Low Molecular Weight Heat Shock Proteins Research Articles

Heat shock protein 22 (HSPB8) limits TGF-β-stimulated migration of osteoblasts

Heat shock proteins (HSPs) are induced in response to various physiological and environmental conditions such as chemical and heat stress, and recognized to function as molecular chaperones. HSP22 (HSPB8), a low-molecular weight HSP, is ubiquitously expressed in many cell types. However, the precise role of HSP22 in bone metabolism remains to be clarified. In the present study, we investigated whether HSP22 is implicated in the transforming growth factor-β (TGF-β)-stimulated migration of osteoblast-like MC3T3-E1 cells. Although protein levels of HSP22 were clearly detected in unstimulated MC3T3-E1 cells, TGF-β failed to induce the protein levels. The TGF-β-stimulated migration was significantly up-regulated by knockdown of HSP22 expression. The cell migration stimulated by platelet-derived growth factor-BB was also enhanced by HSP22 knockdown. SB203580, an inhibitor of p38 mitogen-activated protein kinase, PD98059, an inhibitor of MEK1/2, or SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase had no effects on the TGF-β-induced migration. SIS3, a specific inhibitor of TGF-β-dependent Smad3 phosphorylation, significantly reduced the migration with or without TGF-β stimulation. Smad2, Smad3, Smad4 or Smad7 was not coimmunoprecipitated with HSP22. On the other hand, the TGF-β-induced Smad2 phosphorylation was enhanced by HSP22 down-regulation. The protein levels of TGF-β type II receptor (TGF-β RII) but not TGF-β type I receptor (TGF-β RI) was significantly up-regulated in HSP22 knockdown cells compared with those in the control cells. However, the levels of TGF-β RII mRNA in HSP22 knockdown cells were little different from those of the control cells. Neither TGF-β RI nor TGF-β RII was coimmunoprecipitated with HSP22. SIS3 reduced the amplification by HSP22 knockdown of the TGF-β-stimulated cell migration almost to the basal level. Our results strongly suggest that HSP22 functions as a negative regulator in the TGF-β-stimulated migration of osteoblasts via suppression of the Smad-dependent pathway, resulting from modulating the protein levels of TGF-β RII.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

Comparative proteomic and metabolomic profiling of citrus fruit with enhancement of disease resistance by postharvest heat treatment

BackgroundFrom field harvest to the consumer’s table, fresh citrus fruit spends a considerable amount of time in shipment and storage. During these processes, physiological disorders and pathological diseases are the main causes of fruit loss. Heat treatment (HT) has been widely used to maintain fruit quality during postharvest storage; however, limited molecular information related to this treatment is currently available at a systemic biological level.ResultsMature ‘Kamei’ Satsuma mandarin (Citrus unshiu Marc.) fruits were selected for exploring the disease resistance mechanisms induced by HT during postharvest storage. Proteomic analyses based on two-dimensional gel electrophoresis (2-DE), and metabolomic research based on gas chromatography coupled to mass spectrometry (GC-MS), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) were conducted. The results show resistance associated proteins were up-regulated in heat treated pericarp, such as beta-1, 3-glucanase, Class III chitinase, 17.7 kDa heat shock protein and low molecular weight heat-shock protein. Also, redox metabolism enzymes were down-regulated in heat treated pericarp, including isoflavone reductase, oxidoreductase and superoxide dismutase. Primary metabolic profiling revealed organic acids and amino acids were down-regulated in heat treated pericarp; but significant accumulation of metabolites, including tetradecanoic acid, oleic acid, ornithine, 2-keto-d-gluconic acid, succinic acid, turanose, sucrose, galactose, myo-inositol, glucose and fructose were detected. Noticeably, H2O2 content decreased, while, lignin content increased in heat treated pericarp compared to the control, which might increase fruit resistibility in response to external stress. Also, flavonoids, substances which are well-known to be effective in reducing external stress, were up-regulated in heat treated pericarp.ConclusionsThis study provides a broad picture of differential accumulation of proteins and metabolites in postharvest citrus fruit, and gives new insights into HT improved fruit disease resistance during subsequent storage of ‘Kamei’ Satsuma mandarin. Interpretation of the data for the proteins and metabolites revealed reactive oxygen species (ROS) and lignin play important roles in heat treatment induced fruit resistance to pathogens and physiological disorders.

Suppression by heat shock protein 20 of hepatocellular carcinoma cell proliferation via inhibition of the mitogen‐activated protein kinases and AKT pathways

Heat shock protein (HSP) 20, one of the low-molecular weight HSPs, is known to have versatile functions, such as vasorelaxation. However, its precise role in cancer proliferation remains to be elucidated. While HSP20 is constitutively expressed in various tissues including the liver, we have previously reported that HSP20 protein levels in human hepatocellular carcinoma (HCC) cells inversely correlate with the progression of HCC. In this study, we investigated the role of HSP20 in HCC proliferation. The activities of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), and AKT were negatively correlated with the HSP20 protein levels in human HCC tissues. Since HSP20 proteins were hardly detected in HCC-derived cell lines, the effects of HSP20 expression were evaluated using human HCC-derived HuH7 cells that were stably transfected with wild-type human HSP20 (HSP20 overexpressing cells). In HSP20 overexpressing cells, cell proliferation was retarded, and the activation of the mitogen-activated protein kinases (MAPKs) signaling pathways, including the ERK and JNK, and AKT pathways, as well as cyclin D1 accumulation induced by either transforming growth factor-α (TGFα) or hepatocyte growth factor, were significantly suppressed compared with the empty vector-transfected cells (control cells). Taken together, our findings strongly suggest that HSP20 suppresses the growth of HCC cells via the MAPKs and AKT signaling pathways, thus suggesting that the HSP20 could be a new therapeutic target for HCC.

Heat shock response in olive (Olea europaea L.) twigs: Identification and analysis of a cDNA coding a class I small heat shock protein

The objective of this study was to investigate at the molecular level the heat shock response in olive by identifying sequences coding for heat shock proteins (HSPs). Young twigs of Olea europaea trees (cv Cellina di Nardò) were subjected to different temperature treatments in order to induce the expression of heat shock genes. In order to identify genes induced by heat treatment, we used a PCR-based approach to amplify specific HS cDNAs. Search for low molecular weight HSP sequences was performed in public domain databases in order to design specific primers based upon multisequence alignments. By this approach, we isolated the first full length cDNA encoding a low-molecular weight HSP from O. europaea. This is a class I low molecular weight HSP of 18.3 kDa, which is highly expressed in young twigs subjected to heat stress.

Tissue-Specific Defense and Thermo-Adaptive Mechanisms of Soybean Seedlings under Heat Stress Revealed by Proteomic Approach

A comparative proteomic approach was employed to explore tissue-specific protein expression patterns in soybean seedlings under heat stress. The changes in the protein expression profiles of soybean seedling leaves, stems, and roots were analyzed after exposure to high temperatures. A total of 54, 35, and 61 differentially expressed proteins were identified from heat-treated leaves, stems, and roots, respectively. Differentially expressed heat shock proteins (HSPs) and proteins involved in antioxidant defense were mostly up-regulated, whereas proteins associated with photosynthesis, secondary metabolism, and amino acid and protein biosynthesis were down-regulated in response to heat stress. A group of proteins, specifically low molecular weight HSPs and HSP70, were up-regulated and expressed in a similar manner in all tissues. Proteomic analysis indicated that the responses of HSP70, CPN-60 beta, and ChsHSP were tissue specific, and this observation was validated by immunoblot analysis. The heat-responsive sHSPs were not induced by other stresses such as cold and hydrogen peroxide. Taken together, these results suggest that to cope with heat stress soybean seedlings operate tissue-specific defenses and adaptive mechanisms, whereas a common defense mechanism associated with the induction of several HSPs was employed in all three tissues. In addition, tissue-specific proteins may play a crucial role in defending each type of tissues against thermal stress.

α2 adrenoreceptor agonist regulates protein kinase C‐induced heat shock protein 27 phosphorylation in C6 glioma cells

Dexmedetomidine (Dexmd), a potent and highly specific alpha(2) adrenoreceptor agonist, is an efficient therapeutic agent for sedation. Dexmd has been recently reported to have a neuroprotective effect. Heat shock protein (HSP) 27, a low-molecular weight HSP has been shown to be expressed following cerebral ischemia in astrocytes but not in neurons. HSP27 expression is involved in ischemic tolerance of the brain. This study investigated the effect of Dexmd on HSP27 in rat C6 glioma cells. 12-O-tetradecanoylphorbol-13-actate (TPA), a direct activator of protein kinase C (PKC), stimulated the phosphorylation of HSP27 at Ser82, but not Ser15 in a time-dependent manner. Prostaglandin (PG) E(1) or PGE(2) which activates the adenylyl cyclase-cAMP system as well as forskolin and dibutyryl-cAMP, suppressed the TPA-induced phosphorylation of HSP27. Dexmd reversed the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system. Therefore, these results strongly suggest that Dexmd reverses the suppression of HSP27 phosphorylation by the adenylyl cyclase-cAMP system activation through the inhibition of its system in C6 cells. alpha(2) Adrenoreceptor agonists may therefore show a neuroprotective effect through the modification of HSP27 phosphorylation induced by PKC activation.

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.

Heat shock of cultivated and wild wheats during early seedling stage: growth, cell viability and heat shock proteins

Growth, cell viability and heat shock proteins (HSPs) in Bezostaya-1, Cukurova-86 and Diyarbakir-81 cultivated wheat cultivars and three Aegilops species were investigated. Etiolated seedlings were exposed to 23 degrees C, 32 degrees C, 35 degrees C, 37 degrees C and 38 degrees C for 24 h, and 35 degrees C (24 h) --> 50 degrees C (1 h) and 37 degrees C (24 h) --> (50 degrees C (1 h). At the end of recovery growth periods, the shoot lengths of the genotypes generally decreased significantly at 35, 37 and 38 degrees C. The acquired thermal tolerance (ATT) in intact seedlings was over 50% at 35 degrees C --> 50 degrees C and 37 degrees C --> 50 degrees C, but in cell viability test it ranged from 2.75% (Ae. triuncialis) to 32.87 (Bezostaya-1) at 35 degrees C, and from 2.82% (Ae. triuncialis) to 37.82 (Bezostaya-1) at 37 degrees C. Ae. triuncialis was most sensitive genotype in both ATT determination. In electrophoretic profiles of proteins, while some HSPs were newly synthesized, some normal cellular proteins disappeared at 37 degrees C and 37 degrees C -->50 degrees C compared to 23 degrees C. The number of low molecular weight (LMW) HSPs were more than intermediate- (IMW) and high- (HMW) HSPs. The genotypes had both common (12 HSPs between at least two genotypes) and genotype-specific (33 HSPs) LMW HSPs. The common HSP of 19.8 kDa (pI 6.5) was synthesized in Bezostaya-1, Cukurova-86, Diyarbakir-81, Ae. biuncialis and Ae. umbellulata. Bezostaya-1 is the only genotype that synthesized 12 IMW and 2 HMW HSPs at 37 degrees C --> 50 degrees C. Ae. triuncialis had only two common LMW HSPs [22.1 (pI 7.1) and 24.2 kDa (pI 6.5)].

The Possibility of Novel Antiplatelet Peptides: The Physiological Effects of Low Molecular Weight HSPs on Platelets

Some low molecular mass heat shock proteins (HSPs) appear to act as molecular chaperones, but their exact physiological roles have not been fully elucidated. We reported on a physiological role of HSP20, HSP27 and alphaB-crystallin on platelet function in vitro and ex vivo. HSP20 and alphaB-crystallin inhibited platelet aggregation using human platelets dose-dependently induced by thrombin or botrocetin. On the other hand, HSP27, the other type of low molecular mass HSP, did not affect platelet aggregation. When HSP20 or alphaB-crystallin was injected intravenously as a bolus in hamsters, the development of thrombus after endothelial injury was prevented. Moreover, 9 amino-acid sequences isolated from HSP20 or alphaB-crystallin significantly reduced platelet aggregation induced by TRAP, but not a PAR-4 agonist. These findings strongly suggest that HSP20 or alphaB-crystallin can act intercellularly to regulate platelet functions. Our results may provide the basis for a novel defensive system to thrombus formation in vivo.

Synthesis of soluble heat shock proteins in seminal root tissues of some cultivated and wild wheat genotypes

Effect of heat stress on the synthesis of soluble heat shock proteins (HSPs) and the regrowth in seminal roots of three cultivated and three wild wheat genotypes was examined. In regrowth experiments, 2-d-old etiolated seedlings were exposed to 23 (control), 32, 35, 37 and 38 degrees C for 24 h, and 35 and 37 degrees C (24 h) followed by 50 degrees C (1 h). The lengths of the seminal roots generally decreased significantly at the end of 48 and 72 h recovery growth periods at 35, 37 and 38 degrees C temperature treatments compared with control. Genotypic variability was significant level at all temperature treatments for the seminal root length. Also, genotypic differences for the number of seminal roots were determined among the wheat cultivars and between the wild wheat species and the wheat cultivars at all temperature treatments; but genotypic differences among wild wheat species were only detected at 37-->50 degrees C treatment. Acquired thermotolerance for the seminal root length is over 50% at 37-->50 degrees C treatment. The genotypic variability of soluble heat shock proteins in seminal root tissues were analyzed by two-dimensional electrophoresis (2-DE). Total number of low molecular weight (LMW) HSPs was more than intermediate-(IMW) and high- (HMW) HSPs at high temperature treatments. The most of LMW HSPs which were generally of acidic character ranged between 14.2-30.7 kDa. The genotypes had both common (43 HSP spots between at least two genotypes and 23 HSP spots between 37 and 37-->50 degrees C) and genotype-specific (72 HSP spots) LMW HSPs.

Heat shock proteins of thermophilic and thermotolerant fungi from Taiwan

Fifteen species of thermophilic and four species of thermotolerant fungi were isolated from soil samples collected at various localities in Taiwan. Fourteen species and three strains responded to three h of thermal stress at elevated temperature from 30℃ to 50℃ by synthesizing 30 heat shock proteins (HSPs) with molecular weights ranging between 20-150 kDa. Heat shock treatments at 40℃, resulted in the synthesis of 22 HSPs with molecular weights of 30-150 kDa. Nine of the seventeen fungal species produced a 46 kDa HSPs, seven species a 52 kDa HSP, and five species a 94 kDa HSP. Four of the seventeen species produced two kinds of HSPs with molecular weights of 87 kDa and 40 kDa. Ten fungal strains responded to an elevated temperature of 50℃ and synthesized 12 HSPs of molecular weights ranging from 20 to 92 kDa. Six species produced a 35 kDa HSP, and four species produced HSPs with molecular weights of 30 kDa, 28 kDa and 22 kDa. When subjected to heat shock treatments at 40℃, thermophilic and thermotolerant fungi synthesized mostly high and medium molecular weight HSPs while at 50℃, they synthesized mostly low molecular weight HSPs.

Direct association and translocation of PKC-alpha with calponin.

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Calponin has also been shown to interact with PKC. We have studied the interaction of calponin with PKC-alpha and with the low molecular weight heat-shock protein (HSP)27 in contraction of colonic smooth muscle cells. Particulate fractions from isolated smooth muscle cells were immunoprecipitated with antibodies to calponin and Western blot analyzed with antibodies to HSP27 and to PKC-alpha. Acetylcholine induced a sustained increase in the immunocomplexing of calponin with HSP27 and of calponin with PKC-alpha in the particulate fraction, indicating an association of the translocated proteins in the membrane. To examine whether the observed interaction in vivo is due to a direct interaction of calponin with PKC-alpha, a cDNA of 1.3 kb of human calponin gene was PCR amplified. PCR product encoding 622 nt of calponin cDNA (nt 351-972 corresponding to amino acids 92-229) was expressed as fusion glutathione S-transferase (GST) protein in the vector pGEX-KT. We have studied the direct association of GST-calponin fusion protein with recombinant PKC-alpha in vitro. Western blot analysis of the fractions collected after elution with reduced glutathione buffer (pH 8.0) show a coelution of GST-calponin with PKC-alpha, indicating a direct association of GST-calponin with PKC-alpha. These data suggest that there is a direct association of translocated calponin and PKC-alpha in the membrane and a role for the complex calponin-PKC-alpha-HSP27, in contraction of colonic smooth muscle cells.

Contrasting effects of midazolam on induction of heat shock protein 27 by vasopressin and heat in aortic smooth muscle cells.

We previously showed that vasopressin stimulates the induction of heat shock protein (HSP) 27, a low molecular-weight HSP, through protein kinase C activation in aortic smooth muscle A10 cells. In the present study, we examined the effects of midazolam, an intravenous anesthetic, on the HSP27 induction stimulated by vasopressin, heat, or sodium arsenite (arsenite) in A10 cells. Midazolam inhibited the accumulation of HSP27 induced by vasopressin or 12-O-tetradecanoylphorbol 13-acetate (TPA), a direct activator of protein kinase C. Midazolam also reduced the vasopressin-induced level of the mRNA for HSP27. In contrast, midazolam enhanced the HSP27-accumulation induced by heat or arsenite. Midazolam also enhanced the heat-increased level of the mRNA for HSP27. However, midazolam had no effect on the dissociation of the aggregated form of HSP27 following stimulation by vasopressin, heat, or arsenite. These results suggest that midazolam suppresses vasopressin-stimulated HSP27 induction in vascular smooth muscle cells, and that this inhibitory effect is exerted at a point downstream from protein kinase C. In contrast, midazolam enhanced heat- or arsenite-stimulated HSP27 induction. Thus, midazolam has dual effects on the HSP27 induction stimulated by various stresses in vascular smooth muscle cells.

Increased expression of high but not low molecular weight heat shock proteins in resectable lung carcinoma

Strong expression of high-molecular-weight (HMW) heat-shock proteins (HSP) by lung carcinoma has been documented using immunohistochemistry. Far less is known about the expression of low-molecular-weight (LMW) HSP in lung cancer. We compared the quantitative expression of HMW (HSP-60, HSP-70) and LMW (HSP-27, ubiquitin) HSP in tumor and non-tumor lung tissue obtained from 47 patients undergoing surgical resection of lung carcinoma. HSP levels were determined in cell lysates from tissue samples by ELISA using streptavidin-biotin technology. Results were normalized to total protein content measured by spectrophotometry. Compared to disease-free lung tissue, tumor tissue samples showed higher levels of both HSP-60 (median value: 227 pg versus 96 pg per mg protein ( P<0.001 by Wilcoxon Rank test for paired data) and HSP-70 (median value: 525 ng versus 401 ng per mg protein ( P=0.01 by Wilcoxon Rank test for paired data). Tumor and tumor-free tissues show similar levels of ubiquitin and HSP-27. Neither the survival rate nor the histologic type and extent of cancer are correlated with the observed differences in HSP-60 and HSP-70 expression ( P>0.1 by one way analysis of variance for repeated measures with one between subject factor). Our data confirm, on a quantitative basis, the increased expression of HSP-60 and HSP-70 in non-small-cell lung carcinoma. However, no prognostic value was found to be associated with this over-expression. In contrast, LMW stress proteins such as ubiquitin and HSP-27, although implicated in cellular processes potentially related to malignant transformation, show no increased expression in lung carcinoma.

Ethanol treatment triggers a heat shock‐like response but no thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings

ABSTRACTNon‐lethal heat‐shock (HS) treatment has previously been shown to induce thermotolerance in soybean (Glycine max cv. Kaohsiung No.8) seedlings. This acquired thermotolerance correlates with the de novo synthesis of heat‐shock proteins (HSPs). Interestingly, we found that ethanol treatments also elicited HS‐like responses in aetiolated soybean seedlings at their normal growth temperature of 28 °C. Northern blot analyses revealed that the expression of HS genes hsp17.5, hsp70 and hsc 70 was induced by ethanol. Radioactive amino acids were preferentially incorporated into high molecular weight (HMW) HSPs rather than class I low molecular weight (LMW) HSPs during non‐lethal ethanol treatments. Immunoblot analysis confirmed that no accumulation of class I LMW HSPs occurred after non‐lethal ethanol treatment. Pre‐treatment with a non‐lethal dose of ethanol did not provide thermotolerance, as the aetiolated soybean seedlings could not survive a subsequent heat shock of 45 °C for 2 h. In contrast, non‐lethal HS pre‐treatment, 40 °C for 2 h, conferred tolerance on aetiolated soybean seedlings to otherwise lethal treatments of 7·5% ethanol for 8 h or 10% ethanol for 4 h. These results suggest that plant class I LMW HSPs may play important roles in providing both thermotolerance and ethanol tolerance.

High Temperature Stress Resistance of Escherichia coli Induced by a Tobacco Class I Low Molecular Weight Heat-Shock Protein

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the aminoterminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90°C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90°C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50°C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.

High temperature stress resistance of Escherichia coli induced by a tobacco class I low molecular weight heat-shock protein.

TLHS1 is a class I low molecular weight heat-shock protein (LMW HSP) of tobacco (Nicotiana tabacum). For a functional study of TLHS1, a recombinant DNA coding for TLHS1 with a hexahistidine tag at the amino-terminus was constructed and expressed in Escherichia coli. An expressed fusion protein, H6TLHS1, was purified using a Ni2+ affinity column and a Sephacryl S400 HR column. A polyclonal antibody against H6TLHS1 was produced to follow the fate of H6TLHS1 in E. coli. The fusion protein in E. coli maintained its solubility at a temperature of up to 90 degrees C and most of the proteins in the E. coli cell lysate with H6TLHS1 were prevented from thermally induced aggregation at up to 90 degrees C. We compared the viability of E. coli cells expressing H6TLHS1 to the E. coli cells without H6TLHS1 at a temperature of 50 degrees C. After 8 h of high temperature treatment, E. coli cells with H6TLHS1 survived about three thousand times more than the bacterial cells without H6TLHS1. These results showed that a plant class I LMW HSP, TLHS1, can protect proteins of E. coli from heat denaturation, which could lead to a higher survival rate of the bacterial cells at high temperature.

Irrigation and Seed Quality Development in Rapid-cycling Brassica: Accumulation of Stress Proteins

The accumulation of representatives from three classes of stress proteins [a class 1 low molecular weight heat-shock protein (HSP); a dehydrin (group 2 LEA); and a group 3 LEA protein] were studied during seed development in rapid-cycling brassica [ Brassica campestris (rapa) L.] under different irrigation regimes. Withholding irrigation to plants altered the timing of events during seed development. All three proteins studied accumulated in seeds towards the end of seed development on well-irrigated plants, as seed moisture content declined. As irrigation was withheld from plants progressively earlier, these proteins appeared earlier both in time and in relation to physiological developmental markers, consistent with a protective role. However, seeds became desiccation tolerant during development before they had accumulated appreciable amounts of the two LEA proteins and the timing of accumulation was unaffected by post-harvest seed drying. This suggests that these particular LEA proteins are not absolutely required for desiccation tolerance. Although the HSP studied was also not present in seeds from well-irrigated plants as desiccation tolerance increased, its synthesis was induced by post-harvest drying at this stage of development. However, the amounts of HSP produced during drying subsequently declined later in development, but seeds remained desiccation tolerant. During the period when seeds were desiccation tolerant at least one of the three proteins was present either before drying, or produced in significant quantities upon drying. In general, the proteins studied continued to accumulate in seeds after mass maturity at the same time that potential longevity was increasing. Copyright 1998 Annals of Botany Company

Comparison of the Structure and Expression Pattern for a Low Molecular Weight Heat-Shock Protein cDNA Clone from Nicotiana tabacum

Comparison of the Structure and Expression Pattern for a Low Molecular Weight Heat-Shock Protein cDNA Clone from Nicotiana tabacum