Regulation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Plasma membrane H<Superscript>+</Superscript>-ATPase (Pma1) by Dextrose and Hsp30 during Exposure to Thermal Stress

Pma1p is an essential plasma membrane H⁺-pump in Saccharomyces cerevisiae that pumps out H⁺ at the expense of cellular ATP. Its activity is induced by glucose at 30°C and is inhibited by Hsp30 during exposure to heat shock conditions. To further investigate the regulation of Pma1 function by glucose and Hsp30 during exposure to thermal stress, we estimated Pma1 activity, its protein levels and ser-phosphorylation status in membrane fractions isolated from BY4741 and hsp30Δ cells grown in dextrose and sorbitol at 30°C, and following exposure at 40°C for 30 min. Our results demonstrate that Pma1 activity and protein levels were reduced in Hsp30⁺ cells following exposure to thermal stress in dextrose media. The above was not observed in hsp30Δ cells wherein Pma1 activity did not decrease following exposure to similar conditions. Although Pma1p levels decreased in heat-shocked hsp30Δ cells, it was lower compared to that observed in Hsp30⁺ cells. Total ser-phosphorylation of Pma1 also showed a decrease following exposure to heat shock condition in dextrose media in both BY4741 and hsp30Δ cells. Its levels were also reduced in BY4741 cells upon heat shock treatment in sorbitol unlike that observed in hsp30Δ cells wherein it was increased. Taken together the above indicate that heat shock induced reduction in Pma1 activity and protein levels in dextrose media required Hsp30. To examine functional interactions between dextrose utilization, Hsp30 and the regulation of various aspects of Pma1, we determined if dextrose regulated other functions attributed to Hsp30. Results demonstrate that the deletion of HSP30 rendered cells dependent on dextrose utilization for survival during exposure to lethal heat stress. Our study has hence been able to establish a functional relationship between glucose utilization, Hsp30 function and the regulation of Pma1 activity. Finally, since the deletion of HSP30 renders Pma1p levels and its activity unresponsive to thermal stress in dextrose media, we concluded that Hsp30 is necessary to maintain Pma1 in a regulation competent conformation. Hsp30 may thus act as a chaperone in the S. cerevisiae plasma membrane.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

Rapamycin conditionally inhibits Hsp90 but not Hsp70 mRNA translation in <Emphasis Type="Italic">Drosophila</Emphasis>: implications for the mechanisms of Hsp mRNA translation

Rapamycin inhibits the activity of the target of rapamycin (TOR)-dependent signaling pathway, which has been characterized as one dedicated to translational regulation through modulating cap-dependent translation, involving eIF4E binding protein (eIF4E-BP) or 4E-BP. Results show that rapamycin strongly inhibits global translation in Drosophila cells. However, Hsp70 mRNA translation is virtually unaffected by rapamycin treatment, whereas Hsp90 mRNA translation is strongly inhibited, at normal growth temperature. Intriguingly, during heat shock Hsp90 mRNA becomes significantly less sensitive to rapamycin-mediated inhibition, suggesting the pathway for Hsp90 mRNA translation is altered during heat shock. Reporter mRNAs containing the Hsp90 or Hsp70 mRNAs’ 5′ untranslated region recapitulate these rapamycin-dependent translational characteristics, indicating this region regulates rapamycin-dependent translational sensitivity as well as heat shock preferential translation. Surprisingly, rapamycin-mediated inhibition of Hsp90 mRNA translation at normal growth temperature is not caused by 4E-BP-mediated inhibition of cap-dependent translation. Indeed, no evidence for rapamycin-mediated impaired eIF4E function is observed. These results support the proposal that preferential translation of different Hsp mRNA utilizes distinct translation mechanisms, even within a single species.     

Improved adaptation to heat,cold, and solvent tolerance in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.     

Geographic variation in thermal tolerance and strategies of heat shock protein expression in the land snail <Emphasis Type="Italic">Theba pisana</Emphasis> in relation to genetic structure

Land snails are exposed to conditions of high ambient temperature and low humidity, and their survival depends on a suite of morphological, behavioral, physiological, and molecular adaptations to the specific microhabitat. We tested in six populations of the land snail Theba pisana whether adaptations to different habitats affect their ability to cope with thermal stress and their strategies of heat shock protein (HSP) expression. Levels of Hsp70 and Hsp90 in the foot tissue were measured in field-collected snails and after acclimation to laboratory conditions. Snails were also exposed to various temperatures (32 up to 54 °C) for 2 h and HSP messenger RNA (mRNA) levels were measured in the foot tissue and survival was determined. To test whether the physiological and molecular data are related to genetic parameters, we analyzed T. pisana populations using partial sequences of nuclear and mitochondrial DNA ribosomal RNA genes. We show that populations collected from warmer habitats were more thermotolerant and had higher constitutive levels of Hsp70 isoforms in the foot tissue. Quantitative real-time polymerase chain reaction (PCR) analysis indicated that hsp70 and hsp90 mRNA levels increased significantly in response to thermal stress, although the increase in hsp70 mRNA was larger compared to hsp90 and its induction continued up to higher temperatures. Generally, warm-adapted populations had higher temperatures of maximal induction of hsp70 mRNA synthesis and higher upper thermal limits to HSP mRNA synthesis. Our study suggests that Hsp70 in the foot tissue of T. pisana snails may have important roles in determining stress resistance, while Hsp90 is more likely implicated in signal transduction processes that are activated by stress. In the phylogenetic analysis, T. pisana haplotypes were principally divided into two major clades largely corresponding to the physiological ability to withstand stress, thus pointing to genetically fixed tolerance.     

Differential expression of heat shock protein 70 in relation to stress type in the flatworm <Emphasis Type="Italic">Schmidtea polychroa</Emphasis>

Most heat shock proteins help to cope with stress in organisms ranging from bacteria to vertebrates. Many stress types acting on the intensity of intracellular protein can induce expression of heat shock proteins. Here, we studied changes in expression level of heat shock protein 70 (Hsp70), one of the best investigated stress proteins, in response to five potential stress factors in the planarian flatworm Schmidtea polychroa: (1) homogenized planarian tissue, which releases an alarm substance that signals predation injury, (2) physical damage by puncturing, (3) a simulation of ecological competition by adding a mixture of naturally co-occurring species: one Dendrocoelum and two Polycelis flatworms, one Asellus water louse and one leech, and (4) magnesium chloride, which inhibits regeneration ability. We found that alarm substance (1), physical harm (2), and magnesium chloride (4) led to increased expression of Hsp70, while interspecific competition (3) did not result in elevated Hsp70 expression. There was no difference between the experimental negative control and two temporal controls immediately after collection and just before the experiment. Results show that Schmidtea polychroa is not sensitive to sampling and lab maintenance. However, planarian homogenate, magnesium chloride and physical harm all caused Hsp70-inducing stress. We conclude that Hsp70 quantification is appropriate to study the current stress level in planaria in response to specific conditions.     

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19°C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5°C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A₂ is involved in the heat shock response.     

Isolation of a <Emphasis Type="Italic">Latimeria menadoensis</Emphasis><Emphasis Type="Italic">heat shock protein 70</Emphasis> (<Emphasis Type="Italic">Lmhsp70</Emphasis>) that has all the features of an inducible gene and encodes a functional molecular chaperone

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system. K. W. Modisakeng and M. Jiwaji contributed equally to this study.     

Effect of Putrescine on Low-Temperature Acclimation in <i>Chlamydomonas reinhardtii</i>

Putrescine is reported to be necessary for cold acclimation under low-temperature stress. In this study, the effect of low-temperature on some physiological and biochemical parameters has been investigated using the green algae Chlamydomonas reinhardtii. The lipid peroxidation rate, amount of Rubisco protein, activities of antioxidant enzymes and gene expression of polyamine biosynthesis (odc2, and spd1), heat shock proteins (hsp70c, hsp90a, and hsp90c), and PSII repair mechanisms (psba, rep27, and tba1) were determined to understand the low-temperature response. Exogenous putrescine application significantly increased Rubisco protein concentration and catalase enzyme activities under low-temperature stress. Moreover, real-time RT-PCR results and gene expression analysis showed that polyamine metabolism induced gene expression at low-temperatures in the first 24 h. In the same way, the gene expression of heat shock proteins (hsp70c, hsp90a, and hsp90c) decreased under low-temperature treatment for 72 h; however, application of putrescine enhanced the gene expression in the first 24 h. The results obtained indicated that molecular response in the first 24 h could be important for cold acclimation. The psba and tba1 expressions were reduced under low-temperatures depending on the exposure time. In contrast, the exogenous putrescine enhanced the expression level of the psba response to low-temperature at 24 and 72 h. The results obtained in this study indicate that putrescine could play a role in the PS II repair mechanisms under low-temperature stress.