Heat-induced preferential synthesis and redistribution of HSP 70 and 28 families in Chinese hamster ovary cells

We observed that members of two HSP families (70 and 28 kDa) preferentially redistributed into the nucleus after heating at 45.5 degrees C for 10 min. The rates of synthesis and redistribution of these proteins were different for each member of HSP families during incubation period at 37 degrees C after heat shock. The maximum rates of synthesis of HSP 70 and HSP 28 families, except HSP 28c, were 6-9 hr after heat shock, whereas the maximum rates of redistribution were 3-6 hr after heat shock. These results suggest that the rates of redistribution of these proteins may be dependent on the amount of intracellular proteins as well as the alteration of binding affinity of nucleoproteins following heat shock.     

Regulation and function of small heat shock protein genes during amphibian development

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress such as elevated temperature or exposure to heavy metals or arsenate. Recent interest in shsps has been propelled by the finding that shsp synthesis or mutations are associated with various human diseases. While much is known about shsps in cultured cells, less is known about their expression and function during early animal development. In amphibian model systems, shsp genes are developmentally regulated under both normal and environmental stress conditions. For example, in Xenopus, the shsp gene family, hsp30, is repressed and not heat-inducible until the late neurula/early tailbud stage whereas other hsps are inducible at the onset of zygotic genome activation at the midblastula stage. Furthermore, these shsp genes are preferentially induced in selected tissues. Recent studies suggest that the developmental regulation of these shsp genes is controlled, in part, at the level of chromatin structure. Some shsps including Xenopus and Rana hsp30 are synthesized constitutively in selected tissues where they may function in the prevention of apoptosis. During environmental stress, amphibian multimeric shsps bind to denatured target protein, inhibittheir aggregation and maintain them in a folding-competent state until reactivated by other cellular chaperones. Phosphorylation of shsps appears to play a major role in the regulation of their function.     

Application of the shsp gene,encoding a small heat shock protein,as a food-grade selection marker for lactic acid bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60 degrees C or pH 3.5 and in the ability to grow at 52 degrees C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em(r)) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 x 10(4) and 1.0 x 10(4) CFU/0.5 micro g of DNA, with standard deviations of 0.54 x 10(4) and 0.32 x 10(4), for shsp and Em(r) selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 x 10(4) and 3.8 x 10(3) CFU/0.5 micro g of DNA, with standard deviations of 0.63 x 10(4) and 3.48 x 10(3), for shsp and Em(r) selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

Intracellular localization of Xenopus small heat shock protein, hsp30, in A6 kidney epithelial cells

Small heat shock proteins (shsps) are molecular chaperones that are inducible by environmental stress. In this study, immunocytochemical analysis and laser scanning confocal microscopy revealed that the shsp family, hsp30, was localized primarily in the cytoplasm of Xenopus A6 kidney epithelial cells after heat shock or sodium arsenite treatment. Heat shock-induced hsp30 was enriched in the perinuclear region with some immunostaining in the nucleus but not in the nucleolus. In sodium arsenite-treated cells hsp30 was enriched towards the cytoplasmic periphery as well as showing some immunostaining in the nucleus. At higher heat shock temperatures (35 degrees C) or after 10 microM sodium arsenite treatment, the actin cytoskeleton displayed some disorganization that co-localized with areas of hsp30 enrichment. Treatment of A6 cells with 50 microM sodium arsenite induced a collapse of the cytoskeleton around the nucleus. These results coupled with previous studies suggest that stress-inducible hsp30 acts as a molecular chaperone primarily in the cytoplasm and may interact with cytoskeletal proteins.     

Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.     

Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.     

Genotype-specific heat shock proteins in two maize inbreds

Summary Leaf blade tissue of maize inbred lines B73 and Mo17 was analyzed for intraspecific genetic variability in the heat shock response. The maize inbreds were characterized for acquired thermal tolerance and patterns of heat shock protein synthesis. The leakage conductivity assay of membrane stability during stress indicated that Mol7 possesses greater potential than B73 to acquire thermal tolerance. Poly(A)⁺ RNA, extracted from leaf blades, was translated in vitro in the presence of ³⁵S-methionine and the translation products separated by twodimensional gel electrophoresis. Major genotypic differences were observed in the translation products. Mo 17 synthesized twelve unique heat shock proteins in the 15–18 kD range, but B73 synthesized only three unique heat shock proteins in the same range. DNA polymorphisms were observed between the maize lines using ³²P labeled heat shock protein gene probes.Abbreviations HKT Heat-killing time - HS Heat shock - HSP Heat shock protein - HMW High molecular weight - LMW Low molecular weight Contribution of the College of Agricultural Sciences, Texas Tech University, Journal No. T-4-333     

Sero-epidemiology as a tool to screen populations for exposure to Mycobacterium ulcerans

Background

Previous analyses of sera from a limited number of Ghanaian Buruli ulcer (BU) patients, their household contacts, individuals living in BU non-endemic regions as well as European controls have indicated that antibody responses to the M. ulcerans 18 kDa small heat shock protein (shsp) reflect exposure to this pathogen. Here, we have investigated to what extent inhabitants of regions in Ghana regarded as non-endemic for BU develop anti-18 kDa shsp antibody titers.

Methodology/Principal Findings

For this purpose we determined anti-18 kDa shsp IgG titers in sera collected from healthy inhabitants of the BU endemic Densu River Valley and the Volta Region, which was so far regarded as BU non-endemic. Significantly more sera from the Densu River Valley contained anti-18 kDa shsp IgG (32% versus 12%, respectively). However, some sera from the Volta Region also showed high titers. When interviewing these sero-responders, it was revealed that the person with the highest titer had a chronic wound, which was clinically diagnosed and laboratory reconfirmed as active BU. After identification of this BU index case, further BU cases were clinically diagnosed by the Volta Region local health authorities and laboratory reconfirmed. Interestingly, there was neither a difference in sero-prevalence nor in IS2404 PCR positivity of environmental samples between BU endemic and non-endemic communities located in the Densu River Valley.

Conclusions

These data indicate that the intensity of exposure to M. ulcerans in endemic and non-endemic communities along the Densu River is comparable and that currently unknown host and/or pathogen factors may determine how frequently exposure is leading to clinical disease. While even high serum titers of anti-18 kDa shsp IgG do not indicate active disease, sero-epidemiological studies can be used to identify new BU endemic areas.     

Transforming growth factor-beta 1 rapidly induces Hsp70 and Hsp90 molecular chaperones in cultured chicken embryo cells.

In this report we show that: (1) molecular chaperones in the heat shock protein (hsp) family are a new class of cellular proteins induced by Transforming Growth Factor-beta 1 (TGF beta), a cytokine present in serum, (2) rapid induction of Hsc70 precedes a general increase in protein synthesis and may be a preparatory event, (3) TGF beta is a potent regulator of overall protein synthesis in chicken embryo cells (CEC), and (4) isoforms of Hsp90 with different biochemical properties exist, raising the possibility that they may have different functions. TGF beta can substitute for serum in stimulating synthesis of members of the Hsp90 and Hsp70 families of stress proteins, whereas other cytokines, including PDGF, FGF, and EGF, were not effective nor did they enhance the stimulatory effect of TGF beta on the hsp's. Analysis of the induction of hsp's using one- and two-dimensional polyacrylamide gel electrophoresis indicated that members of the Hsp70 family of molecular chaperones were induced rapidly by TGF beta, reaching maximum rates of accumulation by 5 hours of treatment. Total protein synthesis increased more slowly, undergoing an approximately twofold increase in 24 hours. Using a modified protocol for two-dimensional gel electrophoresis, the Hsp90 protein family was separated into four isoelectric forms, two of which were phosphorylated (Hsp90-2 and -4). These phosphorylated isoforms turned over faster than the unphosphorylated forms of Hsp90. All four isoforms were heat inducible, but only Hsp90-2 and -3 were induced rapidly by TGF beta, again within 5 hours of treatment. The effects of serum on these protein families were similar to those of TGF beta, suggesting that this cytokine may be the serum component primarily responsible for up-regulating members of the Hsp90 and Hsp70 families. We hypothesize that cells rapidly increase their chaperoning capacity for newly synthesized polypeptides in preparation for an increase in the rate of synthesis of proteins up-regulated by TGF beta.     

Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.     

Analysis of the cytosolic hsp70 gene family inZea mays

In this study we have analysed the multigene family coding for the cytoplasmic heat shock 70 kDa proteins (hsp70) inZea mays. Fully degenerate primers were used in a polymerase chain reaction (PCR) to amplify selected regions of the hsp70 genes. Sequence and Southern blot analysis reveals that at least three highly conserved genes exist in maize. In addition, amplification reveals the presence of a conserved intron in all genes examined. Expression analysis shows that the hsp70 genes studied represent members of the inducible and constitutive families. The results obtained may indicate that there are subfamilies of cytoplasmic hsp70 genes expressed in higher plants.     

Heat shock proteins in neurodegenerative disorders and aging

Many members of the heat shock protein family act in unison to refold or degrade misfolded proteins. Some heat shock proteins also directly interfere with apoptosis. These homeostatic functions are especially important in proteinopathic neurodegenerative diseases, in which specific proteins misfold, aggregate, and kill cells through proteotoxic stress. Heat shock protein levels may be increased or decreased in these disorders, with the direction of the response depending on the individual heat shock protein, the disease, cell type, and brain region. Aging is also associated with an accrual of proteotoxic stress and modulates expression of several heat shock proteins. We speculate that the increase in some heat shock proteins in neurodegenerative conditions may be partly responsible for the slow progression of these disorders, whereas the increase in some heat shock proteins with aging may help delay senescence. The protective nature of many heat shock proteins in experimental models of neurodegeneration supports these hypotheses. Furthermore, some heat shock proteins appear to be expressed at higher levels in longer-lived species. However, increases in heat shock proteins may be insufficient to override overwhelming proteotoxic stress or reverse the course of these conditions, because the expression of several other heat shock proteins and endogenous defense systems is lowered. In this review we describe a number of stress-induced changes in heat shock proteins as a function of age and neurodegenerative pathology, with an emphasis on the heat shock protein 70 (Hsp70) family and the two most common proteinopathic disorders of the brain, Alzheimer’s and Parkinson’s disease.     

Heat shock induces intestinal-type alkaline phosphatase in rat IEC-18 cells

We demonstrate a previously unknown regulation for intestinal-type alkaline phosphatase (IAP) as a heat shock protein (HSP). Heat shock to rat intestinal epithelial cells (IEC)-18 at 43 degrees C induced the expression of IAP-I and HSP72 mRNAs time dependently (<60 min) but did not induce expression of IAP-II, tissue nonspecific-type alkaline phosphatase (TNAP), or HSP90 as determined by the RT-PCR method. To confirm the identity of the IAP-I gene, we sequenced the amplification product of IAP-I and found the gene to have 99% homology with the sequence of the IAP-I gene in rat intestine. Under the subculture conditions used, no IAP protein was detected in IEC-18 cells, but it became detectable as a 62-kDa band on a Western blot after heat shock. IAP-I was also induced by sodium arsenite, which generates reactive oxygen species and is an inducer of members of the HSP family. Glutathione suppressed activating protein-1 and cAMP response element-binding protein activation caused by heat shock but did not suppress the expression of IAP-I. These results suggest that cellular stress induces the elevation of IAP-I mRNA and protein synthesis. IAP-I may play an important role as a dephosphorylating enzyme under stress conditions.     

Molecular Cloning the Gene of Small Heat Shock Protein in the Mitochondria and Endoplasmic Reticulum of Tomato

A cDNA library was constructed with the heat shocked tomato (Lycopersicon esculentum Mill.) flowers and then was screened with the probes of mitochondrial and endoplasmic reticulum conservative regions that were cloned by using RT-PCR. The complete cDNAs of mitochondrial and endoplasmic reticulum small heat shock protein ( shsp ) were selected out from the cDNA library. Furthermore, the temperature responses of these shsp genes were determined. Northern hybridization showed that the heat response temperatures of both genes in tomato flower were lower than that in leaf and that mitochondria shsp in leaf was cold-inducible. In this paper, the molecular features of the cloned genes, the causes of the uncommon heat response temperatures of sHSP in flower and the cold inducible character of mitochondria shsp gene in leaf were discussed.      

Heat Shock Protein Synthesis Induced by Methomyl in Maize (Zea mays L.) Seedlings

Exposure of plumules of intact maize seedlings (Zea mays L.) to S-methyl-N-[(methylcarbamoyl)-oxy]thioacetimidate (methomyl) represses synthesis of several polypeptides normally made under control conditions and induces synthesis of polypeptides similar to maize heat shock polypeptides (HSPs). Three of the methomyl-induced polypeptides (18 kilodaltons) are recognized by antibodies raised against 18 kilodalton maize heat shock polypeptides.