Inducible Hsp70 Protein Research Articles

Genetics and ontogeny are key factors influencing thermal resilience in a culturally and economically important bivalve

Increasing seawater temperatures coupled with more intense and frequent heatwaves pose an increasing threat to marine species. In this study, the New Zealand green-lipped mussel, Perna canaliculus, was used to investigate the effect of genetics and ontogeny on thermal resilience. The culturally and economically significant mussel P. canaliculus (Gmelin, 1971) has been selectively-bred in New Zealand for two decades, making it a unique biological resource to investigate genetic interactions in a temperate bivalve species. Six selectively-bred full sibling families and four different ages, from early juveniles (6, 8, 10 weeks post-fertilisation) to sub-adults (52 weeks post-fertilisation), were used for experimentation. At each age, each family was exposed to a three-hour heat challenge, followed by recovery, and survival assessments. The shell lengths of live and dead juvenile mussels were also measured. Gill tissue samples from sub-adults were collected after the thermal challenge to quantify the 70 kDa heat shock protein gene (hsp70). Results showed that genetics, ontogeny and size influence thermal resilience in P. canaliculus, with LT50 values ranging between 31.3 and 34.4 °C for all studied families and ages. Juveniles showed greater thermotolerance compared to sub-adults, while the largest individuals within each family/age class tended to be more heat sensitive than their siblings. Sub-adults differentially upregulated hsp70 in a pattern that correlated with net family survival following heat challenge, reinforcing the perceived role of inducible HSP70 protein in molluscs. This study provides insights into the complex interactions of age and genotype in determining heat tolerance of a key mussel species. As marine temperatures increase, equally complex selection pressure responses may therefore occur. Future research should focus on transcriptomic and genomic approaches for key species such as P. canaliculus to further understand and predict the effect of genetic variation and ontogeny on their survival in the context of climate change.

Quinazoline Based HSP90 Inhibitors: Synthesis, Modeling Study and ADME Calculations Towards Breast Cancer Targeting

A new 2-thioquinazolinones series was designed and synthesized as HSP90 inhibitors based on the structure of hit compound VII obtained by virtual screening approach. Their in vitro anti-proliferative activity was evaluated against three human cancer cell lines rich in HSP90 namely; colorectal carcinoma (HCT-116), and cervical carcinoma (Hela), breast carcinoma (MCF-7). Compounds 5a, 5d, 5e and 9h showed a significant broad spectrum anti-proliferative activity against all tested cell lines. They were characterized by potent effect against breast cancer in particular with IC50 of 11.73, 8.56, 7.35 and 9.48 μM, respectively against Doxorubicin (IC50 4.17 μM). HSP90 ATPase activity inhibition assay were conducted where compound 5d exhibited the best IC50 with 1.58 μM compared to Tanespimycin (IC50 = 2.17 μM). Compounds 5a and 9h showed higher IC50 values of 3.21 and 3.41 μM, respectively. The effects of 5a, 5d and 9h on Her2 (a client proteins of HSP90) and HSP70 were evaluated in MCF-7 cells. All tested compounds were found to reduce Her2 protein expression levels and induce Hsp70 protein expression levels significantly, emphasizing that antibreast cancer effect is a consequence of HSP90 chaperone inhibition. Cell cycle analysis of MCF-7 cells treated with 5d showed cell cycle arrest at G2/M phase 38.89% and pro-apoptotic activity as indicated by annexin V-FITC staining by 22.42%. Molecular docking studies suggested mode of interaction to HSP90 via hydrogen bonding. ADME properties prediction of the active compounds suggested that they could be used as orally absorbed anticancer drug candidates.

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding*[S

Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that, apart from conserved molecular allostery, Hsp70 proteins have retained and adapted the ability to assemble as functionally relevant ATP-bound dimers throughout evolution. Here, we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins, showing that their dimerization propensities differ, with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. Structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40, as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo, supporting its functional role in vivo As human cytosolic Hsp70 can interact with tetratricopeptide repeat (TPR) domain containing cochaperones, we tested the interaction of Hsp70 ATP-dependent dimers with Chip and Tomm34 cochaperones. Although Chip associates with intact Hsp70 dimers to form a larger complex, binding of Tomm34 disrupts the Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests a novel role of TPR domain cochaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.

Design, synthesis and biological evaluation of 7-(aryl)-2,3-dihydro-[1,4]dioxino[2,3-g]quinoline derivatives as potential Hsp90 inhibitors and anticancer agents

A new series of quinoline analogues was designed and synthesized as Hsp90 inhibitors. The cytotoxic activity of the synthesized compounds was evaluated against three human cancer cell lines including MCF-7 (human breast cancer cells), DU145 (human prostate cancer cell lines), and A549 (adenocarcinomic human alveolar basal epithelial cells). Some of our compounds (13a-13f) showed significant cytotoxic activity on MCF-7 cells. The most potent anti-proliferative compounds were also tested against Her2, a client protein of Hsp90. Compound 13d that demonstrated the highest antiproliferative activity in the series, was found the most potent one for both Her2 protein degradation and Hsp70 protein induction as well. Molecular modeling studies displayed possible mode of interaction between this compound and N-terminal ATP-binding site of Hsp90.

HSP70 in human polymorphonuclear and mononuclear leukocytes: comparison of the protein content and transcriptional activity of HSPA genes.

Cell-type specific variations are typical for the expression of different members of the HSP70 family. In circulating immune cells, HSP70 proteins interact with units of signaling pathways involved in the immune responses and may promote cell survival in sites of inflammation. In this work, we compared basal HSP70 expression and stress-induced HSP70 response in polymorphonuclear and mononuclear human leukocytes. The intracellular content of inducible and constitutive forms of HSP70 was analyzed in relation to the transcriptional activity of HSPA genes. Hyperthermia was used as the stress model for induction of HSP70 synthesis in the cells. Our results demonstrated that granulocytes (mainly neutrophils) and mononuclear cells differ significantly by both basal HSP70 expression and levels of HSP70 induction under hyperthermia. The differences were observed at the levels of HSPA gene transcription and intracellular HSP70 content. The expression of constitutive Hsс70 protein was much higher in mononuclear cells consisting of monocytes and lymphocytes than in granulocytes. At the same time, intact neutrophils showed increased expression of inducible Hsp70 protein compared to mononuclear cells. Heat treatment induced additional expression of HSPA genes in leukocytes. The most pronounced increase in the expression was observed in polymorphonuclear and mononuclear leukocytes for HSPA1A/B. However, in granulocytes, the induction of the transcription of the HSPA8 gene encoding the Hsc70 protein was significantly higher than in mononuclear cells. These variations in transcriptional activity of HSPA genes and intracellular HSP70 content in different populations of leukocytes may reflect specified requirements for the chaperone activity in the cells with a distinct functional role in the immune system.

Response of Two Mytilids to a Heatwave: The Complex Interplay of Physiology, Behaviour and Ecological Interactions.

Different combinations of behavioural and physiological responses may play a crucial role in the ecological success of species, notably in the context of biological invasions. The invasive mussel Xenostrobus securis has successfully colonised the inner part of the Galician Rias Baixas (NW Spain), where it co-occurs with the commercially-important mussel Mytilus galloprovincialis. This study investigated the effect of a heatwave on the physiological and behavioural responses in monospecific or mixed aggregations of these species. In a mesocosm experiment, mussels were exposed to simulated tidal cycles and similar temperature conditions to those experienced in the field during a heat-wave that occurred in the summer of 2013, when field robo-mussels registered temperatures up to 44.5°C at low tide. The overall responses to stress differed markedly between the two species. In monospecific aggregations M. galloprovincialis was more vulnerable than X. securis to heat exposure during emersion. However, in mixed aggregations, the presence of the invader was associated with lower mortality in M. galloprovincialis. The greater sensitivity of M. galloprovincialis to heat exposure was reflected in a higher mortality level, greater induction of Hsp70 protein and higher rates of respiration and gaping activity, which were accompanied by a lower heart rate (bradycardia). The findings show that the invader enhanced the physiological performance of M. galloprovincialis, highlighting the importance of species interactions in regulating responses to environmental stress. Understanding the complex interactions between ecological factors and physiological and behavioural responses of closely-related species is essential for predicting the impacts of invasions in the context of future climate change.

Chronology of UPR activation in skeletal muscle adaptations to chronic contractile activity.

The mitochondrial and endoplasmic reticulum unfolded protein responses (UPR(mt) and UPR(ER)) are important for cellular homeostasis during stimulus-induced increases in protein synthesis. Exercise triggers the synthesis of mitochondrial proteins, regulated in part by peroxisome proliferator activator receptor-γ coactivator 1α (PGC-1α). To investigate the role of the UPR in exercise-induced adaptations, we subjected rats to 3 h of chronic contractile activity (CCA) for 1, 2, 3, 5, or 7 days followed by 3 h of recovery. Mitochondrial biogenesis signaling, through PGC-1α mRNA, increased 14-fold after 1 day of CCA. This resulted in 10-32% increases in cytochrome c oxidase activity, indicative of mitochondrial content, between days 3 and 7, as well as increases in the autophagic degradation of p62 and microtubule-associated proteins 1A/1B light chain 3A (LC3)-II protein. Before these adaptations, the UPR(ER) transcripts activating transcription factor-4, spliced X-box-binding protein 1, and binding immunoglobulin protein were elevated (1.3- to 3.8-fold) at days 1-3, while CCAAT/enhancer-binding protein homologous protein (CHOP) and chaperones binding immunoglobulin protein and heat shock protein (HSP) 70 were elevated at mRNA and protein levels (1.5- to 3.9-fold) at days 1-7 of CCA. The mitochondrial chaperones 10-kDa chaperonin, HSP60, and 75-kDa mitochondrial HSP, the protease ATP-dependent Clp protease proteolytic subunit, and the regulatory protein sirtuin-3 of the UPR(mt) were concurrently induced 10-80% between days 1 and 7 To test the role of the UPR in CCA-induced remodeling, we treated animals with the endoplasmic reticulum stress suppressor tauroursodeoxycholic acid and subjected them to 2 or 7 days of CCA. Tauroursodeoxycholic acid attenuated CHOP and HSP70 protein induction; however, this failed to impact mitochondrial remodeling. Our data indicate that signaling to the UPR is rapidly activated following acute contractile activity, that this is attenuated with repeated bouts, and that the UPR is involved in chronic adaptations to CCA; however, this appears to be independent of CHOP signaling.

The role of inducible Hsp70 protein in modulation of neurodegenerative pathology in the nigrostriatal system typical to Parkinson’s disease

The role of inducible Hsp70 protein in modulation of neurodegenerative pathology in the nigrostriatal system typical to Parkinson’s disease

Changes in the heat shock 70 kDa protein level in human neutrophils induced by heat shock

Changes in the content of constitutive and inducible proteins of the family of heat shock 70 kDa proteins (HSP70) caused by heat shock in human neutrophils, white blood cells with an atypically short lifespan, which provide a nonspecific defense of the organism against bacterial pathogens, have been studied. An analysis of the intracellular content of the constitutive and inducible HSP70 proteins by flow cytometry revealed a biphasic dynamics of changes in the protein level, which was characterized by an increase in the protein level immediately after heat shock followed by a decrease within 15–30 min after the termination of heat treatment. Because the inhibitor of protein synthesis cycloheximide did not change the dynamics profile, it was assumed that the increase in the HSP70 level is related not to the de novo synthesis of these proteins but to conformational changes of HSP70 molecules and an increased accessibility of some epitopes for antibody binding. Using a panel of antibodies specific to the N-terminal ATP-binding or the C-terminal substrate-binding domains of the protein, it was shown by cell immunofluorescence and flow cytometry that the heat shock-associated increase in the intracellular HSP70 level results from an increased efficiency of the binding of antibodies recognizing the substrate-binding domain. It was also demonstrated that the decrease in the intracellular HSP70 level after the heat shock, may be partially due to a release into the extracellular space of both the constitutive and inducible HSP70 proteins, which is regulated with the involvement of ABC-transporters.

HSP70-binding protein HSPBP1 regulates chaperone expression at a posttranslational level and is essential for spermatogenesis.

Molecular chaperones play key roles during growth, development, and stress survival. The ability to induce chaperone expression enables cells to cope with the accumulation of nonnative proteins under stress and complete developmental processes with an increased requirement for chaperone assistance. Here we generate and analyze transgenic mice that lack the cochaperone HSPBP1, a nucleotide-exchange factor of HSP70 proteins and inhibitor of chaperone-assisted protein degradation. Male HSPBP1(-/-) mice are sterile because of impaired meiosis and massive apoptosis of spermatocytes. HSPBP1 deficiency in testes strongly reduces the expression of the inducible, antiapoptotic HSP70 family members HSPA1L and HSPA2, the latter of which is essential for synaptonemal complex disassembly during meiosis. We demonstrate that HSPBP1 affects chaperone expression at a posttranslational level by inhibiting the ubiquitylation and proteasomal degradation of inducible HSP70 proteins. We further provide evidence that the cochaperone BAG2 contributes to HSP70 stabilization in tissues other than testes. Our findings reveal that chaperone expression is determined not only by regulated transcription, but also by controlled degradation, with degradation-inhibiting cochaperones exerting essential prosurvival functions.

SIRT1 Knockdown Promotes Neural Differentiation and Attenuates the Heat Shock Response

Neurons have a limited capacity for heat shock protein (HSP) induction and are vulnerable to the pathogenic consequence of protein misfolding and aggregation as seen in age-related neurodegenerative diseases. Sirtuin 1 (SIRT1), an NAD(+) -dependent lysine deacetylase with important biological functions, has been shown to sustain the DNA-binding state of HSF1 for HSP induction. Here we show that differentiation and maturation of embryonic cortical neurons and N2a neuroprogenitor cells is associated with decreases in SIRT1 expression and heat shock-dependent induction of HSP70 protein. Tests of a pharmacological activator and an inhibitor of SIRT1 affirm the regulatory role of SIRT1 in HSP70 induction. Protein cross-linking studies show that nuclear SIRT1 and HSF1 form a co-migrating high molecular weight complex upon stress. The use of retroviral vectors to manipulate SIRT1 expression in N2a cells show that shRNA-mediated knock down of SIRT1 causes spontaneous neurite outgrowth coincident with reduced growth rate and decreased induction of hsp70-reporter gene, whereas SIRT1 over-expression blocks the induced neural differentiation of N2a cells. Our results suggest that decreased SIRT1 expression is conducive to neuronal differentiation and this decrease contributes to the attenuated induction of HSPs in neurons.

Increased NFAT5 expression stimulates transcription of Hsp70 in preeclamptic placentas

ObjectiveWe investigated the expression of heat shock protein 70 (Hsp70), nuclear factor of activated T cells 5 (NFAT5), and hypoxia-induced factor-1α (HIF-1α) in the placentas of normal and preeclamptic pregnancies and in human placental hypoxia models in vitro to examine the regulatory mechanisms of placental Hsp70 expression. MethodsThe expression levels of HIF-1α, NFAT5, and Hsp70 were examined in placental samples from 10 females with preeclampsia and 10 normotensive control patients and in human choriocarcinoma trophoblast cells treated with 1 mM CoCl2 by western blotting. Using models of placental hypoxia, pharmacological inhibition of HIF-1α with chetomin and shRNA knockdown and overexpression of NFAT5 were performed to investigate the roles of HIF-1α and NFAT5 in induction of Hsp70 by placental hypoxia. ResultsThe levels of HIF-1α, NFAT5, and Hsp70 expression were significantly higher in the preeclamptic compared to normal placentas. In the placental hypoxia models, the expression of HIF-1α, NFAT5, and Hsp70 were significantly higher after 3, 6, and 12 h of 1 mM CoCl2 treatment, respectively. Pharmacological inhibition of HIF-1α suppressed the induction of NFAT5 and Hsp70 at the protein level. shRNA knockdown of NFAT5 suppressed the induction of Hsp70 protein and overexpression of NFAT5 stimulated the induction of Hsp70 mRNA and protein in models of human placental hypoxia in vitro. ConclusionHIF-1α positively regulates the induction of NFAT5 and Hsp70 by placental hypoxia and NFAT5 stimulates transcription of Hsp70 in response to placental hypoxia in models of human placental hypoxia in vitro.

Detection of constitutive and inducible HSP70 proteins in formalin fixed human brain tissue

The investigation of formalin fixed and paraffin embedded tissue is a routine method in forensic histology. Since these samples are usually stored for decades they provide a unique tissue bank for different scientific issues. In the past, numerous studies were conducted using different kinds of paraffin embedded tissues. However, it is well known that formalin affects macromolecules and thus might hamper reliable and reproducible molecular experiments.The aim of this study was to find out if the treatment with formalin has a negative effect on different protein detection methods and additionally to define the dimension of those possible deleterious effects.We incubated brain tissue samples in formalin for up to three months. After incubation, the samples were analyzed using immunohistochemistry (IHC) and Western blotting to specifically detect and quantify members of the HSP70 superfamily (heat shock proteins). Our study shows that the Western blot analysis of formalin fixed tissues does not allow a reliable detection of proteins at all, while a reproducible detection by IHC was still possible after one month of incubation.

Low endogenous and chemical induced heat shock protein induction in a 0N3Rtau-expressing Drosophila larval model of Alzheimer's disease.

Reduction of tau phosphorylation and aggregation by manipulation of heat shock protein (HSP) molecular chaperones has received much attention in attempts to further understand and treat tauopathies such as Alzheimer's disease. We examined whether endogenous HSPs are induced in Drosophila larvae expressing human tau (3R-tau) in motor neurons, and screened several chemical compounds that target the HSP system using medium-throughput behavioral analysis to assay their effects on tau-induced neuronal dysfunction in vivo. Tau-expressing larvae did not show a significant endogenous HSP induction response, whereas robust induction of hsp70 was detectable in a similar larval model of polyglutamine disease. Although pan-neuronal tau expression augmented the induction of hsp70 following heat shock, several candidate HSP inducing compounds induced hsp70 protein in mammalian cells in vitro but did not detectably induce hsp70 mRNA or protein in tau expressing larvae. The hsp90 inhibitors 17-AAG and radicicol nevertheless caused a dose-dependent reduction in total human tau levels in transgenic larvae without specifically altering tau hyperphosphorylated at S396/S404. These and several other HSP modulating compounds also failed to rescue the tau-induced larval locomotion deficit in this model. Tau pathology in tau-expressing larvae, therefore, induces weak de novo HSP expression relative to other neurodegenerative disease models, and unlike these disease models, pharmacological manipulation of the hsp90 pathway does not lead to further induction of the heat shock response. Forthcoming studies investigating the effects of HSP induction on tau-mediated dysfunction/toxicity in such models will require more robust, non-pharmacological (perhaps genetic) means of manipulating the hsp90 pathway.

Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm(3). Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8 ± 103.4 mm(2) at the beginning versus 704.8 ± 94.4 mm(3) at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9% ± 4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5 ± 0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

The heat shock response in congeneric land snails (Sphincterochila) from different habitats

Land snails are subject to daily and seasonal variations in temperature and in water availability, and use heat shock proteins (HSPs) as part of their survival strategy. We used experimental heat stress to test whether adaptation to different habitats affects HSP expression in two closely related Sphincterochila snail species, a desert species, Sphincterochila zonata, and a Mediterranean-type species, Sphincterochila cariosa. Our findings show that in S. cariosa, heat stress caused rapid induction of Hsp70 proteins and Hsp90 in the foot and kidney tissues, whereas the desert-inhabiting species S. zonata displayed delayed induction of Hsp70 proteins in the foot and upregulation of Hsp90 alone in the kidney. Our study suggests that Sphincterochila species use HSPs as part of their survival strategy following heat stress and that adaptation to different habitats results in the development of distinct strategies of HSP expression in response to heat, namely the reduced induction of HSPs in the desert-dwelling species. We suggest that the desert species S. zonata relies on mechanisms and adaptations other than HSP induction, thus avoiding the fitness consequences of continuous HSP upregulation.

Synthesis and biological evaluation of 2,4-diaminoquinazoline derivatives as novel heat shock protein 90 inhibitors

Novel 2,4-diaminoquinazoline derivatives originating from a virtual screening approach were designed, synthesized and their biological activities as heat shock protein 90 (Hsp90) inhibitors were evaluated. The prepared compounds exhibited significant anti-proliferative activities against DU-145, HT-29, HCT-116, A375P and MCF-7 cancer cell lines. The selected compounds were tested against Her2, a client protein of Hsp90, and showed significant reduction in Her2 protein expression. Compound 6b was found the most potent, reduced Her2 protein expression levels and induced Hsp70 protein expression levels significantly.

A Novel mouse model of enhanced proteostasis: Full-length human heat shock factor 1 transgenic mice

The heat shock response (HSR) is controlled by the master transcriptional regulator heat shock factor 1 (HSF1). HSF1 maintains proteostasis and resistance to stress through production of heat shock proteins (HSPs). No transgenic model exists that overexpresses HSF1 in tissues of the central nervous system (CNS). We generated a transgenic mouse overexpressing full-length non-mutant HSF1 and observed a 2–4-fold increase in HSF1 mRNA and protein expression in all tissues studied of HSF1 transgenic (HSF1 +/0) mice compared to wild type (WT) littermates, including several regions of the CNS. Basal expression of HSP70 and 90 showed only mild tissue-specific changes; however, in response to forced exercise, the skeletal muscle HSR was more elevated in HSF1 +/0 mice compared to WT littermates and in fibroblasts following heat shock, as indicated by levels of inducible HSP70 mRNA and protein. HSF1 +/0 cells elicited a significantly more robust HSR in response to expression of the 82 repeat polyglutamine-YFP fusion construct (Q82YFP) and maintained proteasome-dependent processing of Q82YFP compared to WT fibroblasts. Overexpression of HSF1 was associated with fewer, but larger Q82YFP aggregates resembling aggresomes in HSF1 +/0 cells, and increased viability. Therefore, our data demonstrate that tissues and cells from mice overexpressing full-length non-mutant HSF1 exhibit enhanced proteostasis.

Arsenite stabilizes HIF-1α protein through p85α-mediated up-regulation of inducible Hsp70 protein expression

Hypoxia-inducible factor-1α (HIF-1α) has been reported to regulate over 100 gene expressions in response to hypoxia and other stress conditions. In the present study, we found that arsenite could induce HIF-1α protein accumulation in both mouse epidermal Cl41 cells and mouse embryonic fibroblasts (MEFs). Knockout of p85α, a regulatory subunit of PI-3K, in MEFs (p85α(-/-)) dramatically decreased the arsenite-induced HIF-1α accumulation, indicating that p85α is crucial for arsenite effects on the stabilization of HIF-1α protein. Our further studies suggest that arsenite could induce inducible Hsp70 expression, and transfection of inducible Hsp70 into p85α(-/-) MEFs could restore HIF-1α protein accumulation. Moreover, the results using EMSA and Supershift assays indicate that p85α is crucial for arsenite-induced activation of the heat-shock transcription factor 1 (HSF-1), which is responsible for transcription of inducible Hsp70. Taken together, p85α-mediated HIF-1α stabilization upon arsenite exposure is specifically through HSF-1 activation and subsequent up-regulation of the inducible Hsp70 expression.

The Nrf2–Keap1 cellular defense pathway and heat shock protein 70 (Hsp70) response. Role in protection against oxidative stress in early neonatal unilateral ureteral obstruction (UUO)

Perturbation of renal tubular antioxidants and overproduction of reactive oxygen species may amplify the proinflammatory state of renal obstruction, culminating in oxidative stress and tubular loss. Here, we analyzed the heat shock protein 70 (Hsp70) response and the function and signal transduction of NF-E2-related protein 2 (Nrf2) transcription factor on oxidative stress modulation in obstruction. Rats were subjected to unilateral ureteral obstruction or sham operation and kidneys harvested at 5, 7, 10, and 14days after obstruction. Hsp70 expression and Nrf2 activity and its downstream target gene products were assessed. After 10 and 14days of obstruction, enhanced lipid peroxidation through higher thiobarbituric acid reactive substances levels and increased oxidative stress resulted in reduced total antioxidant activity and enhanced nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity were demonstrated. This was accompanied by decreased inducible Hsp70 expression and a progressive reduction of nuclear Nrf2 and its target gene products glutathione S-transferase A2 (GSTA2) and NADPH/quinone oxidoreductase 1 (NQO1), whereas the Nrf2 repressor Kelch-like ECH-associated protein-1 (Keap1) was upregulated. By contrast, on early obstruction for 7days, lack of increased oxidative markers associated with higher inducible Hsp70 protein levels and a rapid nuclear accumulation of Nrf2, Keap1 downregulation, and mRNA induction of the identified Nrf2-dependent genes, NQO1 and GSTA2, were shown. For these results, we suggest that the magnitude of cytoprotection in early obstruction depends on the combined contribution of induced activation of Nrf2 upregulating its downstream gene products and Hsp70 response. Impaired ability to mount the biological response to the prevailing oxidative stress leading to renal injury was shown in prolonged obstruction.