Heat-inducible Protein Research Articles

Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.

Thermal conditioning of fifth‐instar Cydia pomonella (Lepidoptera: Tortricidae) affects HSP70 accumulation and insect mortality

Abstract Levels of HSP70 protein of fifth‐instar codling moth [Cydia pomonella (L.) (Lepidoptera: Tortricidae)] are determined after conditioning at 35 °C for different times and also after recovery at 22 °C. Protein samples from larvae conditioned for different times are separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis electrophoresis. Sub‐lethal thermal conditioning at 35 °C for 40 min, 2, 6 and 18 h induces new protein bands in the extracts from treated codling moth larvae. Immunodetection with an antibody to a heat‐inducible HSP70 indicates a stronger reaction after 35 °C for 2, 6 and 18 h than after 35 °C for 40 min or control and, during the recovery period at 22 °C, the level of heat shock protein decreases. Conditioning of fifth‐instar codling moths at 35 °C also induces thermotolerance in the insects and necessitates longer times at a lethal temperature to ensure mortality. Thermotolerance is correlated with the accumulation of heat inducible HSP70 protein.

The heat shock response of Synechocystis sp. PCC 6803 analysed by transcriptomics and proteomics

When cells of the cyanobacterium Synechocystis sp. PCC 6803 are exposed to high temperature they perceive changes in the growth conditions and regulate the expression of genes and synthesize heat-inducible proteins as a response to the heat stress. DNA microarray analysis revealed that genes for chaperonins and proteases, such as groESL1, groEL2, htpG, hspA, and clpB1 were transiently induced after incubation of the cells at 44 degrees C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that the levels of these chaperonins and proteases were elevated after incubation of cells at 44 degrees C for 60 min. These findings indicated that levels of the mRNAs and proteins of chaperonins were well correlated in the cells of Synechocystis. However, the level of elongation factors are mainly regulated at the protein level. These results indicated that acclimation to the heat-shock conditions might be governed by transcriptional and translational regulation in Synechocystis.

Virus Induction of Heat Shock Protein 70 Reflects a General Response to Protein Accumulation in the Plant Cytosol

Different cytoplasmically replicating RNA viruses were shown to induce a specific subset of heat-inducible heat shock protein 70 (HSP70) genes in Arabidopsis (Arabidopsis thaliana). To identify the inducing principle, a promoterreporter system was developed for the facile analysis of differentially responding Arabidopsis HSP70 genes, by infiltration into Nicotiana benthamiana leaves. Through transient expression of individual viral cistrons or through deletion analysis of a viral replicon, we were unable to identify a unique inducer of HSP70. However, there was a positive correlation between the translatability of the test construct and the differential induction of HSP70. Since these data implied a lack of specificity in the induction process, we also expressed a random series of cytosolically targeted Arabidopsis genes and showed that these also differentially induced HSP70. Through a comparison of different promoterreporter constructs and through measurements of the steady-state levels of the individual proteins, it appeared that the HSP70 response reflected the ability of the cytosol to sense individual properties of particular proteins when expressed at high levels. This phenomenon is reminiscent of the unfolded protein response observed when the induced accumulation of proteins in the endoplasmic reticulum also induces a specific suite of chaperones.

Peptidase Activity of the Escherichia coli Hsp31 Chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.

Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti‐Porphyromonas gingivalis heat‐shock protein 60 antisera

The present study has been performed to evaluate Porphyromonas gingivalis heat shock protein (HSP) 60 as a candidate vaccine to protect against multiple putative periodontopathic bacteria. Mouse anti-P. gingivalis HSP antisera demonstrated the elevated IgG antibody titers against the multiple bacteria tested and cross-reacted with heat-induced bacterial proteins of the target bacteria. The antisera also demonstrated a significantly higher opsonophagocytosis function against all the target bacteria than the control sera (P<0.01). We concluded that P. gingivalis HSP 60 could potentially be developed as a vaccine against multiple periodontopathic bacteria.

Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity.

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic alpha-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.

Heat-resistance and heat-shock response in the nosocomial pathogen Enterococcus faecium.

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50 degrees C, and no growth at 52 degrees C or 55 degrees C. In agreement, a marked decrease of general protein synthesis was observed at 52 degrees C, and very light synthesis was detected at 55 degrees C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52 degrees C and, after 2 h of incubation, viable cells were still observed at 70 degrees C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment.

Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release.

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.

Proteomic analysis of the effect of heat stress on hexaploid wheat grain: Characterization of heat-responsive proteins from total endosperm.

High temperatures during grain filling have been reported to be one of the factors that can affect the dough properties and quality characteristics of wheat. Responses to high temperature have been related to changes in protein composition at both quantitative and qualitative levels. The present study was conducted to determine the influence of high temperature during grain filling on the protein composition of bread wheat evaluated by proteomic tools. Plants were grown in the field and transferred to cabinets soon after flowering. They were subjected to two thermal regimes 18 degrees C/10 degrees C (day/night) and 34 degrees C/10 degrees C. Total proteins were extracted from control grains and treated plants at three different post-anthesis stages. The proteins were separated by two-dimensional gel electrophoresis and analysed by Melanie 3 software. Of the total number of mature wheat grain proteins, 37 were identified as significantly changed by heat treatment. Analysis by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry coupled with database searching allowed the characterization of 25 heat-induced proteins and only one heat-decreased protein spot. To learn more about the function of the identified proteins, we examined their expression during treatment.

Identification of proteins involved in the heat stress response of Bacillus cereus ATCC 14579.

To monitor the ability of the food-borne opportunistic pathogen Bacillus cereus to survive during minimal processing of food products, we determined its heat-adaptive response. During pre-exposure to 42 degrees C, B. cereus ATCC 14579 adapts to heat exposure at the lethal temperature of 50 degrees C (maximum protection occurs after 15 min to 1 h of pre-exposure to 42 degrees C). For this heat-adaptive response, de novo protein synthesis is required. By using two-dimensional gel electrophoresis, we observed 31 heat-induced proteins, and we determined the N-terminal sequences of a subset of these proteins. This revealed induction of stress proteins (CspB, CspE, and SodA), proteins involved in sporulation (SpoVG and AldA), metabolic enzymes (FolD and Dra), identified heat-induced proteins in related organisms (DnaK, GroEL, ClpP, RsbV, HSP16.4, YflT, PpiB, and TrxA), and other proteins (MreB, YloH, and YbbT). The upregulation of several stress proteins was confirmed by using antibodies specific for well-characterized heat shock proteins (HSPs) of B. subtilis. These observations indicate that heat adaptation of B. cereus involves proteins that function in a variety of cellular processes. Notably, a 30-min pre-exposure to 4% ethanol, pH 5, or 2.5% NaCl also results in increased thermotolerance. Also, for these adaptation processes, protein synthesis is required, and indeed, some HSPs are induced under these conditions. Collectively, these data show that during mild processing, cross-protection from heating occurs in pathogenic B. cereus, which may result in increased survival in foods.

Analysis of heat-shock transcription factor and element-binding activity.

Stress response is nearly universal. Living organisms respond to the changes in their chemical, physical, and biological environments by synthesizing a group of proteins called stress or heat-shock proteins (). The preferential synthesis of these proteins appears to be involved in induced transient thermal resistance (). It is well known that transcription of heat-shock genes is regulated by heat-shock transcription factors (HSFs), which bind to heat-shock elements (HSEs) located in the promoter region of genes encoding heat-shock proteins (). Heat-shock protein gene promoter contains three heat-shock elements (HSE, 5′-GAAnnTTCnnGAA-3′) (). The HSE is known as a conserved motif present in the promoters of many heat-inducible heat-shock protein genes. Many studies have demonstrated that stress-induced denatured, unfolded, or malfolded proteins trigger the activation of heat shock transcription factors (HSFs) (, , ). In mammalian cells, the activated HSF binds to the HSE and then stimulates transcription.

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae).

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactmcera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situ hybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleae provided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoni and Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.

Hsp70 and thermal pretreatment mitigate developmental damage caused by mitotic poisons in Drosophila.

To assess the ability of the heat-inducible molecular chaperone heat-shock protein 70 (Hsp70) to mitigate a specific developmental lesion, we administered the antimicrotubule drugs vinblastine (VB) and colchicine (COL) to larvae of Drosophila engineered to express differing levels of Hsp70 after heat pretreatment (HP). VB and COL decreased survival during metamorphosis, disrupted development of the adult eye and other structures as well as their precursor imaginal disks, and induced chromosome nondisjunction in the wing imaginal disk as indicated by the somatic mutation and recombination test (SMART) assay. Hsp70-inducing HP reduced many of these effects. For the traits viability, adult eye morphology, eye imaginal disk morphology, cell death in the eye imaginal disks, and single and total mutant clone formation in the SMART assay, HP reduced the impact of VB to a greater extent in Drosophila with 6 hsp70 transgenes than in a sister strain from which the transgenes had been excised. Because the extra-copy strain has higher levels of Hsp70 than does the excision strain but is otherwise almost identical in genetic background to the excision strain, these outcomes are attributable to Hsp70. The hsp70 copy number had a variable interaction with HP and COL administration.

Cloning and characterization of a LON gene homologue from the human pathogen Paracoccidioides brasiliensis.

A LON gene homologue from the human pathogen Paracoccidioides brasiliensis (PbLON) has been cloned, sequenced and characterized. It encodes a putative ATP-dependent proteinase Lon, which in Saccharomyces cerevisisae (PIM1) is a heat-inducible protein involved in the degradation of abnormal or short-lived proteins in the mitochondria. The PbLON ORF is within a 3369 bp fragment interrupted by two introns located in the 3'segment. The 5' and 3' regions flanking the ORF contain sequences which resemble known transcription elements. Several transcription binding factor motifs have also been found, including sites for heat shock/stress response and nitrogen control. The deduced protein consists of 1063 residues containing a mitochondrial import signal at the N-terminus and conserved ATP-binding (GPPGVGKT) and serine catalytic (KDGPSAG) sites. It shares high identity with Lon homologues from S. cerevisiae (73%), Homo sapiens (62%) and Escherichia coli (56%). In P. brasiliensis, an MDJ1 putative gene has also been partially sequenced adjacent to PbLON, possibly sharing divergently orientated promoter elements. This chromosomal organization is interesting, since Mdj1p is a heat shock chaperone essential for substrate degradation by PIM1 in yeast.

Laboratory selection at different temperatures modifies heat-shock transcription factor (HSF) activation in Drosophila melanogaster.

The magnitude and time course of activation of the heat-shock transcription factor (HSF) differ among Drosophila melanogaster lines evolving at 18 degrees C, 25 degrees C or 28 degrees C for more than 20 years. At lower heat-shock temperatures (27-35 degrees C), flies from the 18 degrees C population had higher levels of activated HSF (as detected by an electrophoretic mobility shift assay) than those reared at 25 degrees C and 28 degrees C. At higher temperatures (36 and 37 degrees C), however, the 28 degrees C flies had the highest levels of HSF. These differences persisted after one generation of acclimation at 25 degrees C, suggesting that phenotypic plasticity was limited. In addition, larvae from the 28 degrees C lines activated HSF less rapidly after a 35 degrees C heat shock than those from the 18 degrees C and 25 degrees C populations. These results are similar but not identical to previously reported differences in expression of Hsp70 (the major heat-inducible stress protein in Drosophila melanogaster) among the experimental lines. We conclude that HSF activation evolves rapidly during laboratory culture at diverse temperatures and could play an important role in the evolution of the heat-shock response.

Rheumatoid arthritis-related antigen 47kDa (RA-A47) is a product of colligin-2 and acts as a human HSP47.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

The Chaperone Function of hsp70 Is Required for Protection against Stress-Induced Apoptosis

Cellular stress can trigger a process of self-destruction known as apoptosis. Cells can also respond to stress by adaptive changes that increase their ability to tolerate normally lethal conditions. Expression of the major heat-inducible protein hsp70 protects cells from heat-induced apoptosis. hsp70 has been reported to act in some situations upstream or downstream of caspase activation, and its protective effects have been said to be either dependent on or independent of its ability to inhibit JNK activation. Purified hsp70 has been shown to block procaspase processing in vitro but is unable to inhibit the activity of active caspase 3. Since some aspects of hsp70 function can occur in the absence of its chaperone activity, we examined whether hsp70 lacking its ATPase domain or the C-terminal EEVD sequence that is essential for peptide binding was required for the prevention of apoptosis. We generated stable cell lines with tetracycline-regulated expression of hsp70, hsc70, and chaperone-defective hsp70 mutants lacking the ATPase domain or the C-terminal EEVD sequence or containing AAAA in place of EEVD. Overexpression of hsp70 or hsc70 protected cells from heat shock-induced cell death by preventing the processing of procaspases 9 and 3. This required the chaperone function of hsp70 since hsp70 mutant proteins did not prevent procaspase processing or provide protection from apoptosis. JNK activation was inhibited by both hsp70 and hsc70 and by each of the hsp70 domain mutant proteins. The chaperoning activity of hsp70 is therefore not required for inhibition of JNK activation, and JNK inhibition was not sufficient for the prevention of apoptosis. Release of cytochrome c from mitochondria was inhibited in cells expressing full-length hsp70 but not in cells expressing the protein with ATPase deleted. Together with the recently identified ability of hsp70 to inhibit cytochrome c-mediated procaspase 9 processing in vitro, these data demonstrate that hsp70 can affect the apoptotic pathway at the levels of both cytochrome c release and initiator caspase activation and that the chaperone function of hsp70 is required for these effects.

Heating rate and induced thermotolerance in Mexican fruit fly (Diptera: Tephritidae) larvae, a quarantine pest of citrus and mangoes.

A bioassay and graduated temperature water baths were used to document the induction of thermotolerance in third-instar Mexican fruit fly, Anastrepha ludens (Loew). The 99% lethal time dose for larvae exposed to 44 degrees C core temperatures in artificial fruit is 61.5 min when a slow heating rate (120 min ramp) is applied, but only 41.9 min when a fast heating rate (15 min ramp) is applied. In electrophoretic profiles a heat inducible protein of molecular weight 32 kDa was detected in 76% of the larvae exposed to the slow ramp treatment, but only 42% of the larvae in the fast ramp treatment. Results from this research demonstrate that thermotolerance can be induced under conditions used to commercially disinfest fresh produce and highlight the necessity for specifying heating rates in quarantine treatment schedules.

Heat Shock Response of &lt;I&gt;Bactrocera oleae&lt;/I&gt; (Diptera: Tephritidae): Genes and Proteins

The heat shock response as revealed by heat inducible puffing and protein patterns, as well as the localization of the major heat shock genes on the salivary gland polytene chromosomes of the olive fruit fly, Bactrocera oleae (Gmelin), a major agricultural pest, have been studied. After in situ hybridization of specific DNA sequences to the polytene chromosomes of B. oleae, the loci of the major heat shock genes hsp70, hsp83, and a putative hsp68 were detected. The two latter genes gave unique signals, and hsp70 hybridized to two adjacent discrete bands. Eight puffs were induced after heat shock, two of which were found to be the loci of the hsp70 and hsp83 genes. The newly synthesized polypeptides from the salivary glands of B. oleae, after gradual temperature elevation from 25 to 42°C, were analyzed in denaturing acrylamide gels. The most heat inducible polypeptides were found to exhibit relative electrophoretic mobilities of 83, 70, 69, 27, 26, and 23 kDa. The thermotolerance of B. oleae assumed by the above results is discussed in comparison with that observed in Drosophila species.