Heat shock protein 70 purification and characterization from Cyprinion macrastomus macrastomus and Garra rufa obtusa

Organisms resist stress factors by common mechanisms at cellular and physiological levels. Heat shock proteins (Hsps) play an essential role as a protective mechanism against stressors, and Hsp70 has a central role in the heat shock response. Although several investigations have been made on the cellular functions of Hsps, very little is known about extreme temperature effects on organisms like fish. To address this point, we have studied two fish species, Cyprinion macrostomus macrostomus and Garra rufa obtuse, whose habitat is the Kangal hot spring in Turkey. The hot spring has multiple stress factors; extremely hot temperature, food deprivation, infectious medium and low levels of oxygen. Like other Hsp70s, two Cyprinidae species Hsp70 showed similar ATPase and substrate protein folding activities in vitro. These proteins also showed relatively higher thermal stability. Biochemical characterization of two Hsp70, suggested a Hsp mechanism capable of protecting the cell against stressors even under adverse conditions and in the presence of multiple stress factors.     

Hsp70B' and Hsp72 form a complex in stressed human colon cells and each contributes to cytoprotection

Hsp70B′ is the only major human isoform in the hsp70 family that is strictly stress-inducible, and therefore available to function only in stressed cells. Since Hsp70B′ is evolutionarily closely related to human Hsp72, they are thought to function similarly, but direct evidence of Hsp70B′ function in stressed cells has been lacking. Here we showed that both Hsp70B′ and Hsp72 are essential relatively early after heat stress in the acquisition of cytoprotection by two human colon cell lines. Using flow cytometry to count viable cells, we also showed that cytoprotection is more pronounced in cultures grown at low cell number (LCN), where there is an ample amount of both Hsp70s. siRNA knock-down of either Hsp70B′ or Hsp72 severely handicapped the ability of cells to acquire cytoprotection. Hsp70B′ and Hsp72 were found to form a complex following stress that included the co-chaperone HOP. These results taken together support the hypothesis that Hsp70B′ and Hsp72 play cooperative roles in cell survival of proteotoxic stress. In addition there are implications for chemotherapy protocols and for pathological conditions in which the contributions to cytoprotection of both Hsp70B′ and Hsp72 are modulated by cell numbers or density.     

Effects of environmental stress on intertidal mussels and their sea star predators

Consumer stress models of ecological theory predict that predators are more susceptible to stress than their prey. Intertidal mussels, Mytilus californianus, span a vertical stress gradient from the low zone (lower stress) to the high zone (higher thermal and desiccation stress), while their sea star predators, Pisaster ochraceus, range from the low zone only into the lower edge of the mussel zone. In summer 2003, we tested the responses of sea stars and mussels to environmental stress in an experiment conducted on the Oregon coast. Mussels were transplanted from the middle of the mussel bed to cages in the low and high edges of the mussel bed. Sea star predators were added to half of the mussel cages. Mussels and sea stars were sampled between June and August for indicators of sublethal stress. Mussel growth was measured, and tissues were collected for heat shock protein (Hsp70) analyses and histological analyses of reproduction. Sea stars were weighed, and tissues were sampled for Hsp70 analyses. Mussels in high-edge cages had higher levels of total Hsp70 and exhibited spawning activity earlier in the summer than mussels in the low-edge cages. Sea stars suffered high mortality in the high edge, and low-edge sea stars lost weight but showed no differences in Hsp70 production. These results suggest that stress in the intertidal zone affected the mobile predator more than its sessile prey, which is consistent with predictions of consumer stress models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Overexpression of Zostera japonica heat shock protein gene ZjHsp70 enhances the thermotolerance of transgenic Arabidopsis

Molecular Biology Reports - Heat shock protein 70s (Hsp70s) are major members of the heat shock protein family and play a variety of roles to protect plants against stress. Plant Hsp70s are a...     

Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii

Larvae of the brine shrimp Artemia franciscana serve as important feed in fish and shellfish larviculture; however, they are subject to bacterial diseases that devastate entire populations and consequently hinder their use in aquaculture. Exposure to abiotic stress was shown previously to shield Artemia larvae against infection by pathogenic Vibrio, with the results suggesting a mechanistic role for heat shock protein 70. In the current report, combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, the latter indicated by reduced mortality and lower bacterial load in challenge tests. Resistance to Vibrio improved in the face of declining body mass as demonstrated by measurement of ash-free dry weight. Hypothermic stress only and acute osmotic insult did not promote Hsp70 expression and thermotolerance in Artemia larvae nor was resistance to Vibrio challenge augmented. The data support a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. This possibility is now under investigation, and the work may reveal fundamental properties of crustacean immunity. Additionally, the findings are important in aquaculture where development of procedures to prevent bacterial infection of feed stock such as Artemia larvae is a priority.     

Arabidopsis stromal 70-kDa heat shock proteins are essential for chloroplast development

70 kDa heat shock proteins (Hsp70s) act as molecular chaperones involved in essential cellular processes such as protein folding and protein transport across membranes. They also play a role in the cell’s response to a wide range of stress conditions. The Arabidopsis family of Hsp70s homologues includes two highly conserved proteins, cpHsc70-1 and cpHsc70-2 which are both imported into chloroplasts (Su and Li in Plant Physiol 146:1231–1241, 2008). Here, we demonstrate that YFP-fusion proteins of both cpHsc70-1 and cpHsc70-2 are predominantly stromal, though low levels were detected in the thylakoid membrane. Both genes are ubiquitously expressed at high levels in both seedlings and adult plants. We further show that both cpHsc70-1 and cpHsc70-2 harbour ATPase activity which is essential for Hsp70 chaperone activity. A previously described T-DNA insertion line for cpHsc70-1 (ΔcpHsc70-1) has variegated cotyledons, malformed leaves, growth retardation, impaired root growth and sensitivity to heat shock treatment. In addition, under stress conditions, this mutant also exhibits unusual sepals, and malformed flowers and sucrose concentrations as low as 1% significantly impair growth. cpHsc70-1/cpHsc70-2 double-mutants are lethal. However, we demonstrate through co-suppression and artificial microRNA (amiRNA) approaches that transgenic plants with severely reduced levels of both genes have a white and stunted phenotype. Interestingly, chloroplasts in these plants have an unusual morphology and contain few or no thylakoid membranes. Our data show that cpHsc70-1 and cpHsc70-2 are essential ATPases, have overlapping roles and are required for normal plastid structure.     

Stress and development phenotyping of Hsp101 and diverse other Hsp mutants of Arabidopsis thaliana

Heat shock proteins or Hsps are critical in mounting plant resistance against heat stress. The complex Hsp spectrum of Arabidopsis thaliana plant contains over two hundred proteins belonging to six different families namely Hsp20, Hsp40, Hsp60, Hsp70, Hsp90 and Hsp100. Importantly, the cellular function(s) of most Hsps remains to be established. We aimed at phenotyping of stress and development response of the selected, homozygous hsp mutant lines produced by T-DNA insertional mutagenesis method. The heat stress phenotype was assessed for basal and acquired heat stress response at seed and seedling stages. Distinct phenotype was noted for the hot1-3 mutant (knockout mutant of Hsp101 gene) showing higher heat sensitivity and for the salk_087844 mutant (knockout mutant of Hsc70-2 gene) showing higher heat tolerance than the wild type seedlings. The homozygous cs808162 mutant (mutant of ClpB-p gene encoding for the chloroplast-localized form of Hsp101) did not survive even under unstressed, control condition. salk_064887C mutant (mutant of cpn60β4 gene) showed accelerated development cycling. The hot1-3 mutant apart from showing different heat response, exhibited development lesions like bigger size of seeds, buds, siliques, and pollen compared to the wild type plants. In response to controlled deterioration treatment of seeds, hot1-3 seeds showed higher accumulation of reactive oxygen species molecules, higher rates of protein and lipid oxidation and a faster decline in germination rate as compared to wild type seeds. Our findings show that Hsps perform diverse metabolic functions in plant response to stress, growth, and development.

     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

Genome‐wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress

Heat shock proteins (HSP)are essential molecular chaperones that play important roles in the stress stimulation of insects.Bemisia tabaci,a phloem feeder and invasive species,can cause extensive crop damage through direct feeding and transmission of plant viruses.Here we employed comprehensive genomics approaches to identity HSP superfamily members in the Middle East Asia Minor 1 whitefly genome.In total,we identified 26 Hsp genes,including three Hsp90,17 Hsp70,one Hsp60 and five sHSP (small heat shock protein)genes.The HSP gene superfamily of whitefly is expanded compared with the other five insects surveyed here.The gene structures among the same families are relatively conserved.Meanwhile,the motif compositions and secondary structures of BtHsp proteins were predicted.In addition,quantitative polymerase chain reaction analysis showed that the expression patterns of BtHsp gene superfamily were diverse across different tissues of whiteflies.Most Hsp genes were induced or repressed by thermal stress (40℃)and cold treatment (4℃)in whitefly.Silencing the expression of BtHsp70-6 significantly decreased the survival rate of whitefly under 45℃.All the results showed the Hsps conferred thermo-tolerance or cold-tolerance to whiteflies that protect them from being affected by detrimental temperature conditions.Our observations highlighted the molecular evolutionary properties and the response mechanism to temperature assaults of Hsp genes in whitefly.     

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025–10 μg?ml^?1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.     

Arabidopsis thaliana J-class heat shock proteins: cellular stress sensors

Plants are sessile organisms that have evolved a variety of mechanisms to maintain their cellular homeostasis under stressful environmental conditions. Survival of plants under abiotic stress conditions requires specialized group of heat shock protein machinery, belonging to Hsp70:J-protein family. These heat shock proteins are most ubiquitous types of chaperone machineries involved in diverse cellular processes including protein folding, translocation across cell membranes, and protein degradation. They play a crucial role in maintaining the protein homeostasis by reestablishing functional native conformations under environmental stress conditions, thus providing protection to the cell. J-proteins are co-chaperones of Hsp70 machine, which play a critical role by stimulating Hsp70s ATPase activity, thereby stabilizing its interaction with client proteins. Using genome-wide analysis of Arabidopsis thaliana, here we have outlined identification and systematic classification of J-protein co-chaperones which are key regulators of Hsp70s function. In comparison with Saccharomyces cerevisiae model system, a comprehensive domain structural organization, cellular localization, and functional diversity of A. thaliana J-proteins have also been summarized.     

Effect of allozyme heterozygosity on basal and induced levels of heat shock protein (Hsp70), in juvenile Concholepas concholepas (Mollusca)

Organisms cope physiologically with extreme temperature by producing heat shock proteins (HSPs). Expression of Hsp70 enhances thermal tolerance and represents a key strategy for ectotherms to tolerate elevated temperature in nature. Synthesis of these proteins, together with other physiological responses to elevated temperatures, increases energy demands. A positive association between multiple and single locus heterozygosity (MLH and SLH, respectively) and individual fitness has been widely demonstrated. In molluscs, MLH can decrease routine metabolic rates and improve energetic status. Juvenile Concholepas concholepas live in the intertidal zone and are constantly exposed to temperature fluctuations. Thus, these young individuals are exposed both to thermal risks and the large metabolic costs required to cope with thermal stress. We evaluated the effects of allozyme MLH and SLH on basal (control animals) and induced (stressed animals) levels of the Hsp70 in juveniles C. concholepas. Juveniles (n = 400) were acclimated at 16 °C for 2 weeks; then 100 animals were exposed to 24 °C (stress) and 100 were kept at 16 °C (control) for 2 and 7 days. The variability of 20 loci was analyzed by starch gel electrophoresis. For SLH effects we used 7 polymorphic loci. We quantified expression of Hsp70 by Western blot analyses. Hsp70 expression increased markedly (~ 90%) with temperature. We found a positive association between MLH and basal and induced levels of Hsp70 in the 2-day exposure experiment. Regardless of temperature, Hsp70 levels increased with MLH (r² = 0.7 and 0.9, for basal and induced levels, respectively) reaching maximal levels in juveniles with intermediate and high MLH levels (2 and 3 loci), and decreasing slightly (but not significantly) in juveniles with highest MLH (≥ 4 heterozygous loci). However, after 7 days of exposure to thermal stress, less heterozygous juveniles attained the same levels of Hsp70 than more heterozygous juveniles. Given the faster increment of Hsp70 in C. concholepas juveniles with intermediate-high levels of MLH, these individuals could be less affected by thermal stress in the intertidal zone. We found an association between specific loci genotype and higher Hsp70 levels (basal or induced). In comparison to homozygous juveniles, heterozygous juveniles for several loci showed higher Hsp70. However, these associations were not for the same loci in juveniles exposed to high temperature for 2 and 7 days. This suggests genotypic variation at some allozyme loci could be more important in the period of initial response to high temperature and others can be more important in the response to the chronic temperature stress.     

Synaptic thermoprotection in a desert‐dwelling Drosophila species

Synaptic transmission is a critical mechanism for transferring information from the nervous system to the body. Environmental stress, such as extreme temperature, can disrupt synaptic transmission and result in death. Previous work on larval Drosophila has shown that prior heat‐shock exposure protects synaptic transmission against failure during subsequent thermal stress. This induced thermoprotection has been ascribed to an up‐regulation of the inducible heat‐shock protein, Hsp70. However, the mechanisms mediating natural thermoprotection in the wild are unknown. We compared synaptic thermosensitivity between D. melanogaster and a desert species, D. arizonae. Synaptic thermosensitivity and the functional limits of the related locomotor behavior differed significantly between closely related, albeit ecologically distinct species. Locomotory behavior of wandering third instar D. arizonae larvae was less thermosensitive and the upper temperature limit of locomotory function exceeded that of D. melanogaster by 6°C. Behavioral results corresponded with significantly lower synaptic thermosensitivity at the neuromuscular junction in D. arizonae. Prior heat‐shock protected only D. melanogaster by increasing relative excitatory junctional potential (EJP) duration, the time required for EJP failure at 40°C, and the incidence of EJP recovery following heat‐induced failure. Hsp70 induction profiles following heat‐shock demonstrate up‐regulation of inducible Hsp70 in D. melanogaster but not in D. arizonae. However, expression of Hsp70 under control conditions is greater in D. arizonae. These results suggest that the mechanisms of natural thermoprotection involve an increase in baseline Hsp70 expression. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Inducible and constitutive heat shock gene expression responds to modification of Hsp70 copy number in Drosophila melanogaster but does not compensate for loss of thermotolerance in Hsp70 null flies

Background  

The heat shock protein Hsp70 promotes inducible thermotolerance in nearly every organism examined to date. Hsp70 interacts with a network of other stress-response proteins, and dissecting the relative roles of these interactions in causing thermotolerance remains difficult. Here we examine the effect of Hsp70 gene copy number modification on thermotolerance and the expression of multiple stress-response genes in Drosophila melanogaster, to determine which genes may represent mechanisms of stress tolerance independent of Hsp70.     

Mild heat stress at a young age in<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> leads to increased Hsp70 synthesis after stress exposure later in life

In a number of animal species it has been shown that exposure to low levels of stress at a young age has a positive effect on stress resistance later in life, and on longevity. The positive effects have been attributed to the activation of defence/cleaning systems (heat shock proteins (Hsps), antioxidases, DNA repair) or to effects of a changed metabolic rate, or both. We investigated the effect of mild stress exposures early in life on Hsp70 synthesis after a harder stress exposure later in life in five isofemale lines ofDrosophila melanogaster. Female flies were either exposed to repeated bouts of mild heat stress (3 h at 34‡C) at a young age (days 2, 4 and 6 post-eclosion) or held under standard laboratory conditions. At 16 and 32 days of adult age, respectively, flies were exposed to a high temperature treatment known to induce Hsp70 in the investigated species (1 h at 37‡C). Thereafter, the inducible Hsp70 levels were measured. Our data show a tendency towards increased Hsp70 synthesis with increased age for both ’mild stress’ and ’no stress’ flies. Moreover, the results show that flies exposed to mild stress at a young age synthesized more Hsp70 upon induction, compared to control flies, and that this difference was accentuated at 32 days compared to 16 days of age. Thus, bouts of mild heat stress at a young age impact on the physiological stress response system later in life. This may be caused by an increased ability to react to future stresses. Alternatively, the mild stress exposure at a young age may actually have caused cellular damages increasing the need for Hsp70 levels after stress exposure later in life. The importance of an Hsp70 upregulation (throughout life) in explaining the phenomenon of hormesis is discussed, together with alternative hypotheses, and suggestions for further studies.