18. Interestingly, activation of the heat shock factor 1 (HSF1)/HSPA1A axis, either during HSP90 inhibition or by heat shock, decreased NLRP3 inflammasome activity and reduced secretion of cytokines. CONCLUSION: Our studies indicate that inhibition of HSP90 and activation of HSF1/HSPA1A reduce IL-1ß and IL-18 via decrease in NLRP3/CASP-1 and GSDMD activity in ALD.