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

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

果蝇热激蛋白的研究进展

热休克蛋白(heat shock proteins,HSPs)是生物体受到应激刺激时诱导产生的一组保守性蛋白,普遍存在于各种生物体中。近年来,果蝇Drosophila作为生命科学与人类疾病研究的重要模式生物,其热激蛋白的研究取得了许多新的进展。文章对果蝇热激蛋白的类别、热激蛋白基因的表达调控机制、热激蛋白的分子伴侣功能、调节细胞存亡和影响发育及寿命等相关生物学功能进行综述,并对热激蛋白在神经退行性疾病治疗中的应用前景作展望。     

Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins

Abstract The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli ) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli . One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 × 10⁻⁴. IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.     

The pea lectin gene family contains only one functional gene

Molecular hybridization experiments have shown that the pea genome contains four regions which hybridize with pea lectin cDNA (Kaminski, Buffard, and Strosberg, 1986. Plant Science 46, 111–116). The complete organization of the pea lectin gene family was investigated. Four partial EcoRI genomic libraries were screened with a lectin cDNA (pPS 15–50) covering the entire coding region. Four positive recombinant phages, I 101, I 52, III 51 and IV 22, were isolated and the DNA sequences of the subclones, designated respectively PSL1, PSL2, PSL3 and PSL4, were determined. PSL2, PSL3 and PSL4 are incomplete genes; the presence of several stop codons in the correct reading frames indicate that these genes cannot code for a functional lectin protein. The sequences of PSL1 and pPS 15–50 have identical coding regions. The pea lectin gene has no intervening sequences and is flanked at its 5 region by a sequence containing an exceptionally high A+T content (73%). Eucaryotic consensus sequences such as a TATA box and a polyadenylation signal are also found in the flanking regions of the PSL1 clone.     

A comparative genomic analysis of the small heat shock proteins in Caenorhabditis elegans and briggsae

The small heat shock proteins (sHSPs) are a ubiquitous family of molecular chaperones. We have identified 18 sHSPs in the Caenorhabditis elegans genome and 20 sHSPs in the Caenorhabditis briggsae genome. Analysis of phylogenetic relationships and evolutionary dynamics of the sHSPs in these two genomes reveals a very complex pattern of evolution. The sHSPs in C. elegans and C. briggsae do not display clear orthologous relationships with other invertebrate sHSPs. But many sHSPs in C. elegans have orthologs in C. briggsae. One group of sHSPs, the HSP16s, has a very unusual evolutionary history. Although there are a number of HSP16s in both the C. elegans and C. briggsae genomes, none of the HSP16s display orthologous relationships across these two species. The HSP16s have an unusual gene pair structure and a complex evolutionary history shaped by gene duplication, gene conversion, and purifying selection. We found no evidence of recent positive selection acting on any of the sHSPs in C. elegans or in C. briggsae. There is also no evidence of functional divergence within the pairs of orthologous C. elegans and C. briggsae sHSPs. However, the evolutionary patterns do suggest that functional divergence has occurred between the sHSPs in C. elegans and C. briggsae and the sHSPs in more distantly related invertebrates.     

Multilevel structural characteristics for the natural substrate proteins of bacterial small heat shock proteins

Small heat shock proteins (sHSPs) are ubiquitous molecular chaperones that prevent the aggregation of various non‐native proteins and play crucial roles for protein quality control in cells. It is poorly understood what natural substrate proteins, with respect to structural characteristics, are preferentially bound by sHSPs in cells. Here we compared the structural characteristics for the natural substrate proteins of Escherichia coli IbpB and Deinococcus radiodurans Hsp20.2 with the respective bacterial proteome at multiple levels, mainly by using bioinformatics analysis. Data indicate that both IbpB and Hsp20.2 preferentially bind to substrates of high molecular weight or moderate acidity. Surprisingly, their substrates contain abundant charged residues but not abundant hydrophobic residues, thus strongly indicating that ionic interactions other than hydrophobic interactions also play crucial roles for the substrate recognition and binding of sHSPs. Further, secondary structure prediction analysis indicates that the substrates of low percentage of β‐sheets or coils but high percentage of α‐helices are un‐favored by both IbpB and Hsp20.2. In addition, IbpB preferentially interacts with multi‐domain proteins but unfavorably with α + β proteins as revealed by SCOP analysis. Together, our data suggest that bacterial sHSPs, though having broad substrate spectrums, selectively bind to substrates of certain structural features. These structural characteristic elements may substantially participate in the sHSP–substrate interaction and/or increase the aggregation tendency of the substrates, thus making the substrates more preferentially bound by sHSPs.     

A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers

Conservation of Lethal-leaf spot 1 (Lls1) lesion mimic gene in land plants including moss is consistent with its recently reported function as pheophorbide a oxygenase (Pao) which catalyzes a key step in chlorophyll degradation (Pruzinska et al., 2003). A bioinformatics survey of complete plant genomes reveals that LLS1(PAO) belongs to a small 5-member family of non-heme oxygenases defined by the presence of Rieske and mononuclear iron-binding domains. This gene family includes chlorophyll a oxygenase (Cao), choline monooxygenase (Cmo), the gene for a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic 55) and a novel 52 kDa protein isolated from chloroplasts (Ptc 52). Analysis of gene structure reveals that these genes diverged prior to monocot/dicot divergence. Homologues of LLS1(PAO), CAO, TIC55 and PTC52 but not CMO are found in the genomes of several cyanobacteria. LLS1(PAO), PTC52, TIC55 and a set of related cyanobacterial homologues share an extended carboxyl terminus containing a novel F/Y/W-x(2)-H-x(3)-C-x(2)-C motif not present in CAO. These proteins appear to have evolved during the transition to oxygenic photosynthesis to play various roles in chlorophyll metabolism. In contrast, CMO homologues are found only in plants and are most closely related to aromatic ring-hydroxylating enzymes from soil-dwelling bacteria, suggesting a more recent evolution of this enzyme, possibly by horizontal gene transfer. Our phylogenetic analysis of 95 extant non-heme dioxygenases provides a useful framework for the classification of LLS1(PAO)-related non-heme oxygenases.     

Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas

A given plant species is able to resist most of the potentially pathogenic microorganisms with which it comes in contact. This phenomenon, known as non-host resistance, can be overcome only by a very small number of true pathogens which can use that plant as a host. In some cases, plants have developed mechanisms for overcoming infection by specific races or strains of a true pathogen. This race-specific resistance can be easily manipulated into agronomically important cultivars by plant breeders. We have previously described nine cDNA clones which represent pea genes active during non-host resistance against the fungus Fusarium solani f. sp. phaseoli. In the present work, we have used these cDNAs as probes to compare non-host resistance with race-specific responses of peas against three races of Pseudomonas syringae pv. pisi. Five of the genes most active during non-host resistance were also active in direct correlation with the phenotypic expression of resistance in race-specific reactions of five differential pea cultivars against three races of Pseudomonas syringae pv. pisi.     

Detection of DNA polymorphism among pea cultivars using RAPD technique

An influence of some Random Amplified Polymorphic DNA (RAPD) reaction factors on resulting banding pattern and the ability of RAPD technique to detect DNA polymorphism among six economically important pea cultivars was tested. Relatively high level of DNA polymorphism among peas was observed, using polyacrylamide/urea gels and silver staining. Altogether 13 arbitrarily designed primers produced 313 amplification products. In addition 59 polymorphisms were found. These polymorphisms can serve as potential genetic markers. RAPD data were processed using cluster analysis and plotted as dendrogram. Each tested cultivar was clearly distinguished from the others. Moreover,Pisum sativum andP. sativum subsp.arvense cultivars were separated into 2 different clusters, according to their systematic relationships.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.     

Stimulatory effect of cytokinins and interaction with IAA on the release of lateral buds of pea plants from apical dominance

Lateral buds of pea plants can be released from apical dominance and even be transformed into dominant shoots when repeatedly treated with synthetic exogenous cytokinins (CKs). The mechanism of the effect of CKs, however, is not clear. The results in this work showed that the stimulatory effects of CKs on the growth of lateral buds and the increase in their fresh weights in pea plants depended on the structure and concentration of the CKs used. The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) was stronger than that of 6-benzylaminopurine (6-BA). Indoleacetic acid (IAA) concentration in shoot, IAA export out of the treated apex and basipetal transport in stems were markedly increased after the application of CPPU or 6-BA to the apex or the second node of pea plant. This increase was positively correlated with the increased concentration of the applied CKs. These results suggest that the increased IAA synthesis and export induced by CKs application might be responsible for the growth of lateral shoots in intact pea plants.     

Preliminary study of heat shock proteins in nemerteans

Nemerteans experience varying environmental temperatures during low tide exposures. Inducible heat shock proteins (hsps) have been reported for most organisms following both artificial heat stress and natural environmental temperature variations. This preliminary study reports the presence of hsps in the phylum Nemertea. A lethal temperature of 36 °C was determined for Paranemertes peregrina Coe, 1901. The nemerteans were exposed to a temperature of 34 °C for 2 h. After a 2 h recovery time, the worms were then analyzed for hsps by SDS–PAGE and western immunoblot protocols. Control worms were allowed to acclimate to ambient temperatures (13–15 °C) before hsp analysis. Hsp70 and hsp90 were detected in both the control and heat-shocked worms in highly variable concentrations, and the latter group had significantly elevated hsp70 levels. In addition, the analysis detected different isoforms of hsp70. The detection of hsps indicates a possible role in nemertean physiology during response to thermal stress, and potentially to other environmental challenges.     

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.     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Effect of exogenous hydrogen peroxide on enzymatic and nonenzymatic antioxidants in leaves of young pea plants treated with paraquat

The effects of exogenously applied hydrogen peroxide on the antioxidant system of pea plants were investigated. Ten-day-old pea seedlings were sprayed with 2.5 mM H₂O₂ and 24 h later with 0.2 mM PQ. Samples were taken 0, 2 and 5 h after the start of illumination. The protective effect of H₂O₂ was evaluated by monitoring of parameters related to the damage caused by PQ. The treatment with PQ led to a severe leakage of electrolytes from leaf tissues. Malondialdehyde level increased in PQ treated plants, but remained unchanged in H₂O₂ pre-treated ones after 5 h of illumination. Increased catalase and glutathione-S-transferase activity was observed in pea plants treated with H₂O₂ and PQ. Ascorbate peroxidase activity decreased significantly after paraquat application, but pre-treatment with H₂O₂ prevented ascorbate peroxidase inhibition to some extent. Increased guaiacol peroxidase activity was detected after H₂O₂ application. PQ application caused a drastic decline in the levels of thiol-group bearing compounds, reduced glutathione and ascorbate, while the quantity of oxidized glutathione and dehydroascorbate were increased. The results presented on changes in enzymatic and nonenzymatic antioxidants suggest that preliminary H₂O₂ application to pea plants treated with PQ, alleviates the toxic effects of the herbicide.