Heat shock protein expression enhances heat tolerance of reptile embryos

The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes.     

Photosynthetic acclimation to high temperatures associated with heat tolerance in creeping bentgrass

Photosynthetic responses to increasing temperatures play important roles in regulating heat tolerance. The objectives of this study were to determine photosynthetic acclimation to increasing temperatures for creeping bentgrass (Agrostis stolonifera L.) and to examine changes in major photosynthetic components (photosynthetic pigments, photochemical efficiency, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity, and activation state of rubisco) involved in heat responses of photosynthesis. 'Penncross' was exposed to 20, 25, 30, and 35 degrees C for 7d at each temperature (acclimated) before being exposed to 40 degrees C for 28d or directly exposed to 40 degrees C for 28d from 20 degrees C (non-acclimated) in growth chambers. Leaf net photosynthetic rate (Pn), photochemical efficiency, rubisco activity, rubisco activation state, chlorophyll content, and carotenoid content decreased when grasses were subjected to severe heat stress at 40 degrees C for 28d. The declines in rubisco activity and activation state were most dramatic among different photosynthetic components examined in this study. Heat-acclimated plants were able to maintain significantly higher Pn, the content of chlorophyll and carotenoid, and the level of rubisco activity and activation state during subsequent exposure to severe heat stress, compared to non-acclimated plants. These results suggested that photosynthetic acclimation to increasing temperatures contributed to creeping bentgrass tolerance to severe heat stress, which was associated with the maintenance of both higher light-harvesting capacity and carbon fixation activity during heat stress.     

Heat shock protein synthesis and thermal tolerance in wheat

Plants respond to high temperature stress by the synthesis of an assortment of heat shock proteins that have been correlated with an acquired thermal tolerance to otherwise lethal temperatures. This study was conducted to determine whether genotypic differences in acquired thermal tolerance were associated with changes in the pattern of heat shock protein synthesis. The pattern of heat shock protein synthesis was analyzed by ³⁵S-methionine incorporation in wheat (Triticum aestivum L.) varieties exhibiting distinct levels of acquired thermal tolerance. Significant quantitative differences between the cultivars Mustang and Sturdy were observed in the HSP exhibiting apparent molecular weights of 16, 17, 22, 26, 33, and 42 Kilodaltons. Genotypic differences in the synthesis of the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase were observed at 34°C. Two-dimensional electrophoretic analysis revealed unique proteins (16, 17, and 26 kilodaltons) in the thermal tolerant variety Mustang that were absent in the more thermal sensitive variety Sturdy. These results provide a correlation between the synthesis of specific low molecular weight heat shock proteins and the degree of thermal tolerance expressed following exposure to elevated temperatures.     

Heat shock protein 27 stimulates recovery of RNA and protein synthesis following a heat shock

Constitutive expression of human hsp27 resulted in a 100-fold increase in survival to a single lethal heat shock in CHO cells without effecting the development of thermotolerance. A possible mechanism for the thermoprotective function of hsp27 may be increased recovery of protein synthesis and RNA synthesis following a heat shock. A lethal heat shock (44°C, 30 min) results in a 90% reduction in the rate of protein synthesis in non-tolerant cells. Control transfected cells recovered protein synthesis to a pre-heat shock rate 10 h after the heat shock; while cell lines that constitutively express human hsp27 recovered 6 h after the heat shock. Thermotolerant cells had a 50% reduction in protein synthesis, which recovered within 7 h following the heat shock. The same lethal heat shock (44°C, 30 min) reduced RNA synthesis by 60% in the transfected cell lines, with the controls recovering in 7 h; while the hsp27 expressing cell lines recovered within 5 h. Thermotolerant cells had a 40% reduction in RNA synthesis and were able to recover within 4 h. The enhanced ability of hsp27 to facilitate recovery of protein synthesis and RNA synthesis following a heat shock may provide the cell with a survival advantage. J. Cell. Biochem. 66:153–164, 1997. © 1997 Wiley-Liss Inc.     

The heat shock protein HSP70 and heat shock cognate protein HSC70 contribute to antimony tolerance in the protozoan parasite leishmania

Antimony-containing drugs are still the drugs of choice in the treatment of infections caused by the parasite Leishmania. Resistance to antimony is now common in some parts of the world, and several mechanisms of resistance have been described. By transfecting cosmid banks and selecting with potassium antimonyl tartrate (SbIII), we have isolated a cosmid associated with resistance. This cosmid contains 2 copies of the heat shock protein 70 (HSP70) and 1 copy of the heat shock cognate protein 70 (HSC70). Several data linked HSP70 to antimony response and resistance. First, several Leishmania species, both as promastigotes and amastigotes, increased the expression of their HSP70 proteins when grown in the presence of 1 or 2 times the Effect Concentration 50% of SbIII. In several mutants selected for resistance to either SbIII or to the related metal arsenite, the HSP70 proteins were found to be overexpressed. This increase was also observed in revertant cells grown for several passages in the absence of SbIII, suggesting that this increased production of HSP70 is stable. Transfection of HSP70 or HSC70 in Leishmania cells does not confer resistance directly, though these transfectants were better able to tolerate a shock with SbIII. Our results are consistent with HSP70 and HSC70 being a first line of defense against SbIII until more specific and efficient resistance mechanisms take over.     

Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp21

Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25° in relation to each other, suggesting a role for global dynamics in dodecamer function.     

Analysis of conserved domains identifies a unique structural feature of a chloroplast heat shock protein

Summary A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts that this region forms an amphipathic -helix with high conservation of methionine residues on the hydrophobic face and 100% conservation of residues on the hydrophilic face. This structure is similar to three helices, termed methionine bristles, which are found in a methionine-rich domain of a 54 kDa protein component of signal recognition particle (SRP54). The conservation of regions I and II among LMW cytoplasmic and chloroplast HSPs suggests that these HSPs perform related functions in different cellular compartments. However, identification of the methionine bristle domain suggests that chloroplast HSPs also have unique functions or substrates within the special environment of the chloroplast or other plastids.Abbreviations HS heat shock - HSP heat shock protein - LMW low molecular weight     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.     

Heat shock protein 27 and alpha B-crystallin can form a complex, which dissociates by heat shock.

We have previously demonstrated that in non-oncogenic adenovirus-transformed baby rat kidney cells a complex of hsp27 and a 22-kDa protein is present, which is lacking in oncogenic cells (Zantema, A., de Jong, E., Lardenoije, R., and van der Eb, A. J. (1989) J. Virol. 63, 3368-3375). Here we show that the 22-kDa protein is identical to alpha B-crystallin. The complex of hsp27 and alpha B-crystallin is also found in some other (non-transformed) cells. However, in most cells tested only hsp27 and no alpha B-crystallin is synthesized. Gel filtration studies show that both proteins are present almost exclusively in a 700-kDa complex. Heat treatment makes the complex fall apart, which is accompanied by a change in the conformation of alpha B-crystallin. Upon recovery, complexes are formed again from both pre-existing and newly synthesized proteins.     

Changes in heat shock protein synthesis and heat sensitivity during mouse thymocyte development

Heat shock protein synthesis was examined in mouse thymocytes at three stages of development: early embryonic thymocytes, which are CD4^?CD8^?, adult thymocytes, which are primarily CD4⁺CD8⁺, and mature spleen T cells, which are CD4⁺CD8^? or CD4^?CD8⁺. After either a 41°C or 42°C heat shock, the synthesis of the maior heat-inducible protein (hsp68) was elevated during the first hour of recovery but then decreased abruptly in thymocytes from adult mice. In contrast, the synthesis of hsp68 continued for up to 4 h after heating embryonic mouse thymocytes or mature spleen T cells. The more rapid termination ofthe heat shock response in the adult thymocytes was not the result of eitherless heat damage or more rapid repair since the recovery of general protein synthesis was more severely delayed in these cells. As well, the double positive CD4⁺CD8⁺ cells were more sensitive to hyperthermia than either the double negative CD4^?CD8^? or single positive CD4⁺CD8^? or CD4^?CD8⁺ cells. Exposure of fetal thymus organ cultures to elevated temperature revealed that the double negative thymocytes were able to survive and differentiate normally following a heat shock treatment that was lethal for the double positive thymocytes. Exposure of thymocytes from adult mice to elevated temperatures induced apoptotic cell death. This was evident by the cleavage of DNA into oligonucleosome-sized fragments. Quantitation of the extent of DNA fragmentation and the number of apoptotic cells by flow cytometry demonstrated that the extent of apoptotic cell death was related to the severity of the heat stress. Double positive (CD4+CD8+) thymocytes are selected on the basis of their T-cell antigen receptor (TCR). Most of these cells are negatively selected and die within the thymus by an active process of cell deletion known as apoptosis. Restricting hsp synthesis in response to stress might be essential during developmental processes in which cell maturation is likely to result in death rather than functional differentiation. © 1993Wiley-Liss, Inc.     

Correlated evolution of chloroplast heat shock protein expression in closely related plant species

Interspecific variation in chloroplast low molecular weight (cLMW) HSP (heat shock protein) expression was examined with respect to phylogeny, species specific leaf area, chlorophyll fluorescence, and mean environmental conditions within species ranges. Eight species of Ceanothus (Rhamnaceae) were heat shocked for 4 h at several different temperatures. Leaf samples were collected immediately after the heat shock, and cLMW HSP expression was quantified using Western blots. At 45°C species from the subgenus Cerastes had significantly greater cLMW HSP expression than species from the subgenus Ceanothus. Specific leaf area was negatively correlated with cLMW HSP expression after the 45°C heat treatment. In addition, chlorophyll fluorescence (F(v)/F(m)) 1 h after the heat shocks was positively correlated with cLMW HSP expression. Contrary to our prediction, there was no correlation between July maximum temperature within species ranges and cLMW HSP expression. These results suggest that evolutionary differentiation in cLMW HSP expression is associated with leaf physiological parameters and related aspects of life history, yet associations between climatic conditions within species ranges and cLMW HSP expression require further study.     

Overexpression of heat shock protein gene PfHSP21.4 in Arabidopsis thaliana enhances heat tolerance

Nuclear-encoded chloroplast small heat shock proteins (Cp-sHSPs) play important roles in plant stress tolerance due to their abundance and diversity. Their functions in Primula under heat treatment are poorly characterized. Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4 was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4 transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and peroxidase. In addition, the induced expression of HSP101, HSP70, ascorbate peroxidase and Δ1-pyrroline-5-carboxylate synthase under heat stress was more pronounced in transgenic plants than in wild-type plants. These results support the positive role of PfHSP21.4 in response to heat stress in plants.     

A heat shock protein localized to chloroplasts is a member of a eukaryotic superfamily of heat shock proteins.

We have isolated cDNA clones from soybean and pea that specify nuclear-encoded heat shock proteins (HSPs) which localize to chloroplasts. The mRNAs for these HSPs are undetectable at control temperatures, but increase approximately 150-fold during a 2-h heat shock. Hybridization-selection followed by in vitro translation demonstrates that these HSPs are synthesized as precursor proteins which are processed by the removal of 5-6.5 kd during import into isolated chloroplasts. The nucleotide sequence of the cDNAs shows the derived amino acid sequences of the mature pea and soybean proteins are 79% identical. While the predicted transit peptide encoded by the pea cDNA has some characteristics typical of transit sequences, including high Ser content, multiple basic residues and no acidic residues, it lacks two domains proposed to be important for import and maturation of other chloroplast proteins. The carboxy-terminal region of the chloroplast HSP has significant homology to cytoplasmic HSPs from soybean and other eukaryotes. We hypothesize that the chloroplast HSP shares a common structural and functional domain with low mol. wt HSPs which localize to other parts of the cell, and may have evolved from a nuclear gene.     

Isolation and characterization of a cDNA clone encoding a cognate 70-kDa heat shock protein of the chloroplast envelope.

The translocation of proteins into the endoplasmic reticulum, the mitochondrion, and the chloroplast has recently been shown to involve homologues of the highly conserved 70-kDa heat shock protein (HSP70) family. In this study, we have isolated and sequenced a full-length cDNA clone encoding a cognate 70-kDa heat shock protein of the spinach chloroplast envelope (SCE70). The cDNA insert is 2,535 base pairs long and codes for 653 amino acid residues of a protein with a predicted molecular mass of 71,731 daltons. The deduced amino acid sequence shows a high degree of homology with HSP70 proteins from other organisms. Southern genomic and RNA analyses reveal different hybridization patterns than that observed for a heat-inducible 70-kDa protein gene. The protein synthesized from the SCE70 cDNA insert co-migrates with a 70-kDa polypeptide of the chloroplast envelope following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis and import studies indicate that SCE70 is associated with the chloroplast outer envelope. The import data suggest that SCE70 is targeted to the envelope membrane via a pathway different from other plastidic precursors but similar to that recently reported for outer envelope proteins SOE1 and OM14.     

Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas

Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.     

Heat shock protein 70 is translocated to lipid droplets in rat adipocytes upon heat stimulation

In mammalian cells, lipid storage droplets contain a triacylglycerol and cholesterol ester core surrounded by a phospholipid monolayer into which a number of proteins are imbedded. These proteins are thought to be involved in modulating the formation and metabolic functions of the lipid droplet. In this study, we show that heat stress upregulates several heat shock proteins (Hsps), including Hsp27, Hsp60, Hsp70, Hsp90, and Grp78, in primary and differentiated adipocytes. Immunostaining and immunoblotting data indicate that among the Hsps examined, only Hsp70 is induced to redirect to the lipid droplet surface in heat-stressed adipocytes. The thermal induction of Hsp70 translocation to lipid droplet does not typically happen in a temperature- or time-dependent manner and occurs abruptly at 30-40 min and rapidly achieves a steady state within 60 min after 40 degrees C stress of adipocytes. Though Hsp70 is co-localized with perilipin on the lipid droplets in stressed adipocytes, immunoprecipitation experiments suggest that Hsp70 does not directly interact with perilipin. Alkaline treatments indicate that Hsp70 associates with the droplet surface through non-hydrophobic interactions. We speculate that Hsp70 might noncovalently associate with monolayer microdomains of the lipid droplet in a manner similar to its interaction with lipid bilayer moieties composed of specific fatty acids. As an acute and specific cellular response to the heat stimulation, accumulation of Hsp70 on adipocytes lipid droplets might be involved in stabilizing the droplet monolayer, transferring nascent proteins to the lipid droplets, or chaperoning denatured proteins on the droplet for subsequent refolding.