Production of stress-inducible form of heat-shock protein 70 in mouse peritoneal adherent cells afterin Vivo infection byFrancisella tularensis

Heat-shock proteins (hsp) are ubiquitously produced molecules which participate in the protection of cells from environmental perturbation. Moreover, the members of the heat-shock protein 60 (hsp60) and 70 (hsp70) families play an important role in pathogen-host interactions. We studiedin vivo production of the 70-kDa heat-shock proteins in the extract of peritoneal exudate cells (PEC) from mice injected intraperitoneally with an attenuated vaccine strain (LVS) ofFrancisella tularensis. We found a differential production of a highly stress-inducible member of the hsp70 family, designated hsp72, in three inbred strains of mice exhibiting either resistance or susceptibility toF. tularensis LVS infection. Whereas in tularemia-resistant mice hsp72 was even expressed in PEC without injection of bacteria and its production further increased on day 3 and slowly declined on days 5 and 7 after injection, in susceptible mice hsp72 production was highly inducble and restricted only to day 3 afterin vivo infection. Further analysis of hsp72 expression revealed intracellular hsp72 accumulation and its preferential production by peritoneal adherent cells.     

Common antigenicity of mouse 42 degrees C-specific heat-shock protein with mouse HSP 105

Mammalian cells incubated at 42 degrees C synthesize a specific heat-shock protein at 42 degrees C (42 degrees C-hsp) that is not induced by heat-shock at 45 degrees C or by other stresses that induce major heat shock proteins (Hatayama et al. (1986) Biochem. Biophys. Res. Commun. 137, 957-963). Antibody raised against a heat-shock protein with molecular weight of 105,000 (hsp 105) purified from mouse FM 3A cells cross-reacted to the 42 degrees C-hsp of the same cells. The antibody reacted only weakly to hsp 105 and 42 degrees C-hsp of human HeLa cells. These results suggested that hsp 105 and 42 degrees C-hsp have the same antigenic determinant, and that 42 degrees C-hsp may have a structure similar to that of hsp 105.     

The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.     

Specific antibodies against the stress-inducible 72-kDa protein, a member of the heat-shock protein hsp70, in healthy human subjects.

1. On Western blot analysis, serum IgG from a healthy human subject reacted with a stress-induced protein, having an apparent molecular mass of 70 kDa, from PC12 cells. 2. This serum IgG also reacted with hsp70 (70-kDa heat-shock protein) purified from the bovine brain. 3. In 5 out of 34 healthy human subjects, IgG antibodies against hsp70 were detected. 4. These antibodies were directed against the stress-inducible 72-kDa protein, but did not cross-react with the constitutive 73-kDa protein.     

The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 alpha at two NH2-terminal threonine residues

The 90-kDa heat-shock protein, hsp90, is an abundant cytoplasmic protein that can be phosphorylated in vitro by a double-stranded (ds) DNA-activated protein kinase found in cells from several species. Here we show that the dsDNA-activated protein kinase from human HeLa cells phosphorylates 2 threonine residues in the sequence PEETQTQDQPME at the amino terminus of human hsp90 alpha. Hsp90 beta, which is 97% identical to hsp90 alpha but lacks both amino-terminal threonines, is not phosphorylated by the dsDNA-activated protein kinase. Mouse hsp86 and rabbit hsp90 alpha are homologous to human hsp90 alpha; both heterologous proteins are phosphorylated at the same amino-terminal threonines by the human dsDNA-activated protein kinase.     

Tissue specificity of the heat-shock response in maize

The tissue specificity of the heat-shock response in maize was investigated. The ability to synthesize heat shock proteins (hsp) at 40°C, as well as the intensity and duration of that synthesis, was analyzed in coleoptiles, scutella, green and etiolated leaves, suspension-cultured cells, germinating pollen grains, and primary root sections at different stages of development. One-dimensional sodium dodecyl sulfate gel electrophoresis of extracted proteins revealed that most of the tissues synthesized the typical set of 10 hsp, but that the exact characteristics of the response depended upon the tissue type. While elongating portions of the primary root exhibited a strong heat shock response, the more mature portions showed a reduced ability to synthesize hsp. Leaves, whether green or etiolated, excised or intact, constitutively synthesized a low level of hsp at 25°C, and high levels could be induced at 40°C. Suspension-cultures of Black Mexican sweet corn synthesized, besides the typical set of hsp, two additional polypeptides. In contrast to all the other tissues, germinating pollen grains could not be induced to synthesize the typical set of hsp but did synthesize two new polypeptides of 92 and 56 kD molecular weight.

The heat shock response was transient for most of the tissues which synthesized the standard set of hsp. Hsp synthesis was detected up to 2 to 3 hours, but not at 10 hours of continuous 40°C treatment. The exception was suspension cultured cells, in which hsp synthesis showed only a slight reduction after 10 hours at 40°C. Tissue-specific differences in the heat-shock response suggest that there are differences in the way a given tissue is able to adapt to high temperature.

We have confirmed the previous suggestion that maize hsp do not accumulate in substantial quantities. Using two-dimensional gel analysis, hsp could be detected by autoradiography but not by sensitive silver staining techniques.

     

Accumulation of heat shock protein 72 (hsp 72) in postimplantation rat embryos after exposure to various periods of hyperthermia (40 degrees -43 degrees C) in vitro: evidence that heat shock protein 72 is a biomarker of heat-induced embryotoxicity.

A monoclonal antibody to the 72 kDa heat shock protein and Western blot analysis were used to determine the induction, accumulation and turnover of hsp 72 after day 10 rat embryos were exposed to elevated temperatures (40 degrees-43 degrees C) for various lengths of time (2.5 minutes to 18 hours). Embryos exposed to temperatures that exceed the normal culture temperature (37 degrees C) by 4 degrees C or more for as little as 2.5 minutes (43 degrees C) or 15 minutes (41, 42 degrees C) synthesized and accumulated detectable amounts of heat-inducible hsp 72. Hsp 72 could not be detected by Western blot analysis of proteins from embryos cultured at 40 degrees C or below. Once induced, hsp 72 can be detected in embryos for 24-48 hours after they are removed from the hyperthermic conditions and returned to normothermic conditions. Our results also indicate that hsp 72 is induced by all hyperthermic exposures that induce alterations in rat embryo growth and development; therefore, hsp 72 is a potential biomarker for heat-induced embryotoxicity.     

Human 60-kDa heat-shock protein: a danger signal to the innate immune system

Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-alpha; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-gamma in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation.     

Subcellular localization of the 84,000 dalton heat-shock protein in mouse neuroblastoma cells: evidence for a cytoplasmic and nuclear location

Using affinity-purified antibodies, the 84,000 dalton heat-shock protein (hsp) has been localized in mouse N2A neuroblastoma cells by immunocytochemical techniques. Immunofluorescence microscopy showed that hsp84 was present both in the cytoplasm and in the nucleus. The nucleoli were found to be unlabelled. Immunogold labelling on ultrathin cryosections revealed that hsp84 was evenly distributed throughout the entire cytoplasm. No preferential association of hsp84 with the plasma membrane or with membranes from organelles was observed. In the nucleus the hsp84 was present in both the euchromatin and heterochromatin. In the nucleolus only the fibrillar part was labelled and virtually no gold particles were observed in the granular part. A long-term hyperthermic treatment of 3 h at 42.5 degrees C was found to induce an accumulation of hsp84 inside the nucleus. No alterations in hsp84 distribution were observed during a treatment of the cells with 75 microM sodium arsenite for 3 h. Drastic alterations were observed in the nucleoli after both stress treatments. The granular part had totally disappeared and only remnants of the fibrillar part which contained hsp84, were found. Besides the nuclear accumulations of hsp84 during heat shock, no additional changes in the hsp84 location in stressed cells were observed. During a recovery from the heat shock by replacing the cells at 37 degrees C, a decrease in the nuclear location of hsp84 was observed, indicating the reversibility of this process. The significance of these results for the role of hsp84 in normal and in stressed cells is discussed.     

Control of ribosome biosynthesis in plant cell cultures under heat-shock conditions. Ribosomal RNA

The immediate block of ribosome biosynthesis in heat-shocked tomato cell cultures is primarily caused by the complete inhibition of pre-rRNP processing. Depending on the heat-shock conditions synthesis of pre-rRNP goes on, though at a reduced level. Synthesis and/or preservation of pre-rRNP during heat shock as well as its efficient processing in the recovery period are thoroughly improved by preconditioning of cells to the hyperthermic treatment. Such preinduced cultures are characterized by their content of preformed heat-shock proteins, whose dominant representative (hsp 70) becomes highly enriched in the characteristic granular rRNP material observed in nucleoli of heat-shocked cells. This is shown by immune fluorescence staining and microautoradiography.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

In Euglena gracilis, a heat-shock protein related to hsc73 is constitutive and stress inducible

Using monoclonal antibodies directed against different cytoplasmic isoforms of hsp70 proteins, namely, the constitutive hsc73 and the inducible hsp72 isoforms, we found that one isoform related to hsc73 was present in Euglena gracilis. This hsc73-like protein is expressed with a higher rate of synthesis in cells growing under heat shock than in control cells. Moreover, in cadmium-resistant cells, cultured at normal growth temperature, the rate of synthesis of this protein is constitutively increased. These results indicate that a heat-shock protein related to hsc73 is present in an ancestral eukaryote, Euglena gracilis, and that this protein may be constitutive and stress inducible as well.     

Relationship between heat-shock protein synthesis and thermotolerance in rainbow trout fibroblasts

Summary The role of heat-shock protein synthesis in the development of thermotolerance by rainbow trout fibroblasts was examined. During the first 6 h after being shifted from 22°C to 28°C, cells of the rainbow trout fibroblast line, RTG-2, rapidly synthesized the major heat-shock proteins (hsps), hsps 87, 70 and 27, and developed tolerance to 32°C. After 24 h at 28°C hsp synthesis was drastically reduced but thermotolerance was maintained. If these thermotolerant cells were shifted to 32°C, hsp synthesis continued at a very low level, but if they were subsequently returned to 22°C, synthesis of hsps 70 and 27 was induced again. The addition of actinomycin D during the first 6 h at 28°C prevented hsp synthesis and the development of thermotolerance. The presence of actinomycin D during the incubation of thermotolerant cultures at 32°C blocked the reinitiation of hsps synthesis at 22°C but had no effect on survival. Therefore, the hsps that accumulated at 28°C were sufficient to allow cells to survive a subsequent thermal stress at 32°C.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-β-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli β-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific β-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

Synaptophysin-containing microvesicles transport heat-shock protein hsp60 in insulin-secreting beta cells

58–62 kDa heat-shock proteins (hsp60) are molecular chaperonins involved in the process of protein folding, transmembrane translocation and assembly of oligomeric protein complexes. In eukaryotic cells hsp60 proteins have been found in mitochondria and chloroplasts. However, we have recently documented that, in addition to mitochondria, a hsp60-like protein is present in secretory granules of insulin-secreting beta cells. The pathway by which hsp60 is targeted to secretory granules was unknown. Here we report the existence of microvesicles involved in the transport of hsp60 protein. Immunoelectron microscopy of serial thin-sections of beta cells directly visualized stages associated with hsp60 delivery: attachment of microvesicles to a secretory granule, fusion with the secretory granule membrane and release of hsp60 molecules. Further biochemical and immunological analysis of microvesicles revealed the presence in their membrane of synaptophysin, a major component of synaptic-like microvesicles (SLMV) of neuroendocrine cells. Double immunogold labelling with antibodies to synaptophysin and hsp60 demonstrated co-localization of both proteins in the same microvesicles. Moreover, fusion of synaptophysin-positive microvesicles leaves synaptophysin incorporated, at least transiently, to secretory granule membranes. These findings suggest that, in beta cells, synaptic-like vesicles are involved in the transport and delivery of hsp60 and represent a novel pathway for protein transport and secretion.