Heat Shock Effects on the Circadian Rhythm of Protein Synthesis and Phosphorylation of Ribosomal Proteins in Gonyaulax polyedra

Circadian changes in protein synthesis and phosphorylation of ribosomal and cytoplasmic proteins in the marine dinoflagellate Gonyaulax polyedra were analyzed by radioactive labeling and polyacrylamide gel electrophoresis. Maximal rates of protein synthesis were found during the subjective night and minimal rates during the subjective day. Protein synthesis was inhibited by heat shock to a different extent at different circadian phases—maximally during the subjective night. Heat shock proteins (HSPs) having molecular weights of approximately 105, 89, 83, 66, 35, and 18 kDa were induced by these treatments. Induction of HSP89 and HSP35 showed circadian differences with maximal synthesis rates at CT 15, whereas most HSPs maintained a constant constitutive and induced synthesis. Recovery of normal protein synthesis after heat shock occurred faster during the subjective night than during the subjective day. Ribosomal proteins with molecular weights of 16 and 18 kDa were highly phosphorylated by [³⁵S] thio gamma adenosine triphosphate during day phase in a light-dark cycle or at CT 6 in constant dim light and labeled only to a minor degree during night phase or at CT 18. A ribosome-associated protein (35 kDa) was labeled during the day and not during the night, but after heat shock during both day and night. In the 200,000 g cytosolic fraction, a 35-kDa protein was found to be more intensely labeled at night than during the day phase after heat shock. The results of this study show a correlation between circadian changes in the overall protein synthesis and ribosomal protein phosphorylation. The rhythm of protein synthesis and phosphorylation of a ribosome-associated protein are drastically altered by heat shock and dependent on the circadian phase.     

The combined effect of salt stress and heat shock on proteome profiling in Suaeda salsa

Under natural conditions or in the field, plants are often subjected to a combination of different stresses such as salt stress and heat shock. Although salt stress and heat shock have been extensively studied, little is known about how their combination affects plants. We used proteomics, coupled with physiological measurements, to investigate the effect of salt stress, heat shock, and their combination on Suaeda salsa plants. A combination of salt stress and heat shock resulted in suppression of CO₂ assimilation and the photosystem II efficiency. Approximately 440 protein spots changed their expression levels upon salt stress, heat shock and their combination, and 57 proteins were identified by MS. These proteins were classified into several categories including disease/defense, photosynthesis, energy production, material transport, and signal transduction. Some proteins induced during salt stress, e.g. choline monooxygenase, chloroplastic ATP synthase subunit beta, and V-type proton ATPase catalytic subunit A, and some proteins induced during heat shock, e.g. heat shock 70 kDa protein, probable ion channel DMI1, and two component sensor histidine kinase, were either unchanged or suppressed during a combination of salt stress and heat shock. In contrast, the expression of some proteins, including nucleoside diphosphate kinase 1, chlorophyll a/b binding protein, and ABC transporter I family member 1, was specifically induced during a combination of salt stress and heat shock. The potential roles of the stress-responsive proteins are discussed.     

Heat shock inhibits and activates different protein degradation pathways and proteinase activities in Neurospora crassa

Abstract In Neurospora crassa , heat shock treatment inhibits proteolytic activity. ATP-independent proteinases were analysed after polyacrylamide gel electrophoresis using renaturing gelatine gels. Proteinases of 24, 29, and 130 kDa were shown to be inhibited by heat shock and were further characterized as to their properties. A major part of the heat shock-induced inhibition is probably due to suppression of de novo synthesis of proteinases as deduced from experiments with cycloheximide. During several hours of recovery from heat shock, the inhibition of overall protein degradation and ATP-independent proteinases is reversed. Azocasein assays as well as pulse-chase experiments further showed that ATP-dependent protein degradation is only slightly affected by heat shock. Two ATP-binding proteinases of about 60 and 160 kDa even show an increased activity after heat shock. The degradation rate of heat shock proteins is inhibited by heat shock treatment, indicating that they are degraded by ATP-independent proteinases. Western blot analysis of a ∼40-kDa degradation product of HSP70 containing its amino terminal portion revealed a reduction in the amount of this peptide after heat shock.     

Common responses of cultured soybean cells to 2,4-D starvation and fungal elicitor treatment

The patterns of in vivo protein synthesis in soybean cell suspensions were compared by polyacrylamide gel electrophoresis after the cells had been submitted to different stress conditions : treatment with Phytophthora megasperma (Pmg) cell wall elicitors, 2,4-D starvation and heat shock (HS) temperatures. Changes in protein synthesis patterns induced after elicitation of cell suspensions or after infection of soybean hypocotyls by Pmg were found to be similar to changes brought about by auxin starvation of the cells. Changes common to both stress situations involve a prominent 17 kDa peptide family and 27, 29, 35 and about 45 kDa peptides. Moreover, defense reactions, i.e. glyceollin accumulation and synthesis of chalcone synthase (CHS) were also strongly stimulated in auxin-starved cells. On the contrary, although characteristic sets of low molecular weight heat shock (HS) proteins were synthesized by cells grown at 37°C, no clear similarity was observed with peptides characteristic of auxin-starved cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - Pmg Phytophthora megasperma Drechs f.sp.glycinea - HS heat shock - PR pathogenesis-related - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - IEF isoelectrofocusing - iP isoelectric point - kDa kilodalton - P17 17 kDa peptide group of soybean cells cultured in vitro - CHS chalcone synthase     

Heat Shock Proteins of Pigeon Pea (Cajanus cajan)

The proteins synthesized in response to higher temperature ina tropical legume-plant pigeon pea (Cajanus cajari) have beenstudied using polyacrylamide gel electrophoresis. Ten to twelveheat shock proteins (hsps) of molecular weights ranging from15 to 81 kDa are synthesized by excised roots when temperatureis raised by 10?C above their normal growth temperature (30?C).The heat shock response is rapid and the presence of hsps canbe detected just 30 min after raising the temperature. Hspscan not be seen in stained gels, and their presence can onlybe monitored by fluorography. The results indicate the transientnature of their synthesis. Most of the hsps are of nuclear origin,however, at least two of them, 18 and 60 kDa proteins appearto be synthesized in mitochondria. (Received September 2, 1987; Accepted February 3, 1988)     

Characterisation of chloroplast heat shock proteins in young leaves of C4 monocotyledons

In vivo radiolabeling of chloroplast proteins in grain sorghum (Sorghum bicolor L. cv. Texas 610) leaves and their separation by one-dimensional electrophoresis revealed at least 6 heat shock proteins (HSPs) between 24 and 94 kDa. of which the 24 kDa protein was the most prominent. All of these chloroplast heat shock proteins were found exclusively in the stroma. The 24 kDa heat shock protein, upon closer examination using two-dimensional electrophoresis proved to be two similarly-sized heat shock polypeptides with identical molecular masses and level of radiolahel incorporation, hut slightly different in isoeiectric points, suggesting isomers. Separation of stromal heat shock proteins synthesised in two other C₄ monocotyledons ( Punicum miliaceum L. and Umchloa panictrides L.) revealed similar putative isomers. each of 24 kDa. Several other, previously unidentified, heat shock proteins between 22 and 38 kDa were also observed in all three species. In P. miliaceum. the most prominent HSP was the pair of 24 kDa proteins, whereas in U. panicoides. it was a group of 35 to 38 kDa HSPs that was most abundant. In vivo chlorophyll fluorescence measurements showed that no sustained impairment to photosynthetic efficiency had occurred for each species after the heat stress regime. However, when cytoplasmic protein synthesis was inhibited during the high temperature treatment, a dramatic decrease was observed in photosynthetic efficiency, suggesting a possible protective role for chloroplast heat shock proteins. It was also shown that a single chloroplast HSP complex of around 380 kDa was observed in the stroma of both 5. bicolor and P. miliaceum leaves in vivo. This was in contrast to the smaller HSP complex (200–265 kDa) observed in previous studies on chloroplast heat shock proteins in C_j species.     

Identification of Differentially Expressed Water‐insoluble Proteins in the Encystment Process of Colpoda cucullus by Two‐dimensional Electrophoresis and LC‐MS/MS Analysis

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h–1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water‐insoluble proteins by two‐dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween‐80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60‐kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55‐kDa protein (p55; actin) and a 49‐kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50‐kDa protein (p50d; α‐tubulin), a 25‐kDa protein (p25; α‐tubulin) and a 52‐kDa protein (p52c; β‐tubulin) was enhanced.     

Qualitative changes in protein synthesis in germinating pollen of Lilium longiflorum after a heat shock

Abstract Germination and protein synthesis of lily pollen subjected to a short heat shock after imbibition are strongly inhibited. The proteins synthesized after the heat shock were analysed by polyacrylamide gel electrophoresis. The patterns obtained from heat-treated pollen are strikingly different from those of control samples. The difference is nearly completely climinated by a high concentration of proline in the incubation medium. This proline effect correlates with the protection of pollen germination from high temperature by the amino acid.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of the heat shock response inEnterococcus faecalis

We have characterized the general properties of the heat shock response of the Gram-positive hardy bacteriumEnterococcus faecalis. The heat resistance (60°C or 62.5°C, 30 min) of log phase cells ofE. faecalis grown at 37°C was enhanced by exposing cells to a prior heat shock at 45°C or 50°C for 30 min. These conditioning temperatures also induced ethanol (22%, v/v) tolerance. The onset of thermotolerance was accompanied by the synthesis of a number of heat shock proteins. The most prominent bands had molecular weights in the range of 48 to 94kDa. By Western blot analysis two of them were found to be immunologically related to the well known DnaK (72 kDa) and GroEL (63 kDa) heat shock proteins ofEscherichia coli. Four other proteins showing little or no variations after exposure to heat are related to DnaJ, GrpE and Lon (La)E. coli proteins and to theBacillus subtilis ⁴³ factor. Ethanol (2% or 4%, v/v) treatments elicited a similar response although there was a weaker induction of heat shock proteins than with heat shock.     

Protein expression analysis of Halobacillus dabanensis D-8T subjected to salt shock

To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and Imagemaster 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.     

Heat Shock Protein Produced by Helicobacter pylori

The cells of Helicobacter pylori were suspended in the medium containing³⁵S-methionine. After a heat shock of the cells at 42 C for 5, 10, and 30 min, the production of proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Out of many proteins produced by the cells, only 66 kDa protein production was dramatically increased by heat treatment. The N-terminal amino acid sequence of 66 kDa protein was quite similar to that of 62 kDa and 54 kDa proteins previously suggested as heat shock protein (HSP) of H. pylori based on the reaction with polyclonal and monoclonal antibodies against HSP 60 family proteins produced by other bacteria. Therefore, it was concluded that H. pylori produces the 66 kDa protein as its major heat shock protein which belongs to HSP 60 family.     

Induction of stress proteins in anoxic and hyperthermicSpodoptera frugiperda cells

In this study, we compare stress protein induction in anoxic and hyperthermicSpodoptera frugiperda cells. Anoxia transiently induces a cluster of heat shock proteins at 71 and 72 kDa. This is a subset of a larger group of stress proteins induced by heat shock. Several heat shock proteins reported in this study were previously undetected inS. frugiperda. With these additional proteins, the stress response of hyperthermicS. frugiperda closely resembles that ofDrosophila melanogaster. Prior investigations of stress protein induction during oxygen deprivation focused on mammalian cells. In sharp contrast to these cells, anoxicS. frugiperda cells neither induce glucose-regulated proteins nor suppress the heat shock family of 71/72 kDa proteins. These findings provide insight into the virtually unexplored area of stress protein induction in anoxic insect cells. In addition, they help to explain the effects of oxygen deprivation on heterologous protein yield from virally infected insect cells and to develop an oxygenregulated promoter for stably transformed insect cells.Abbreviations DO dissolved oxygen concentration - GRP's glucose-regulated proteins - HSP's heat shock proteins - ORP's oxygen-regulated proteins - PAGE polyacrylamide gel electrophoresis - Sf9 Spodoptera frugiperda cells     

Pattern of Proteins after Heat Shock and UV-B Radiation of some Temperate Marine Diatoms and the Antarctic Odontella weissflogii

Synthesis of stress proteins after heat shock and different periods of UV-B radiation were investigated with marine diatom species from the North Sea Ditylum brightwellii, Lithodesmium variabile, Odontella sinensis, Thalassiosira rotula and the Antarctic diatom Odontella weissfloggii from the Weddell Sea. Algae were grown in an artifical sea-water medium under controlled laboratory conditions: light/dark regime of 12:12 h (7.2 W m^?2), normal air (0.035 vol.% CO₂) and 18° or 4 °C. All the tested diatom species can produce heat shock proteins (HSPS) of the 70 kDa family by in vivo labelling with [³⁵S]-methionine. The same results were obtained for Odontella sinensis, Ditylum brightwellii and Odontella weissflogii by estimation of the in vitro translation products with poly-A-mRNA isolated from these organisms. However, Odontella weissflogii, a species relatively insensitive to UV-B irradiance, did not synthesize UV-induced HSPS, whereas the UV-sensitive diatom Odontella sinensis, as well as Lithodesmium variabile, produced all the observed HSPS after UV-B exposure. In addition, a protein of 43 kDa was found after UV-B irradiance of the temperate Odontella sinensis. The temperate marine diatom Thalassiosira rotula synthesized 70 kDa and 5 7 kDa proteins after a heat shock and a UV-B exposure of 2 h, but a 40 kDa protein could not be detected, whereas a 60 kDa protein was found after 2 h UV-B exposure. The results are discussed in view of a possible adaptation of O. weissflogii to an enhanced UV dose.     

Effect of High Temperature on Protein Expression in Strawberry Plants

Strawberry plants (Fragaria×ananassa Duch.) cvs. Nyoho and Toyonoka were exposed to temperatures of 20, 33, and 42 °C for 4 h, and protein patterns in leaves and flowers was analyzed by 2-dimensional polyacrylamide gel electrophoresis and immunoblotting. In leaves and flowers of both cultivars, the content of most proteins decreased, but a few new proteins appeared in response to heat stress. These heat shock proteins (Hsps) were detected in the range of 19 – 29 kDa in leaves, and 16 – 26 kDa in flowers. The intensity of a 43 kDa protein spot increased in response to heat stress in Nyoho flowers, but not in Toyonoka flowers. The peaHsp17.7 antibody recognized one band at approximately 26 kDa in leaves, and two bands at approximately 16 and 17 kDa in flowers of both cultivars. These results show that the effects of heat stress on Hsp synthesis in strawberry plants differ between plant organs and between cultivars.     

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.     

Utilization of fluorescent chimeras for investigation of heterooligomeric complexes formed by human small heat shock proteins

Fluorescent chimeras composed of enhanced cyan (or enhanced yellow) fluorescent proteins (ECFP or EYFP) and one of the four human small heat shock proteins (HspB1, HspB5, HspB6 or HspB8) were expressed in E. coli and purified. Fluorescent chimeras were used for investigation of heterooligomeric complexes formed by different small heat shock proteins (sHsp) and for analysis of their subunit exchange. EYFP-HspB1 and ECFP-HspB6 form heterooligomeric complex with apparent molecular weight of ∼280 kDa containing equimolar quantities of both sHsp. EYFP-HspB5 and ECFP-HspB6 formed heterogeneous oligomeric complexes. Fluorescent proteins inside heterooligomeric complexes formed by HspB1/HspB6 and HspB5/HspB6 chimeras are closely located, making possible effective fluorescence resonance energy transfer (FRET). Neither the wild type HspB8 nor its fluorescent chimeras were able to form stable heterooligomeric complexes with the wild type HspB1 and HspB5. Homo- and hetero-FRET was used for analysis of subunit exchange of small heat shock proteins. The apparent rate constant of subunit exchange was temperature-dependent and was higher for HspB6 forming small oligomers than for HspB1 forming large oligomers. Replacement induced by homologous subunits was more rapid than the replacement induced by heterologous subunits of small heat shock proteins. Fusion of fluorescent proteins might affect oligomeric structure of small heat shock proteins, however fluorescent chimeras can be useful for investigation of heterooligomeric complexes formed by sHsp and for analysis of kinetics of their subunit exchange.     

RelA-independent (p)ppGpp accumulation and heat shock protein induction after salt stress in Bacillus subtilis

Abstract Sodium chloride treatment triggered the accumulation of (p)ppGpp in the Bacillus subtilis relA ⁺ strain IS58 as well as in its relaxed counterpart IS56 . Besides this relA -independent (p)ppGpp induction the GTP and ATP pools decreased dramatically.
In previous papers we found a direct correlation between (p)ppGpp accumulation and stress protein induction. In B. subtilis relA the (p)ppGpp accumulation was accompanied by the induction of general stress proteins whose synthesis rates were also enhanced by heat stress, amino acid limitation or oxygen starvation. Specific heat shock proteins were not induced by salt stress.
We suggest that these general stress proteins are induced under non-growing conditions in general.