Protein Synthesis in Maize during Anaerobic and Heat Stress

Protein accumulation and protein synthesis were investigated during anaerobic stress and heat shock in maize seedlings (Zea mays L.). Antibodies against alcohol dehydrogenase (ADH) and cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) were used to investigate the expression of the genes encoding these proteins during stress treatment. ADH1 protein accumulation is shown to increase about 10-fold in the root after 24 hours of anaerobic treatment. The Gpc gene products are separable into two size classes: the slow mobility GAPC1 and GAPC2 (GAPC1/2), and the faster GAPC3 and GAPC4 (GAPC3/4). The GAPC1/2 antigen did not increase at all, whereas the GAPC3/4 antigen increased less than fourfold. The proteins synthesized in the root during aerobic and anaerobic conditions were compared, and GAPC3/4 was identified as an anaerobic polypeptide. In vitro translations were used to estimate the levels of different mRNAs in roots following anaerobiosis, recovery from anaerobiosis, and heat shock. This was compared with the in vivo protein synthesis rates in roots labeled under identical conditions. In vivo labeling indicates that GAPC and ADH are not heat shock proteins. Although both GAPC3/4- and ADH1-translatable mRNA levels increase about 10-fold during anaerobiosis, in vivo labeling of these proteins (relative to total protein synthesis) is further enhanced, leading to a selective translation effect for ADH1 of threefold, and for GAPC3/4 of sixfold. In contrast, anoxia causes no change in GAPC1/2-translatable mRNA levels or in vivo labeling. As an additional comparison, β-glucosidase mRNA levels are found to be constant during anoxia, but in vivo synthesis decreases.     

Developmental control of stress stimulons in Streptomyces coelicolor revealed by statistical analyses of global gene expression patterns

Stress-induced regulatory networks coordinated with a procaryotic developmental program were revealed by two-dimensional gel analyses of global gene expression. Four developmental stages were identified by their distinctive protein synthesis patterns using principal component analysis. Statistical analyses focused on five stress stimulons (induced by heat, cold, salt, ethanol, or antibiotic shock) and their synthesis during development. Unlike other bacteria, for which various stresses induce expression of similar sets of protein spots, in Streptomyces coelicolor heat, salt, and ethanol stimulons were composed of independent sets of proteins. This suggested independent control by different physiological stress signals and their corresponding regulatory systems. These stress proteins were also under developmental control. Cluster analysis of stress protein synthesis profiles identified 10 different developmental patterns or "synexpression groups." Proteins induced by cold, heat, or salt shock were enriched in three developmental synexpression groups. In addition, certain proteins belonging to the heat and salt shock stimulons were coregulated during development. Thus, stress regulatory systems controlling these stimulons were implicated as integral parts of the developmental program. This correlation suggested that thermal shock and salt shock stress response regulatory systems either allow the cell to adapt to stresses associated with development or directly control the developmental program.     

Heat shock response during development of the malaria vector Anopheles stephensi (Culicidae: Diptera)

The pattern of synthesis of heat shock proteins (HSP) and thermotolerance to elevated temperatures during the development of the malaria vector Anopheles stephensi normally reared at 28 +/- 2 degrees C was studied using SDS-PAGE. In total twelve heat shock proteins (i.e. 31, 33, 38, 43, 44, 51, 57, 62, 69, 71, 113 and 121 kD were induced by heat shock during various stages of development. Eight polypeptides (HSP during one or other of the instars) appeared during normal development of the adult, which showed very little response towards heat shock. Only two polypeptides (57 and 69 kD) were induced while the 22.5 kD protein disappeared during adult life. The HSP 62 and 71 kD induced during the larval stages showed a sharp decline in quantity in male and female adults upon heat shock. Three HSP (31, 43 and 44 kD) were induced in pupae due to heat shock. The synthesis of HSP in A. stephensi was correlated with the various morphological and physiological events occurring during development.     

Characterization of a small heat shock protein, Mx Hsp16.6, of Myxococcus xanthus

A number of heat shock proteins in Myxococcus xanthus were previously identified by two-dimensional (2D) gel electrophoresis. One of these protein was termed Mx Hsp16.6, and the gene encoding Mx Hsp16.6 was isolated. Mx Hsp16.6 consists of 147 amino acid residues and has an estimated molecular weight of 16,642, in accordance with the apparent molecular mass in the 2D gel. An alpha-crystallin domain, typically conserved in small heat shock proteins, was found in Mx Hsp16.6. Mx Hsp16.6 was not detected during normal vegetative growth but was immediately induced after heat shock. Expression of the hsp16.6 gene was not induced by other stresses, such as starvation, oxidation, and high osmolarity. Mx Hsp16.6 was mostly localized in particles formed after heat shock and precipitated by low-speed centrifugation. Furthermore, Mx Hsp16.6 was detected in highly electron-dense particles in heat-shocked cells by immunoelectron microscopy, suggesting that it forms large complexes with heat-denatured proteins. An insertion mutation in the hsp16.6 gene resulted in lower viability during heat shock and lower acquired thermotolerance. Therefore, it is likely that Mx Hsp16.6 plays critical roles in the heat shock response in M. xanthus.     

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 and cold shock in Deinococcus radiodurans

On the basis of acquired thermotolerance and cryotolerance, the optimal heat shock and cold shock temperatures have been determined for Deinococcus radiodurans. A heat shock at 42°C maximized survival at the lethal temperature of 52°C and a cold shock at 20°C maximized survival after repeated freeze-thawing. Enhanced survival from heat shock was found to be strongly dependent on growth stage, with its greatest effect shortly after phase. Increased synthesis of a total of 67 proteins during heat shock and 42 proteins during cold shock were observed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and autoradiography. Eight of the most highly induced heat shock proteins shown by 2D PAGE were identified by MALDI-MS as Hsp20, GroEL, DnaK, SodA, Csp, Protease I and two proteins of unknown function.     

Differential responses of stress proteins, antioxidant enzymes, and photosynthetic efficiency to physiological stresses in the Florida red tide dinoflagellate, Karenia brevis

This study identifies stress proteins and antioxidant enzymes that may play a role in the survival strategies of the Florida red tide dinoflagellate, Karenia brevis. Heat shock protein 60 (Hsp 60), mitochondrial small heat shock protein (mitosHsp), chloroplastic small heat shock protein (chlsHsp), Mn superoxide dismutase (SOD), and Fe SOD were first identified by Western blotting. The induction of these proteins in laboratory cultures in response to elevated temperatures, hydrogen peroxide, lead, or elevated light intensities was next assessed. In parallel, F(V)/F(M), a measurement of photosynthetic efficiency and common proxy of cellular stress, was determined. Hsp 60, Fe SOD, and Mn SOD were induced following exposure to elevated temperatures, hydrogen peroxide, or lead. MitosHsp responded only to heat, whereas chlsHsp responded only to H(2)O(2)-induced stress. The expression of stress proteins and antioxidant enzymes appears to be a more sensitive indicator of heat or chemically induced stresses than F(V)/F(M). However, F(V)/F(M) decreased significantly in response to elevated light intensities that did not induce the expression of stress proteins. These results identify for the first time stress proteins and antioxidant enzymes in K. brevis, provide evidence for differential sensitivity of cellular organelles to various sources of stress, and confirm the presence of conserved stress responses observed across phyla in a dinoflagellate.     

Gene regulation during anaerobiosis in soya roots

The proteins of soybean roots undergoing anaerobiosis can be grouped into three classes. Class 1 proteins are induced severalfold and at least 28 of these were identified by in vivo labeling. These proteins include the enzymes alcohol dehydrogenase (ADH), fructose aldolase, pyruvate decarboxylase, phosphoglucomutase, and lactate dehydrogenase. Class 2 proteins include such enzymes as glucose phosphate isomerase, sucrase, and malate dehydrogenase; their specific activity remains constant in aerobiosis or anaerobiosis. The third class of proteins includes those enzymes such as peroxidase whose activity decreases more than 90% after just 1 day in anaerobiosis. Immunoblotting coupled with two-dimensional chromatography of in vitro translated plant extracts demonstrated that ADH level during anaerobiosis is controlled by its mRNA concentration. Little or no mRNA for ADH was detected in aerobically grown roots. This suggests that the increased level of ADH activity is due to de novo synthesis of the mRNA rather than activation of a sequestered mRNA or superactivation of the protein.     

Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.     

On the Mechanism of Action of H2O2 in the Cellular Stress

We propose a hypothesis according to which the reactive and reduced species of oxygen could be the intracellular inducers of the stress (or “heat-shock”) response. This hypothesis is based on the following observations on Drosphila cells: -a) the return to normoxia after 24 h anaerobiosis is suficient to induce the synthesis of the ‘heat shock’ proteins without elevation of temperature together with a rapid increase of O₂ consumption; -b) hydrogen peroxide introduced in the culture medium induces the early transcrip-tional activation of the ‘heat shock’ genes (maximal after 5 minutes); -c) hydrogen peroxide added to cellular extracts in vitro (thus acting as an intracellular metabolite) activates instantaneously the binding capacity of a ‘heat shock’ factor to a DNA ‘heat shock’ regulatory element. Thus, hydrogen peroxide, and possibly other reactive reduced species ofoxygen, could trigger the onset of the stress (or ‘heat shock’) response.     

Differential induction of heat shock, SOS, and oxidation stress regulons and accumulation of nucleotides in Escherichia coli.

Heat and various inhibitory chemicals were tested in Escherichia coli for the ability to cause accumulation of adenylylated nucleotides and to induce proteins of the heat shock (htpR-controlled), the oxidation stress (oxyR-controlled), and the SOS (lexA-controlled) regulons. Under the conditions used, heat and ethanol initiated solely a heat shock response, hydrogen peroxide and 6-amino-7-chloro-5,8-dioxoquinoline (ACDQ) induced primarily an oxidation stress response and secondarily an SOS response, nalidixic acid and puromycin induced primarily an SOS and secondarily a heat shock response, isoleucine restriction induced a poor heat shock response, and CdCl2 strongly induced all three stress responses. ACDQ, CdCl2, and H2O2 each stimulated the synthesis of approximately 35 proteins by factors of 5- to 50-fold, and the heat shock, oxidation stress, and SOS regulons constituted a minor fraction of the overall cellular response. The pattern of accumulation of adenylylated nucleotides during these treatments was inconsistent with a simple role for these nucleotides as alarmones sufficient for triggering the heat shock response, but was consistent with a role in the oxyR-mediated response.     

Rac1 regulates heat shock responses by reorganization of vimentin filaments: identification using MALDI-TOF MS.

Rac1 has been implicated in a wide variety of biological processes, including actin remodeling and various signaling cascades. Here we have examined whether Rac1 might be involved in heat shock-induced cell signaling. We found that Rat2 stable cells expressing a dominant negative Rac1 mutant, RacN17 (Rat2-RacN17), were significantly more tolerant to heat shock than control Rat2 cells, and simultaneously inhibited the activation of SAPK/JNK by heat shock compared to control Rat2 cells. However, no discernible effect was observed in typical heat shock responses including total protein synthesis and heat shock protein synthesis. To identify the proteins involved in this difference, we separated the proteins of both Rat2 and Rat2-RacN17 cell lines after heat shock using two-dimensional gel electrophoresis and identified the differentially expressed proteins by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) after in-gel trypsin digestion. Differentially expressed proteins between two cell lines were identified as vimentin. Rat2-RacN17 cells showed significant changes in vimentin as well as marked changes in vimentin reorganization by heat shock. The vimentin changes were identified as N-terminal head domain cleavage. These results suggest that Rac1 plays a pivotal role in the heat shock-induced signaling cascade by modifying intermediate vimentin filaments.     

Development profile of the heat shock response in early embryos of Drosophila

Drosophila melanogaster embryos reared at 22 degrees C were subjected to a mild heat shock (40 min at 37 degrees C) at various ages in order to determine whether there are changes in the heat shock response during embryogenesis. The effects of the heat shock were measured by assaying (1), subsequent developmental abnormalities (2), developmental time (3), hatchability, and (4), the ability to synthesize the heat shock proteins as assayed by 35S-methionine pulse labeling followed by protein separations using both one-and two-dimensional polyacrylamide gel electrophoresis. Our data show that, first, proteins with molecular weights similar to those of six of the seven major heat shock proteins are normally found in the embryo at control temperatures (22 degrees C); second, that the pregastrula embryo (stages 2-6) is not capable of displaying any aspect of the heat shock response upon treatment, although it may possess all of the so-called heat shock proteins; third, that the complete heat shock response is acquired very rapidly by early gastrula embryos; and fourth, that the heat shock treatment brings about developmental delays and/or abnormalities, depending on the developmental stage of the embryo at the time of the treatment. These developmental abnormalities appear to stem from the failure of early embryos to completely inhibit their synthesis of non-heat-shock proteins. In the light of these findings, it becomes important not to base conclusions about the putative presence of a heat shock response in a particular tissue or developmental stage solely on the presence or absence of the heat shock proteins.     

Role of RpoH, a heat shock regulator protein, in Escherichia coli carbon starvation protein synthesis and survival.

Escherichia coli starvation proteins include several heat shock proteins whose induction by heat is controlled by the minor sigma factor, sigma 32. The level of sigma 32 increased in wild-type E. coli upon starvation, and three sigma 32-controlled heat shock proteins (DnaK, GroEL, and HtpG) were not induced during starvation in an isogenic delta rpoH strain, which is unable to synthesize sigma 32. Thus, sigma 32 plays a role in the induction of these proteins during both heat shock and starvation. The delta rpoH strain was more sensitive to starvation but could develop starvation-mediated cross protection against heat and oxidation.     

A normal mitochondrial protein is selectively synthesized and accumulated during heat shock in Tetrahymena thermophila.

We have identified and purified a 58-kilodalton protein of Tetrahymena thermophila whose synthesis during heat shock parallels that of the major heat shock proteins. This protein, hsp58, was found in both non-heat-shocked as well as heat-shocked cells; however, its concentration in the cell increased approximately two- to threefold during heat shock. The majority of hsp58 in both non-heat-shocked and heat-shocked cells was found by both cell fractionation studies and immunocytochemical techniques to be mitochondrially associated. During heat shock, the additional hsp58 was found to selectively accumulate in mitochondria. Nondenatured hsp58 released from mitochondria of non-heat-shocked or heat-shocked cells sedimented in sucrose gradients as a 20S to 25S complex. We suggest that this protein may play a role in mitochondria analogous to the role the major heat shock proteins play in the nucleus and cytosol.     

Alcohol stress,membranes, and chaperones

Ethanol, which affects all body organs, exerts a number of cytotoxic effects, most of them independent of cell type. Ethanol treatment leads to increased membrane fluidity and to changes in membrane protein composition. It can also interact directly with membrane proteins, causing conformational changes and thereby influencing their function. The cytotoxic action may include an increased level of oxidative stress. Heat shock protein molecular chaperones are ubiquitously expressed evolutionarily conserved proteins which serve as critical regulators of cellular homeostasis. Heat shock proteins can be induced by various forms of stresses such as elevated temperature, alcohol treatment, or ischemia, and they are also upregulated in certain pathological conditions. As heat shock and ethanol stress provoke similar responses, it is likely that heat shock protein activation also has a role in the protection of membranes and other cellular components during alcohol stress.     

Proteome analysis on differentially expressed proteins of the fat body of two silkworm breeds, Bombyx mori, exposed to heat shock exposure

Proteomes of heat tolerant (multivoltine) and heat susceptible (bivoltine) silkworms (Bombyx mori) in response to heat shock were studied. Detected proteins from fat body were identified by using MALDI-TOF/TOF spectrometer, MS/MS, and MS analysis. Eight proteins, including small heat shock proteins (sHSPs) and HSP70, were expressed similarly in both breeds, while 4 protein spots were expressed specifically in the bivoltine breed and 12 protein spots were expressed specifically in the multivoltine breed. In the present proteomics approach, 5 separate spots of sHSP proteins (HSP19.9, HSP20.1, HSP20.4, HSP20.8, and HSP21.4) were identified. Protein spot intensity of sHSPs was lower in the multivoltine breed than in the bivoltine breed after the 45°C heat shock treatment, while the difference between two breeds was not significant after the 41°C heat shock treatment. These results indicated that some other mechanisms might be engaged in thermal tolerance of multivotine breed except for the expression of sHSP and HSP70. There were visible differences in the intensity of heat shock protein expression between male and female, however, differences were not statistically significant.