Autophagy and Hsp70 activation alleviate oral epithelial cell death induced by food-derived hypertonicity.

Stable intracellular and intercellular osmolarity is vital for all physiological processes. Although it is the first organ that receives food, the osmolarity around the mouth epithelium has never been systematically investigated. We found that oral epithelial cells are a population of ignored cells routinely exposed to hypertonic environments mainly composed of saline, glucose, etc. in vivo after chewing food. By using cultured oral epithelial cells as an in vitro model, we found that the hypotonic environments caused by both high NaCl and high glucose induced cell death in a dose- and time-dependent manner. Transcriptomics revealed similar expression profiles after high NaCl and high glucose stimulation. Most of the common differentially expressed genes were enriched in "mitophagy" and "autophagy" according to KEGG pathway enrichment analysis. Hypertonic stimulation for 1 to 6 h resulted in autophagosome formation. The activation of autophagy protected cells from high osmolarity-induced cell death. The activation of Hsp70 by the pharmacological activator handelin significantly improved the cell survival rate after hypertonic stimulation. The protective role of Hsp70 activation was partially dependent on autophagy activation, indicating a crosstalk between Hsp70 and autophagy in hypertonic stress response. The extract of the handelin-containing herb Chrysanthemum indicum significantly protected oral epithelial cells from hypertonic-induced death, providing an inexpensive way to protect against hypertonic-induced oral epithelial damage. In conclusion, the present study emphasized the importance of changes in osmolarity in oral health for the first time. The identification of novel compounds or herbal plant extracts that can activate autophagy or HSPs may contribute to oral health and the food industry.

The up-regulation of two identified wound healing specific proteins-HSP70 and lysozyme in regenerated Eisenia fetida through transcriptome analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Disposed earthworm has been used to treat various common ailments including burns, arthritis, itching, and inflammation for thousands of years in China. As their remarkable ability to fully regenerate in a scar-free manner, regenerated tissue homogenate of amputated Eisenia fetida (E. fetida) have been considered as an excellent wound repair therapy in our previous study. We have demonstrated that regenerated earthworm (G-90') can perform higher wound repair ability to non-regeneration tissue (G-90) through significant promotion of cutaneous wound repair in mice after their administration into wound beds. OBJECTIVE: In the present study, we aimed to reveal the mechanism of G-90' and to explore a potential wound healing accelerated strategy. METHODS AND RESULTS: Two functional proteins- HSP70 and lysozyme in G-90' were confirmed by cross-identification of LC-MS/MS and transcriptome analyses. Followed with semi-quantitative PCR and western blot, their expression were validated to up-regulate in 3-day regenerated tissues (G-90'). CONCLUSION: This study implies the therapeutic potency of G-90' for wound recovery and provides a new strategy to assess other natural materials targeting wound healing with the tail-amputated E .fetida as a model organism.

Inducing Muscle Heat Shock Protein 70 Improves Insulin Sensitivity and Muscular Performance in Aged Mice.

Heat shock proteins (HSPs) are molecular chaperones with roles in longevity and muscular preservation. We aimed to show elevating HSP70 improves indices of health span. Aged C57/BL6 mice acclimated to a western diet were randomized into: geranylgeranylacetone (GGA)-treated (100 mg/kg/d), biweekly heat therapy (HT), or control. The GGA and HT are well-known pharmacological and environmental inducers of HSP70, respectively. Assessments before and after 8 weeks of treatment included glycemic endpoints, body composition, and muscular endurance, power, and perfusion. An HT mice had more than threefold, and GGA mice had a twofold greater HSP70 compared with control. Despite comparable body compositions, both treatment groups had significantly better insulin sensitivity and insulin signaling capacity. Compared with baseline, HT mice ran 23% longer than at study start, which was significantly more than GGA or control. Hanging ability (muscular endurance) also tended to be best preserved in HT mice. Muscle power, contractile force, capillary perfusion, and innervation were not different. Heat treatment has a clear benefit on muscular endurance, whereas HT and GGA both improved insulin sensitivity. Different effects may relate to muscle HSP70 levels. An HSP induction could be a promising approach for improving health span in the aged mice.

Alanyl-glutamine and glutamine plus alanine supplements improve skeletal redox status in trained rats: involvement of heat shock protein pathways.

AIMS: We hypothesized that oral l-glutamine supplementations could attenuate muscle damage and oxidative stress, mediated by glutathione (GSH) in high-intensity aerobic exercise by increasing the 70-kDa heat shock proteins (HSP70) and heat shock factor 1 (HSF1). MAIN METHODS: Adult male Wistar rats were 8-week trained (60-min/day, 5 days/week) on a treadmill. During the last 21 days, the animals were supplemented with either l-alanyl-l-glutamine dipeptide (1.5 g/kg, DIP) or a solution containing the amino acids l-glutamine (1g/kg) and l-alanine (0.67 g/kg) in their free form (GLN+ALA) or water (controls). KEY FINDINGS: Plasma from both DIP- and GLN+ALA-treated animals showed higher l-glutamine concentrations and reduced ammonium, malondialdehyde, myoglobin and creatine kinase activity. In the soleus and gastrocnemius muscle of both supplemented groups, l-glutamine and GSH contents were increased and GSH disulfide (GSSG) to GSH ratio was attenuated (p<0.001). In the soleus muscle, cytosolic and nuclear HSP70 and HSF1 were increased by DIP supplementation. GLN+ALA group exhibited higher HSP70 (only in the nucleus) and HSF1 (cytosol and nucleus). In the gastrocnemius muscle, both supplementations were able to increase cytosolic HSP70 and cytosolic and nuclear HSF1. SIGNIFICANCE: In trained rats, oral supplementation with DIP or GLN+ALA solution increased the expression of muscle HSP70, favored muscle l-glutamine/GSH status and improved redox defenses, which attenuate markers of muscle damage, thus improving the beneficial effects of high-intensity exercise training.

Heat shock increases expression of NAD(P)H:quinone oxidoreductase (NQO1), mediator of beta-lapachone cytotoxicity, by increasing NQO1 gene activity and via Hsp70-mediated stabilisation of NQO1 protein.

NAD(P)H:quinone oxidoreductase (NQO1) mediates cell death caused by the novel anti-cancer drug beta-lapachone (beta-lap). Therefore, beta-lap sensitivity of cells is positively related to the level of cellular NQO1. Heat shock up-regulates NQO1 expression in cancer cells, thereby enhancing the clonogenic cell death caused by beta-lap. The mechanisms by which heat shock elevates NQO1 expression were investigated in the present study using human A549 lung cancer cells and human MDA-MB-231 breast cancer cells. When MDA-MB-231(NQO1+) cells stably transfected with NQO1 were heated at 42 degrees C for 1 h the expression of NQO1 and the sensitivity of the cells to beta-lap progressively increased during the 24-48 h post-heating period. Heating increased NQO1 transcription by cis-acting elements such as xenobiotic response element and antioxidant response element located in the NQO1 gene promoter region. The turnover of NQO1 protein in heated cells was much slower than in unheated cells. NQO1 and heat shock protein 70 (Hsp70) co-precipitated and co-localised in cells before and after heating, demonstrating the close association of these two proteins in the cells. These results suggest that NQO1 is stabilised by the Hsp70 molecular chaperone. It is concluded that the prolonged increase in NQO1 expression after heat shock is due to increased NQO1 transcription, and also increased Hsp70-mediated NQO1 stabilisation.

Diminished reproductive potential of male mice in response to selenium-induced oxidative stress: involvement of HSP70, HSP70-2, and MSJ-1.

The oxidative stress imposed by nutritional variations in selenium (Se) has plausible role in reproductive toxicology and affects the reproductive potential. Also, the expression of heat shock proteins (HSPs) is a highly regulated event throughout the process of spermatogenesis and is modulated by stressful stimuli. This prompted us to investigate the possibility that Se-induced oxidative stress may affect the fertility status by altering the expressions of the constitutive and inducible HSP70 proteins, having crucial role in spermatogenesis. Different Se status-deficient, adequate, and excess, male Balb/c mice were created by feeding yeast-based Se-deficient diet (group I) and deficient diet supplemented with Se as sodium selenite at 0.2 and 1 ppm Se (group II and III) for a period of 8 weeks. After completion of the diet-feeding schedule, a significant decrease in the Se and glutathione peroxidase (GSH-Px) levels was observed in the Se-deficient group (I), whereas Se-excess group (III) demonstrated an increase. Increased levels of reactive oxygen species, malondialdehyde, and alterations in the redox status in both groups I and III indicated oxidative-stressed conditions. There was an overall reduced fertility status in mice supplemented with Se-deficient and Se-excess diet. The mRNA and protein expression of HSP70 was found to be elevated in these two groups, whereas the expression patterns of HSP70-2 and MSJ-1 demonstrated a reverse trend. In vitro CDC2 kinase assay showed reduced kinase activity in group I and group III. These findings suggest that Se-induced oxidative stress by differentially regulating various HSP70s can affect its downstream factors having crucially important role in differentiation of germ cells and completion of spermatogenesis. Therefore, it can provide an insight into the mechanism(s) by which the oxidative stress-induced reproductive toxicity can lead to increased apoptosis/growth arrest and infertility. This will thus add new dimensions to the molecular mechanism underlying the human male infertility and open new vistas in the development of various chemo-preventive methods.

Acute acidification or amiloride treatment suppresses the ability of Hsp70 to inhibit heat-induced apoptosis.

Inhibition of stress-induced apoptosis by the molecular chaperone protein Hsp70 is a contributing factor in tumorigenesis and suppression of this ability could increase the effectiveness of anti-tumor therapy. Tumor cells exist in an acidic environment and acute acidification can sensitize tumor cells to heat-induced cell death. However, the ability of Hsp70 to prevent apoptosis under these conditions has not been examined. The effect of acute acidification on heat-induced apoptosis was examined in a human T-cell line with tetracycline-regulated Hsp70 expression. Apoptosis was inhibited in cells exposed to hyperthermia in acidic media when examined 6 h after the heat stress, but resumed if cells were returned to physiological pH during this recovery period. Long-term proliferation assays showed that acute acidification sensitized cells to heat-induced apoptosis. Hsp70 expressing cells were also sensitized and this was correlated with a reduced ability to suppress the activation of JNK (c-jun N-terminal kinase), Bax and caspase-3. Further sensitization could be achieved with the NHE1 (Na(+)/H(+) exchanger) inhibitor HMA (5-(N, N-hexamethylene) amiloride), which potentiated JNK activation in heat-shocked cells. These results demonstrate that the ability of Hsp70 to suppress apoptosis is compromised when cells are exposed to hyperthermia in an acidic environment, which is correlated with an impaired ability to inhibit JNK activation.

Mitochondrial dysfunction, free radical generation and cellular stress response in neurodegenerative disorders.

Protein conformational diseases, such as Alzheimer's, Parkinson's and Huntington's, affect a large portion of aging population. The pathogenic dysfunctional aggregation of proteins in non-native conformations is associated with metabolic derangements and excessive production of reactive oxygen species. Reduction of cellular expression and activity of antioxidant proteins result in increased oxidative stress. Free-radicals derived from mitochondrial dysfunction and from the cyclooxygenase enzyme activity play a role in oxidative damage of brain. Cyclooxygenase also mediates in neuro-inflammation by the production of pro-inflammatory prostaglandins which contribute to brain injury. The pathogenic role of cyclooxygenase has been demonstrated in Alzheimer and Parkinson diseases. The brain responses to detect and control diverse forms of stress are accomplished by a complex network of "longevity assurance processes" integrated to the expression of genes termed vitagenes. Heat shock proteins are a highly conserved system responsible for the preservation and repair of correct protein conformation. Heme oxygenase-1, a inducible and redox-regulated enzyme, is currently considered as having an important role in cellular antioxidant defense. A neuroprotective effect, due to its heme degrading activity, and tissue-specific pro-oxidant effects, due to its products CO and free iron, are under debate. There is a current interest in dietary compounds that can inhibit, retard or reverse the multi-stage pathophysiology of Alzheimer disease, with a chronic inflammatory response, brain injury and beta-amyloid associated pathology. Curcumin and ferulic acid, two powerful antioxidants, the first from the curry spice turmeric and the second a major constituent of fruit and vegetables, have emerged as strong inducers of the heat shock response. Food supplementation with curcumin and ferulic acid is considered a nutritional approach to reduce oxidative damage and amyloid pathology in Alzheimer disease.

Involvement of multitargets in paeoniflorin-induced preconditioning.

Paeoniflorin (PF) is the principal component of Paeoniae radix prescribed in traditional Chinese medicine. The delayed neuroprotection induced by PF preconditioning and its underlying mechanisms were investigated in rat middle cerebral artery occlusion (MCAO) and reperfusion model. At a dosage of 20 or 40 mg/kg, PF preconditioning 48 h before MCAO followed by 24-h reperfusion significantly reduced the mortality and infarct volume and reversed the neurological deficits caused by ischemia. Likewise, the ameliorative effects on mortality, infarct size, and neurological impairment induced by MCAO emerged as well when PF was administered 24 h, 48 h, or 5 days before MCAO at the dose of 20 mg/kg. Furthermore, comparative proteomics analysis was adopted to identify the differentially expressed proteins induced by PF preconditioning itself. The relative levels of 42 proteins were altered after PF preconditioning, among which 20 were elevated and 22 reduced. In summary, A(1) receptor-regulator of G protein signaling-K(ATP) signaling, arachidonic acid cascade, nitric oxide system, markers of neuronal damage, mitochondrial damage-related molecules, and the mitogen-activated protein kinase and nuclear factor-kappaB pathway are associated with the mechanisms of PF preconditioning.

Characterization of a recombinant adenovirus vector encoding heat-inducible feline interleukin-12 for use in hyperthermia-induced gene-therapy.

Interleukin-12 (IL-12) is a pro-inflammatory cytokine that has shown great promise as a therapeutic agent in experimental models of infectious disease and cancer. However, it is also a highly toxic molecule and for that reason has not been accepted readily into the clinic. A replication-deficient adenoviral vector was designed to deliver the feline interleukin-12 gene into tumour cells. The interleukin-12 gene has been placed under control of a heat inducible promoter, human heat shock promoter 70b, with the intent of spatially and temporally controlling the expression of IL-12, thus limiting its toxicity. In vitro, the transfection efficiency of the adenoviral vector, the effect of multiplicity of infection and the production of biologically active feline IL-12 were studied in the infected cells in response to a range of temperatures. This adenoviral vector will be a useful tool to examine the effects of intra-tumoural IL-12 delivery in a spontaneously occurring feline soft tissue sarcoma model.

Comparison of the anti-tumor effects of various whole-body hyperthermia protocols: correlation with HSP 70 expression and composition of splenic lymphocytes.

Whole-body hyperthermia (WBH) has been used as an adjunct approach to radio-/ chemotherapy for tumor therapy for many years. However, the molecular mechanism underlying the enhancement of tumor control is not clearly understood. It has been hypothesized that WBH might activate immune system by inducing the expression of heat shock proteins (HSPs), which are thought to facilitate the presentation of tumor-specific antigens. In the present work, we examined the effects of various thermal doses of WBH on tumor growth delay and HSP70 levels in tumors on C57BL/6 mice, as well as on splenic lymphocyte subpopulations. The maximal WBH effect (about 40% decrease in tumor weight) was achieved by a 2-hour WBH treatment everyday at 40.0 degrees C. By using this treatment schedule, the populations of CD3+/CD4+ T cells and CD3+/CD8+ T cells increased by 4 and 3 times, respectively, at the end of WBH treatment period. When the length of day-by-day WBH treatment was longer than 2 hours or the frequency of WBH treatment was lower than once a day, the effect of tumor growth delay and the population of CD3+ T lymphocyte in spleen increase were discounted. On the other hand, the HSP70 levels in tumor nodules rose continuously as the WBH treating time increased, but the populations of NK cells in spleen did not change significantly. The results suggest that an increased CD3+ T lymphocyte population is closely related to the anti-tumor effect of WBH, which might be a useful marker for effectiveness of hyperthermia. However, neither the levels of HSP70 nor the NK cell populations in spleen appear to correlate to tumor control.

Preconditioned somatothermal stimulation on median nerve territory increases myocardial heat shock protein 70 and protects rat hearts against ischemia-reperfusion injury.

OBJECTIVE: This study was designed to test the hypotheses that local somatothermal stimulation on the left median nerve territory increases myocardial heat shock protein 70 and that preconditioning of rats with local somatothermal stimulation protects the hearts against subsequent ischemia-reperfusion injury. METHODS: Local somatothermal stimulation was brought about by means of application of a heating rod over and above the left median nerve territory (1.5 cm proximal to the palm crease) in male Sprague-Dawley rats. After rats were treated with local somatothermal stimulation, the gene expression of heat shock protein 70 in regional muscle, heart, and liver was assessed by means of Western blotting and reverse transcription-polymerase chain reaction. In addition, durations of arrhythmia, mortality rates, and mitochondrial functions were compared between groups preconditioned with or without local somatothermal stimulation followed by subsequent myocardial ischemia-reperfusion injury. RESULTS: The results showed that the gene expression of heat shock protein 70 was upregulated in the muscle beneath the area of local somatothermal stimulation, as well as in the heart, although not in the liver. When animals were preconditioned with local somatothermal stimulation on the left median nerve territory followed by subsequent ischemia-reperfusion injury of the heart, there were significant decreases of creatine kinase level from the heart, duration of arrhythmia, mortality rate, and improved mitochondrial respiratory function compared with that seen in those without local somatothermal stimulation preconditioning. CONCLUSION: We conclude that local heat stress preconditioning on the left median nerve territory has a potential cardioprotective effect against subsequent ischemia-reperfusion injury.

Up-regulation of glutamate receptors in nucleus tractus solitarii underlies potentiation of baroreceptor reflex by heat shock protein 70.

Whereas induction of the 70-kDa heat shock protein (HSP70) in the nucleus tractus solitarii (NTS), the terminal site in the brain stem for primary baroreceptor afferents, augments baroreceptor reflex (BRR) response, the underlying cellular and molecular mechanism is essentially unexplored. In Sprague-Dawley rats, we evaluated the hypothesis that HSP70 may potentiate BRR response by up-regulating the molecular synthesis and functional expression of glutamate receptors in the NTS. Animals subjected to brief hyperthermic heat shock (HS; 42 degrees C for 15 min) exhibited augmented expression of NR1 or NR2A subunit of N-methyl-D-aspartate (NMDA) receptors, GluR1 or GluR4 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors and KA1 subunit of kainate receptors in the NTS. Intriguingly, this up-regulation of glutamate receptors was preceded by an increase in HSP70 expression at the NTS. The HS-induced augmentation in responsiveness of barosensitive NTS neurons to transient hypertension or potentiation of BRR response was discernibly blunted by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Bilateral microinjection into the NTS of an antisense hsp70 oligonucleotide (50 pmol) before HS significantly suppressed the induced expression of HSP70 or the increase in glutamate receptor subunits in the dorsal medulla and discernibly attenuated the potentiation of BRR response. Control microinjection into the NTS of sense or scrambled hsp70 oligonucleotide (50 pmol) was ineffective. These findings suggest that HSP70 induced by HS may enhance BRR response by up-regulating the molecular synthesis and functional expression of NR1 or NR2A subunit of NMDA receptors and GluR1, GluR4, or KA1 subunit of non-NMDA receptors in the NTS.

Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas.

BACKGROUND AND AIM: Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues. METHODS: In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, beta adrenergic stimulation) stress. RESULTS: Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis. CONCLUSIONS: These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.

Role of priming stresses and Hsp70 in protection from ischemia-reperfusion injury in cardiac and skeletal muscle.

Ischemia-reperfusion injury limits the survival of muscle involved in tissue trauma or transfers during microsurgical reconstruction. Priming stresses such as ischemic preconditioning or mild hyperthermia have frequently been associated with improved survival of ischemic-reperfused cardiac muscle, such protection coinciding with induction of the stress-related heat shock protein 70 (Hsp70). Little is known about the response of skeletal muscle to priming stresses. This review summarizes the current knowledge on the use of priming stresses as protective strategies against the consequences of ischemia-reperfusion in cardiac and skeletal muscle and the potential role of Hsp70.

[The protective effect of thermal treatment before irradiation on CFUs from bone marrow of mice: a potential involvement of heat shock proteins]. / Zashchitnyi éffekt predradiatsionnoi termicheskoi obrabotki myshei na KOEs ikh kostnogo mozga: vozmozhnoe uchastie belkov teplovogo shoka.

It was studied on mice how prior whole body hyperthemia affects a colony-forming ability of bone marrow after gamma-irradiation. It was found that heating of the animals (42 degrees C, 10 min) 18-22 h before their total irradiation (4 Gy) increases 2-fold the level of CFUs8 and CFUs12 determined in the spleen exotest. The induced radioresistance correlated with accumulation of heat shock proteins, HSP70 and HSP25, in tissues of preheated mice. Injection of quercetin (a selective inhibitor of the heat shock protein synthesis) 0.5 h before the heating fully abolished both the subsequent heat shock protein accumulation and the rise in CFUs populations as compared with control. It is suggested that heat shock proteins, whose expression increases in response to hyperthermia, can play a role of endogenous radioprotectors. Possible mechanisms of their protective action under irradiation are discussed.

Effect of DnaK and DnaJ proteins deprivation on Escherichia coli response to starvation.

E. coli defects in response to nutritional starvation caused by DnaK and DnaJ proteins deprivation are examined. The ability of delta dnaKdnaJ mutant to survive carbon, nitrogen and phosphorus starvation is highly impaired while delta dnaJ mutant is characterized by the diminished survival of phosphorus starvation only. delta dnaKdnaJ mutant grows slowly utilizing maltose and glycerol and delta dnaJ mutant utilizes glycerol inefficiently. The growth on alternate nitrogen sources is comparable to wild-type strain.

Inhibition of heat-shock protein 70 induction in intestinal cells overexpressing cyclooxygenase 2.

BACKGROUND & AIMS: The cyclooxygenase (COX) enzymes catalyze the initial step of prostaglandin formation; the inducible form, COX-2, plays a role in inflammation. Heat-shock protein 70 (hsp70) is a stress-responsive gene important for cell survival; induction of hsp70 appears to be mediated, in part, by the prostaglandin pathway. We determined the effect of COX-2 overexpression on hsp70 induction in rat intestinal epithelial (RIE) cells. METHODS: RIE cells transfected with COX-2 complementary DNA oriented in the sense (RIE-S) or antisense (RIE-AS) direction were subjected to a heat shock; RNA and protein were harvested and analyzed by Northern and Western blots, respectively. Gel shift assays were performed to assess DNA binding. RESULTS: Both hsp70 messenger RNA and HSP70 protein levels were increased in the RIE-AS cells, whereas induction was markedly inhibited in the RIE-S cells after heat shock. Inhibition of heat-shock factor binding was noted in RIE-S cells, suggesting that heat-shock transcription factor regulation may explain the inhibition of hsp70. The COX-2 selective inhibitor, NS-398, reversed the effects of COX-2 overexpression. CONCLUSIONS: The results support a functional role for the prostaglandin/COX pathway in the induction of hsp70. The findings underscore a potential regulatory mechanism involving an inverse relationship between COX-2 expression and hsp70 induction.

Chaperone coexpression plasmids: differential and synergistic roles of DnaK-DnaJ-GrpE and GroEL-GroES in assisting folding of an allergen of Japanese cedar pollen, Cryj2, in Escherichia coli.

Plasmids that can be used for controlled expression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly or recombinant proteins in Escherichia coli. A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when L-arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable when it was produced in E. coli K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor sigma 32 revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of sigma 32 at certain temperatures.

A mathematical model of the hsp70 regulation in the cell.

A mathematical model of the regulation process of the heat shock protein hsp70 in the cell is presented. The model describes the damaging effect of elevated temperature on proteins; the interaction of free hsp70 with injured proteins and its chaperone role in nascent protein translation; the relation between the amount of free hsp70 and the formation of the activated trimer form of the heat shock factor protein (HSF); the binding of activated HSF with the heat shock elements on the DNA; the transcription of mRNA of hsp70 and the synthesis of hsp70. The reaction of the model to a temporal rise in temperature shows an initial decline and a subsequent sharp rise to an ultimately increased level of free hsp70 in the cell. The response of the model to both a single and two consecutive heat shocks appears to closely resemble experimental data on hsp70 synthesis. This general agreement demonstrates the structure of the model to be sound and suitable as a basis for further modelling the complex tolerance mechanism of the cell.