Heat-shocked Cells Research Articles

Insights on early response to acute heat shock of bovine mammary epithelial cells through a multimethod approach

Heat stress is a significant challenge in dairy cattle herds, affecting milk production and quality, and generating important changes at the cellular level. Most in vitro research on heat shock (HS) effects on dairy cow mammary cells was focused on medium-long-term effects. In recent years, Fourier transform-infrared (FT-IR) micro-spectroscopy has been increasingly used to study the effects of several external stresses on different cell lines, down to the level of single cellular components, such as DNA/RNA, lipids, and proteins. In this study, the possible changes at the biochemical and molecular level induced by acute (30 min-2 h) HS in bovine mammary epithelial (BME-UV1) cells were investigated. The cells were exposed to different temperatures, thermoneutral (TN, 37 °C) and HS (42 °C), and FT-IR spectra were acquired to analyse the effects of HS on biochemical characteristics of BME-UV1 cellular components (proteins, lipids, and DNA/RNA). Moreover, cell viability assay, reactive oxygen species production, and mRNA expression of heat shock proteins (HSPA1A, HSP90AA1, GRP78, GRP94) and antioxidant genes (SOD1, SOD2) by RT-qPCR were also analysed. The FT-IR results showed a change already at 30 min of HS exposure, in the content of long-chain fatty acids, which probably acted as a response to a modification of membrane fluidity in HS cells compared with TN cells. After 2 h of HS exposure, modification of DNA/RNA activity and accumulation of aggregated proteins was highlighted in HS cells. The gene expression analyses showed the overexpression of HSPA1A and HSP90AA1 starting from 30 min up to 2 h in HS cells compared with TN cells. At 2 h of HS exposure, also the overexpression of GRP94 was observed in HS cells. Acute HS did not affect cell viability, reactive oxygen species level, and SOD1 and SOD2 gene expression of BME-UV1 cells. According to the results obtained, cells initiate early defence mechanisms in case of acute HS and probably this efficient response capacity may be decisive for tolerance to heat stress of dairy cattle.

JRK binds satellite III DNA and is necessary for the heat shock response.

JRK is a DNA-binding protein of the pogo superfamily of transposons, which includes the well-known centromere binding protein B (CENP-B). Jrk null mice exhibit epilepsy, and growth and reproductive disorders, consistent with its relatively high expression in the brain and reproductive tissues. Human JRK DNA variants and gene expression levels are implicated in cancers and neuropsychiatric disorders. JRK protein modulates β-catenin-TCF activity but little is known of its cellular functions. Based on its homology to CENP-B, we determined whether JRK binds centromeric or other satellite DNAs. We show that human JRK binds satellite III DNA, which is abundant at the chromosome 9q12 juxtacentromeric region and on Yq12, both sites of nuclear stress body assembly. Human JRK-GFP overexpressed in HeLa cells strongly localises to 9q12. Using an anti-JRK antiserum we show that endogenous JRK co-localises with a subset of centromeres in non-stressed cells, and with heat shock factor 1 following heat shock. Knockdown of JRK in HeLa cells proportionately reduces heat shock protein gene expression in heat-shocked cells. A role for JRK in regulating the heat shock response is consistent with the mouse Jrk null phenotype and suggests that human JRK may act as a modifier of diseases with a cellular stress component.

EIF4F is a thermo-sensing regulatory node in the translational heat shock response

Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using invitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.

Exosomes Derived from Heat-shocked Tumor Cells Promote In vitro Maturation of Bone Marrow-derived Dendritic Cells.

Dendritic cells (DCs), professional antigen-presenting cells that process and deliver antigens using MHC II/I molecules, can be enhanced in numerous ways. Exosomes derived from heat-shocked tumor cells (HS-TEXs) contain high amounts of heat-shock proteins (HSPs). HSPs, as chaperons, can induce DC maturation. This study aimed to investigate whether HS-TEXs can promote DC maturation. To generate DC, bone marrow-derived cells were treated with Interleukin-4 and GM-CSF. Exosomes were isolated from heat-treated CT-26 cells. The expression level of HSP in exosomes was checked by western blot and the increase in the expression of this protein was observed. Then, HS-TEXs were co-cultured with iDCs to determine DC maturity, and then DCs were co-cultured with lymphocytes to determine DC activity. Our results showed that DCs treated with HS-TEXs express high levels of molecules involved in DC maturation and function including MHCII, CD40, CD83, and CD86. HS-TEXs caused phenotypic and functional maturation of DCs. In addition, flow cytometric results reflected a higher proliferative response of lymphocytes in the iDC / Tex + HSP group. HS-TEXs could be used as a strategy to improve DC maturation and activation.

Simultaneous Inhibition of Heat Shock Response and Autophagy with Bimetallic Mesoporous Nanoparticles to Enhance Mild‐Temperature Photothermal Therapy

Mild‐temperature photothermal therapy (MPTT) is a promising tumor therapeutic modality because it can avoid the damage of normal tissues near the tumor caused by excessive heat. However, its therapeutic effect is severely impaired because tumor cells can develop heat resistance and self‐repair by activating heat shock response and cell autophagy. Herein, a tannic acid–iron ion metal organic framework‐coated, chloroquine (CQ)‐loaded mesoporous PdPt nanosystem (TF‐CQ@mPdPt) is developed to enhance MPTT by simultaneous suppression of heat shock response and autophagy. TF‐CQ@mPdPt exhibits good peroxidase (POD)‐mimic activity and photothermal performance. As a result, the reactive oxygen species generated by POD‐mediated decomposition of endogenous hydrogen peroxide damage mitochondria, leading to limitation of adenosine triphosphate supply, which suppresses the upregulation of heat shock proteins of tumor cells during MPTT, making tumor cell more sensitive to heat stress. Concurrently, CQ released from TF‐CQ@mPdPt during MPTT inhibits cell autophagy, thereby interrupting the self‐repair pathway of tumor cells. Consequently, TF‐CQ@mPdPt‐mediated MPTT significantly enhances its therapeutic effect, effectively inhibiting tumor progression in 4T1 tumor‐bearing mice. This study presents a novel strategy to enhance MPTT by simultaneously suppressing heat shock response and autophagy.

Multiparametric analysis of etoposide exposed mesenchymal stem cells and Fanconi anemia cells: implications in development of secondary myeloid malignancy.

Secondary acute myeloid leukemia (sAML) may develop following a prior therapy or may evolve from an antecedent hematological disorder such as Fanconi Anemia (FA). Pathophysiology of leukemic evolution is not clear. Etoposide (Eto) is a chemotherapeutic agent implicated in development of sAML. FA is an inherited bone marrow (BM) failure disease characterized by genomic instability and xenobiotic susceptibility. Here, we hypothesized that alterations in the BM niche may play a critical/driver role in development of sAML in both conditions. Expression of selected genes involved in xenobiotic metabolism, DNA double-strand break response, endoplasmic reticulum (ER) stress, heat shock response and cell cycle regulation were determined in BM mesenchymal stem cells (MSCs) of healthy controls and FA patients at steady state and upon exposure to Eto at different concentrations and in recurrent doses. Expression of CYPA1, p53, CCNB1, Dicer1, CXCL12, FLT3L and TGF-Beta genes were significantly downregulated in FA-MSCs compared with healthy controls. Eto exposure induced significant alterations in healthy BM-MSCs with increased expression of CYP1A1, GAD34, ATF4, NUPR1, CXCL12, KLF4, CCNB1 and nuclear localization of Dicer1. Interestingly, FA-MSCs did not show significant alterations in these genes upon Eto exposure. As opposed to healthy MSCs DICER1 gene expression and intracellular localization was not altered on FA BM-MSCs after Eto treatment. These results showed that Eto is a highly potent molecule and has pleiotropic effects on BM-MSCs, FA cells show altered expression profile compared to healthy controls and Eto exposure on FA cells shows differential profile than healthy controls.

Exploring the Potential of Ultrasound Therapy to Reduce Skin Scars: An In Vitro Study Using a Multi-Well Device Based on Printable Piezoelectric Transducers.

Excessive skin scarring affects over 100 million patients worldwide, with effects ranging from cosmetic to systemic problems, and an effective treatment is yet to be found. Ultrasound-based therapies have been used to treat a variety of skin disorders, but the exact mechanisms behind the observed effects are still unclear. The aim of this work was to demonstrate the potential of ultrasound for the treatment of abnormal scarring by developing a multi-well device based on printable piezoelectric material (PiezoPaint™). First, compatibility with cell cultures was evaluated using measurements of heat shock response and cell viability. Second, the multi-well device was used to treat human fibroblasts with ultrasound and quantify their proliferation, focal adhesions, and extracellular matrix (ECM) production. Ultrasound caused a significant reduction in fibroblast growth and ECM deposition without changes in cell viability or adhesion. The data suggest that these effects were mediated by nonthermal mechanisms. Interestingly, the overall results suggest that ultrasound treatment would a be beneficial therapy for scar reduction. In addition, it is expected that this device will be a useful tool for mapping the effects of ultrasound treatment on cultured cells.

The Mediator subunit MED12 promotes formation of HSF1 condensates on heat shock response element arrays in heat-shocked cells.

Upon heat shock, activated heat shock transcription factor 1 (HSF1) binds to the heat shock response elements (HSEs) in the promoters of mammalian heat shock protein (HSP)-encoding genes and recruits the preinitiation complex and coactivators, including Mediator. These transcriptional regulators may be concentrated in phase-separated condensates around the promoters, but they are too minute to be characterized in detail. We herein established HSF1-/- mouse embryonic fibroblasts harbouring HSP72-derived multiple HSE arrays and visualized the condensates of fluorescent protein-tagged HSF1 with liquid-like properties upon heat shock. Using this experimental system, we demonstrate that endogenous MED12, a subunit of Mediator, is concentrated in artificial HSF1 condensates upon heat shock. Furthermore, the knockdown of MED12 markedly reduces the size of condensates, suggesting an important role for MED12 in HSF1 condensate formation.

HSP27 modulates tumoural immune evasion by enhancing STAT3-mediated upregulation of PD-L1 and NLRC5 in ovarian cancer.

Multiple preclinical studies have demonstrated that the addition of hyperthermia (HT) to immunotherapy could enhance tumour immunogenicity and stimulate an antitumour immune response, primarily via heat shock proteins (HSPs). However, antitumour immune responses are often impeded by immune evasion mechanisms, such as the overexpression of programmed death-ligand1 (PD-L1) and the loss of major histocompatibility complex class 1 (MHC-1) expression. In this context, we sought to investigate the effect of HT on PD-L1 and NOD-like receptor family CARD domain containing 5 (NLRC5) identified as the key transcriptional activator of MHC-1 genes, and their interaction in ovarian cancer. A coculture of ovarian cancer cell lines (IGROV1 and SKOV3) with peripheral blood mononuclear cells was set up. Then, culture media conditioned with IGROV1 or SKOV3 subjected to HT was tested on untreated cell cultures. Knocking down heat shock protein B1 (HSPB1 or HSP27), heat shock protein A1 (HSPA1 or HSP70), and pharmacological inhibition of STAT3 phosphorylation were performed. Subsequently, we measured expression levels of PD-L1, NLRC5, and proinflammatory cytokines. The correlation between PD-L1 and NLRC5 expression in ovarian cancer was evaluated using the Cancer Genome Atlas database. We found that HT produces a concomitant decrease in PD-L1 and NLRC5 expression in coculture. Notably, however, the conditioned media by heat-shocked cells increases their expression. HSP27 knockdown can reverse this increase. Adding STAT3 phosphorylation inhibitor significantly enhanced the expression inhibition of PD-L1 and NLRC5 induced by HSP27 silencing. Correlation analysis showed a positive correlation in ovarian cancer between NLRC5 and PD-L1. These findings demonstrate that HSP27 modulates PD-L1 and NLRC5 expression through the activation of a common regulator 'STAT3'. Moreover, the positive correlation between PD-L1 and NLRC5 led us to conclude that the upregulation of PD-L1 and the downregulation of MHC class I are two mutually exclusive mechanisms of immune evasion in ovarian cancer.

PlantPhoneDB: A manually curated pan-plant database of ligand-receptor pairs infers cell-cell communication.

Ligand-receptor pairs play important roles in cell-cell communication for multicellular organisms in response to environmental cues. Recently, the emergence of single-cell RNA-sequencing (scRNA-seq) provides unprecedented opportunities to investigate cellular communication based on ligand-receptor expression. However, so far, no reliable ligand-receptor interaction database is available for plant species. In this study, we developed PlantPhoneDB (https://jasonxu.shinyapps.io/PlantPhoneDB/), a pan-plant database comprising a large number of high-confidence ligand-receptor pairs manually curated from seven resources. Also, we developed a PlantPhoneDB R package, which not only provided optional four scoring approaches that calculate interaction scores of ligand-receptor pairs between cell types but also provided visualization functions to present analysis results. At the PlantPhoneDB web interface, the processed datasets and results can be searched, browsed, and downloaded. To uncover novel cell-cell communication events in plants, we applied the PlantPhoneDB R package on GSE121619 dataset to infer significant cell-cell interactions of heat-shocked root cells in Arabidopsis thaliana. As a result, the PlantPhoneDB predicted the actively communicating AT1G28290-AT2G14890 ligand-receptor pair in atrichoblast-cortex cell pair in Arabidopsis thaliana. Importantly, the downstream target genes of this ligand-receptor pair were significantly enriched in the ribosome pathway, which facilitated plants adapting to environmental changes. In conclusion, PlantPhoneDB provided researchers with integrated resources to infer cell-cell communication from scRNA-seq datasets.

26S proteasomes become stably activated upon heat shock when ubiquitination and protein degradation increase

Heat shock (HS) promotes protein unfolding, and cells respond by stimulating HS gene expression, ubiquitination of cell proteins, and proteolysis by the proteasome. Exposing HeLa and other cells to 43 °C for 2 h caused a twofold increase in the 26S proteasomes' peptidase activity assayed at 37 °C. This increase in activity occurred without any change in proteasome amount and did not require new protein synthesis. After affinity-purification from HS cells, 26S proteasomes still hydrolyzed peptides, adenosine 5'-triphosphate, and ubiquitinated substrates more rapidly without any evident change in subunit composition, postsynthetic modification, or association with reported proteasome-activating proteins. After returning HS cells to 37 °C, ubiquitin conjugates and proteolysis fell rapidly, but proteasome activity remained high for at least 16 h. Exposure to arsenite, which also causes proteotoxic stress in the cytosol, but not tunicamycin, which causes endoplasmic reticulum stress, also increased ubiquitin conjugate levels and 26S proteasome activity. Although the molecular basis for the enhanced proteasomal activity remains elusive, we studied possible signaling mechanisms. Proteasome activation upon proteotoxic stress required the accumulation of ubiquitinated proteins since blocking ubiquitination by E1 inhibition during HS or arsenite exposure prevented the stimulation of 26S activity. Furthermore, increasing cellular content of ubiquitin conjugates at 37 °C by inhibiting deubiquitinating enzymes with RA190 or b-AP15 also caused proteasome activation. Thus, cells respond to proteotoxic stresses, apparently in response to the accumulation of ubiquitinated proteins, by activating 26S proteasomes, which should help promote the clearance of damaged cell proteins.

Abstract 1009: A novel C-terminal HSP90 inhibitor NCT-58 targets cancer stem-like properties and suppresses migratory ability in triple-negative breast cancer cells

Abstract Background: Although several N-terminal HSP90 inhibitors have been investigated in clinical trials, none have yet been approved for clinical use due to issues including induction of the heat shock response (HSR), off-target effects and toxicity. A major issue for N-terminal inhibitors is induction of the heat shock response (HSR) which triggers cell survival. In the present study, we investigated the effects of NCT-58, a rationally-designed novel HSP90 inhibitor that instead targets the C-terminal domain, and evaluated its capacity to induce apoptosis and target cancer stem-like properties in triple-negative breast cancer. Materials and Methods: The effect of NCT-58 on TNBC cell lines in vitro was evaluated with cell viability, apoptosis, heat shock response and cell migration. Cancer stem-like properties were examined by Aldefluor positivity, CD44+/CD24- stem-like population and mammosphere formation assays. Results: NCT-58 induces apoptosis in TNBC cells without triggering the heat shock response (HSR) due to its targeting of the C-terminal region. This is accompanied by potent and simultaneous degradation of AKT, MEK and STAT3. Importantly, NCT-58 kills not only the rapidly proliferating tumor cells and but also effectively eradicates the breast cancer stem-like population (BCSCs). The latter phenomena are accompanied by reductions in the activity of stem/progenitor marker ALDH1 and the CD44+/CD24- stem-like population and as well as impairment of mammosphere formation. Furthermore, NCT-58 markedly impairs cell migratory ability, coinciding with collapse of HSP90 client cytoskeletal proteins including vimentin and F-actin in TNBC cells in vitro. It is noteworthy that NCT-58 exhibits more cytotoxic to tumor cells but minimally cytotoxicity to non-malignant cells. Conclusion: These findings suggest that NCT-58 may represent an effective therapeutic approach for the simultaneous targeting of HSP90 and its client oncoproteins for the treatment of molecularly heterogeneous TNBC. Citation Format: Soeun Park, Jung Min Park, Minsu Park, Dongmi Ko, Seongjae Kim, Juyeon Seo, Kee Dal Nam, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. A novel C-terminal HSP90 inhibitor NCT-58 targets cancer stem-like properties and suppresses migratory ability in triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1009.

HSFs drive transcription of distinct genes and enhancers during oxidative stress and heat shock.

Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 localizes to a unique set of promoters and enhancers to trans-activate oxidative stress-specific genes. Taken together, we show that HSFs function as multi-stress-responsive factors that activate distinct genes and enhancers when encountering changes in temperature and redox state.

BH3-Only Proteins Noxa and Puma Are Key Regulators of Induced Apoptosis.

Apoptosis is an evolutionarily conserved and tightly regulated cell death pathway. Physiological cell death is important for maintaining homeostasis and optimal biological conditions by continuous elimination of undesired or superfluous cells. The BH3-only pro-apoptotic members are strong inducers of apoptosis. The pro-apoptotic BH3-only protein Noxa activates multiple death pathways by inhibiting the anti-apoptotic Bcl-2 family protein, Mcl-1, and other protein members leading to Bax and Bak activation and MOMP. On the other hand, Puma is induced by p53-dependent and p53-independent apoptotic stimuli in several cancer cell lines. Moreover, this protein is involved in several physiological and pathological processes, such as immunity, cancer, and neurodegenerative diseases. Future heat shock research could disclose the effect of hyperthermia on both Noxa and BH3-only proteins. This suggests post-transcriptional mechanisms controlling the translation of both Puma and Noxa mRNA in heat-shocked cells. This study was also the chance to recapitulate the different reactional mechanisms investigated for caspases.

Oxidant/Antioxidant Status Is Impaired in Sepsis and Is Related to Anti-Apoptotic, Inflammatory, and Innate Immunity Alterations.

Oxidative stress is considered pivotal in the pathophysiology of sepsis. Oxidants modulate heat shock proteins (Hsp), interleukins (IL), and cell death pathways, including apoptosis. This multicenter prospective observational study was designed to ascertain whether an oxidant/antioxidant imbalance is an independent sepsis discriminator and mortality predictor in intensive care unit (ICU) patients with sepsis (n = 145), compared to non-infectious critically ill patients (n = 112) and healthy individuals (n = 89). Serum total oxidative status (TOS) and total antioxidant capacity (TAC) were measured by photometric testing. IL-6, -8, -10, -27, Hsp72/90 (ELISA), and selected antioxidant biomolecules (Ζn, glutathione) were correlated with apoptotic mediators (caspase-3, capsase-9) and the central anti-apoptotic survivin protein (ELISA, real-time PCR). A wide scattering of TOS, TAC, and TOS/TAC in all three groups was demonstrated. Septic patients had an elevated TOS/TAC, compared to non-infectious critically ill patients and healthy individuals (p = 0.001). TOS/TAC was associated with severity scores, procalcitonin, IL-6, -10, -27, IFN-γ, Hsp72, Hsp90, survivin protein, and survivin isoforms -2B, -ΔΕx3, -WT (p < 0.001). In a propensity probability (age-sex-adjusted) logistic regression model, only sepsis was independently associated with TOS/TAC (Exp(B) 25.4, p < 0.001). The AUCTOS/TAC (0.96 (95% CI = 0.93–0.99)) was higher than AUCTAC (z = 20, p < 0.001) or AUCTOS (z = 3.1, p = 0.002) in distinguishing sepsis. TOS/TAC, TOS, survivin isoforms -WT and -2B, Hsp90, IL-6, survivin protein, and repressed TAC were strong predictors of mortality (p < 0.01). Oxidant/antioxidant status is impaired in septic compared to critically ill patients with trauma or surgery and is related to anti-apoptotic, inflammatory, and innate immunity alterations. The unpredicted TOS/TAC imbalance might be related to undefined phenotypes in patients and healthy individuals.

Ultrasmall AgBiSe2 nanodots for CT/thermal imaging-guided photothermal tumor therapy in the NIR-II biowindow.

Stimulus-responsive ternary chalcogenide nanomaterials are regarded as promising 'all-in-one' nanotheranostics agents on account of their tunable band structures and multi-metal intrinsic properties. Herein, ultrasmall AgBiSe2 nanodots are prepared by a simple thermal injection method. It shows a narrow band gap of 0.91 eV and high absorption coefficient in the NIR-II biowindow, resulting in excellent photothermal performance. Under the irradiation of a 1064 nm laser, AgBiSe2 can induce the overexpression of intracellular heat shock protein (Hsp70) and cell apoptosis to inhibit the growth of tumor cells. The strong signal from CT/thermal imaging also provides guidance for tumor diagnosis. Importantly, AgBiSe2 can be rapidly excreted from the body, thus avoiding long term toxicity. This study presents the first biomedical application of AgBiSe2 nanodots in cancer treatment and extends the development of ternary chalcogenide-based semiconductor nanomedicine.

More Related Gene Pathways to Vincristine-Induced Death Events in a Human T-Acute Lymphoblastic Leukemia Cell Line.

Acute lymphoblastic leukemia (ALL) is common in children but rare in adults. Vincristine (VCR) is one of the drugs used at the beginning of treatment. Some genes are resistant to VCR in B-ALL. Here, we examined the effect of VCR on gene expression changes in a T-ALL cell line, Jurkat. The MTT method was used to determine the IC50 in Jurkat cells treated with different concentrations of VCR for 48 and 72 hours. Total RNA was isolated from the cells and cDNA was prepared. The Human Cancer Drug Target PCR Array kit was used to evaluate the 84 gene expression changes in Jurkat cells. Protein-protein interaction was analyzed by STRING software. We identified 66 differentially expressed genes as comparison to untreated cells. The response to VCR-induced apoptotic events was remarkable in the pathways of heat shock protein, topoisomerases, protein kinases, cathepsins and cell cycle. In other pathways, there were resistant genes as well as sensitive genes to VCR treatment. Some proteins like HSP90AA1 and ESR1 had determining associations with other proteins. The results suggest VCR target genes in T-ALL cells may be beneficial biomarkers for ALL treatment and can be used to select appropriate synergistic drugs for VCR.

Down-Regulation of Yeast Helicase Ded1 by Glucose Starvation or Heat-Shock Differentially Impairs Translation of Ded1-Dependent mRNAs.

Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for mRNAs with structured 5′UTRs. Recent evidence suggests that the condensation of Ded1 in mRNA granules down-regulates Ded1 function during heat-shock and glucose starvation. We examined this hypothesis by determining the overlap between mRNAs whose relative translational efficiencies (TEs), as determined by ribosomal profiling, were diminished in either stressed WT cells or in ded1 mutants examined in non-stress conditions. Only subsets of the Ded1-hyperdependent mRNAs identified in ded1 mutant cells exhibited strong TE reductions in glucose-starved or heat-shocked WT cells, and those down-regulated by glucose starvation also exhibited hyper-dependence on initiation factor eIF4B, and to a lesser extent eIF4A, for efficient translation in non-stressed cells. These findings are consistent with recent proposals that the dissociation of Ded1 from mRNA 5′UTRs and the condensation of Ded1 contribute to reduced Ded1 function during stress, and they further suggest that the down-regulation of eIF4B and eIF4A functions also contributes to the translational impairment of a select group of Ded1 mRNA targets with heightened dependence on all three factors during glucose starvation.

Caspase-mediated cleavage of miRNA processing proteins Drosha, DGCR8, Dicer, and TRBP2 in heat-shocked cells and its inhibition by HSP70 overexpression.

Cells respond to stress through adaptive mechanisms that limit cellular damage and prevent cell death. MicroRNAs act as regulators of stress responses and stress can impact the functioning of miRNA biogenesis pathways. We were interested in the effect that severe proteotoxic stress capable of inducing apoptosis may have on miRNA biogenesis and the impact of the molecular chaperone protein HSP70 under these conditions. We found that the miRNA processing enzymes Drosha and Dicer and their accessory proteins DGCR8 and TRBP2 are cleaved by caspases in apoptotic cells. Overexpression of HSP70 prevented caspase activation and the degradation of these processing proteins. Caspase cleavage of TRBP2 was mapped to amino acid 234 which separates the two dsRNA-binding domains from the C-terminal Dicer interacting domain. Overexpression of TRBP2 was found to increase miRNA maturation, while expression of either of the fragments generated by caspase cleavage impaired maturation. These results indicate that inactivation of miRNA biogenesis is a critical feature of apoptosis and that cleavage of TRBP2, rather than simply a loss of function, serves to create positive acting inhibitors of pre-miRNA maturation.

Quantitative proteomics identifies the universally conserved ATPase Ola1p as a positive regulator of heat shock response in Saccharomyces cerevisiae

The universally conserved P-loop ATPase Ola1 is implicated in various cellular stress response pathways, as well as in cancer and tumor progression. However, Ola1p functions are divergent between species, and the involved mechanisms are only poorly understood. Here, we studied the role of Ola1p in the heat shock response of the yeast Saccharomyces cerevisiae using a combination of quantitative and pulse labeling-based proteomics approaches, in vitro studies, and cell-based assays. Our data show that when heat stress is applied to cells lacking Ola1p, the expression of stress-protective proteins is enhanced. During heat stress Ola1p associates with detergent-resistant protein aggregates and rapidly forms assemblies that localize to stress granules. The assembly of Ola1p was also observed in vitro using purified protein and conditions, which resembled those in living cells. We show that loss of Ola1p results in increased protein ubiquitination of detergent-insoluble aggregates recovered from heat-shocked cells. When cells lacking Ola1p were subsequently relieved from heat stress, reinitiation of translation was delayed, whereas, at the same time, de novo synthesis of central factors required for protein refolding and the clearance of aggregates was enhanced when compared with wild-type cells. The combined data suggest that upon acute heat stress, Ola1p is involved in the stabilization of misfolded proteins, which become sequestered in cytoplasmic stress granules. This function of Ola1p enables cells to resume translation in a timely manner as soon as heat stress is relieved.