Heat-shock response in Legionella pneumophila

The heat-shock response of Legionella pneumophila was examined by radiolabelling bacterial cell proteins with [35S]methionine following a temperature shift from 30 to 42 degrees C. Five heat-shock proteins were identified as having molecular masses of 17, 60, 70, 78, and 85 kilodaltons (kDa). The 85- and 60-kDa proteins were equally distributed between supernatant and pellet fractions following ultracentrifugation at 100,000 x g, the 70- and 78-kDa proteins were found primarily in the supernatant, and the 17-kDa protein was found primarily in the pellet. Synthesis of subsets of the heat-shock proteins could be stimulated by novobiocin, patulin, or puromycin. Ethanol, an effector of the heat-shock response in other microorganisms, had little effect on L. pneumophila, even at the highest concentration tolerated by the bacterial cells (1.9%). Finally, the 60-kDa heat-shock protein of L. pneumophila was immunologically cross-reactive with a polyclonal antibody prepared to the Escherichia coli groEL protein. However, a mouse monoclonal antibody reactive with the 60-kDa protein of all legionellae tested did not cross-react with the E. coli groEL protein, suggesting that the Legionella 60-kDa protein contains common and unique epitopes.     

Insulin-like Growth Factor-I Induces Phosphorylation in Leishmania {Leishmania) mexicana Promastigotes and Amastigotes

Protein phosphorylation controls major steps of proliferation and differentiation in eukaryotic cells. However there are few studies done in protozoa particularly when being triggered by external stimuli. In this paper we have examined the tyrosine- and serine/threonine-phosphorylated proteins in both promastigote and amastigote-like forms of Leishmania (Leishmania) mexicana stimulated with insulin-like growth factor (IGF)-I. Stimulation with IGF-I induces major tyrosine phosphorylation of a 185-kDa protein in promastigotes and 60- and 40-kDa proteins in amastigotes. Analysis of total phosphorylation revealed additional sets of phosphorylated proteins: a 110-kDa protein band in promastigotes and two other proteins of 120 and 95 kDa in the amastigote-like forms. To further analyze the IGF-I-mediated response we compared it with the phosphorylation pattern obtained with a known inducer of protein kinase C, phorbol myristate acetate. This analysis showed overlapping phosphorylation of most of the proteins but mainly of the 185- and 110-kDa proteins in the promastigotes and the 95-. 60- and 40-kDa proteins in the amastigote-like forms. We thus conclude that there are phosphorylation-dependem pathways in Leishmania parasites induced by IGF-I that are stage-specific.     

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

Acidic extracellular environment induces only a subset of heat-shock proteins in primary mouse kidney cell cultures

Exposure of primary mouse kidney cell cultures to acidic medium (pH 5.5) induced the expression of a 70 kilodalton (kDa) protein. This protein was identified as the major inducible heat-shock protein 70 (hsp70) by immunoprecipitation with anti-hsp70 serum and Northern blot analysis with a hsp70 cDNA probe. Maximum induction of the 70-kDa protein at pH 5.5 after 240 min was about 30% of that observed after 60 min of thermal treatment at 43 degrees C. In addition, there was an apparent induction of the glucose-regulated proteins (GRPs) of 76-78 and 98-100 kDa, but not of the other hsps. This subset induction of the heat-shock response by acidic medium suggests that different mechanisms are responsible for the induction of the various families of hsps.     

Heat-shock response of the upper intertidal barnacle Balanus glandula: thermal stress and acclimation

In the intertidal zone in the Pacific Northwest, body temperatures of sessile marine organisms can reach 35 degrees C for an extended time during low tide, resulting in potential physiological stress. We used immunochemical assays to examine the effects of thermal stress on endogenous Hsp70 levels in the intertidal barnacle Balanus glandula. After thermal stress, endogenous Hsp70 levels did not increase above control levels in B. glandula exposed to 20 and 28 degrees C. In a separate experiment, endogenous Hsp70 levels were higher than control levels when B. glandula was exposed to 34 degrees C for 8.5 h. Although an induced heat-shock response was observed, levels of conjugated ubiquitin failed to indicate irreversible protein damage at temperatures up to 34 degrees C. With metabolic labeling, we examined temperature acclimation and thermally induced heat-shock proteins in B. glandula. An induced heat-shock response of proteins in the 70-kDa region (Hsp70) occurred in B. glandula above 23 degrees C. This heat-shock response was similar in molting and non-molting barnacles. Acclimation of B. glandula to relatively higher temperatures resulted in higher levels of protein synthesis in the 70-kDa region and lack of an upward shift in the induction temperature for heat-shock proteins. Our results suggest that B. glandula may be well adapted to life in the high intertidal zone but may lack the plasticity to acclimate to higher temperatures.     

Hypoxia Induces Synthesis of a Novel 22-kDa Protein in Neonatal Rat Oligodendrocytes

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunologic and structural characterization of the dominant 66- to 73-kDa antigens of Borrelia burgdorferi

The 66- to 73-kDa proteins of Borrelia burgdorferi are dominant immunogens and expressed in all strains of B. burgdorferi. The humoral response to these Ag occurs relatively early during the course of infection. Two-dimensional Western blot analysis of this group of Ag revealed them to consist of a tetrad of proteins with apparent molecular mass of 66, 68, 71, and 73 kDa. Furthermore, in this study we demonstrate the 66-kDa protein to be a potent inducer of lymphoproliferation in the patient immune to B. burgdorferi. Monospecific polyclonal antibodies and mAb demonstrate that each of these proteins was immunologically distinct. However, direct amino acid sequence of the 66- and 68-kDa Ag was almost identical and had a high level of sequence similarity to the GroEL heat-shock protein (Hsp60) of Escherichia coli and the 60-kDa immunodominant protein of Treponema pallidum. The amino terminal sequence of the 71- and 73-kDa proteins of B. burgdorferi was almost identical and these proteins had remarkable sequence similarity to the DnaK heat-shock protein of E. coli (Hsp70). It appears likely, therefore, that proteins related to the heat-shock family are potent immunogens of B. burgdorferi.     

Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins

Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper.     

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase.

Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat-shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat-shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form.     

HSP 26 and 27 are phosphorylated in response to heat shock and ecdysterone in Drosophila melanogaster cells

Protein phosphorylation has been studied in Drosophila melanogaster 8.9 K cells following heat shock. By in vivo double labelling with [35S]-methionine and [32P]-orthophosphate, we observed that two proteins are newly phosphorylated among the 26,000-27,000 dalton heat-shock proteins group. These two proteins are also phosphorylated after ecdysterone treatment, albeit at a lower level. That this phosphorylation event is induced by two different treatments, i.e. ecdysterone, a key steroid hormone of development, and heat-shock, a cellular stress suggests a possible common pathway for those two events and an important function for the phosphorylated heat-shock proteins.     

Limited stress response in Streptococcus pneumoniae.

In Streptococcus pneumoniae, heat shock induces the synthesis of 65-, 73-, and 84-kDa proteins, and ethanol shock induces a 104-kDa protein. In this study, the 65-, 84-, and 104-kDa proteins were identified as members of the GroEL, ClpL and alcohol dehydrogenase families, respectively, and the general properties of the stress response of S. pneumoniae to several other stresses were characterized. However, several stresses which are known to induce stress responses in Escherichia coli and Bacillus subtilis failed to induce any high molecular weight heat-shock proteins (HSPs) such as GroEL and DnaK homologues. A minor temperature shift from 30 to 37 C triggered induction of the homologues of DnaK and GroEL of E. coli. These features may provide a foundation for evaluating the role of heat-shock proteins relative to the physiology and pathogenesis of pneumococcus.     

Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides.

A new and rapid purification procedure has been developed for the mammalian 70,000-dalton (70-kDa) heat-shock (or stress) proteins. Both the constitutive 73-kDa protein and the stress-induced 72-kDa protein have been purified by a two-step procedure employing DE52 ion-exchange chromatography followed by affinity chromatography on ATP-agarose. The two proteins, present in approximately equal amounts in either the 12,000 X g supernatant or pellet of hypotonically lysed heat-shock-treated HeLa cells, were found to copurify in relatively homogenous form. The purified proteins were covalently labeled with the fluorescent dye tetramethylrhodamine isothiocyanate, and the fluorescently labeled proteins were introduced back into living rat embryo fibroblasts via microinjection. The microinjected cells maintained at 37 degrees C showed only diffuse nuclear and cytoplasmic fluorescence. After heat-shock treatment of the cells, fluorescence was observed throughout the nucleus and more prominently within the nucleolus. This result is consistent with our earlier indirect immunofluorescence studies which showed a nuclear and nucleolar distribution of the endogenous 72-kDa stress protein in heat-shock-treated mammalian cells. The result also indicates that, for at least the 72-kDa protein, (i) the protein has been purified in apparently "native" form and (ii) its nucleolar distribution is stress dependent.     

Purification and characterization of the 16-kDa heat-shock-responsive protein from the thermophilic cyanobacterium Synechococcus vulcanus, which is an alpha-crystallin-related, small heat shock protein.

A 16-kDa protein, one of the major proteins that accumulates upon heat-shock treatment in the thermophilic cyanobacterium Synechococcus vulcanus, was purified to apparent homogeneity. The N-terminal and internal amino acid sequences of the protein exhibited a homology to the alpha-crystallin-related, small heat shock proteins from other organisms. The protein was designated HspA. Size-exclusion chromatography and nondenaturing gel electrophoresis demonstrated that HspA formed a large homo-oligomer consisting of 24 subunits. It prevented the aggregation of porcine malic dehydrogenase at 45 degrees C and 50 degrees C and citrate synthase at 50 degrees C. The activity of the malic dehydrogenase, however, was not protected under these heat-shock conditions or reactivated after a shift in temperature from 45 or 50 degrees C to 21 degrees C. HspA was able to enhance the refolding of chemically denatured rabbit muscle lactate dehydrogenase in an ATP-independent manner. A homologue to the 16-kDa protein was also found to be induced upon heat-shock treatment in the mesophilic cyanobacterium Synechocystis sp. PCC 6803.     

Lack of heat-shock response in preovulatory mouse oocytes

The response to heat (hs response) of preovulatory mouse oocytes was compared with that of mouse granulosa cells and characterized in regard to in vitro resumption of meiosis, amino acid incorporation into total protein, and qualitative analysis of protein synthesized before and after the shock. Granulosa cells displayed a hs response typical of other mammalian systems. When incubated at 43 degrees C for 20-40 min, these cells maintained a normal level of amino acid incorporation into total protein, responded to stress by new synthesis of 33- and 68-kDa heat-shock proteins (hsps), and enhanced synthesis of 70-kDa heat-shock cognate protein (hsc70) and of 89- and 110-kDa hsps. In contrast to granulosa cells, preovulatory mouse oocytes were very sensitive to hyperthermia. Incubation at 43 degrees C for 20-40 min strongly inhibited oocyte resumption of meiosis and protein synthesis and did not induce a new or enhanced synthesis of hsps. Unstressed preovulatory mouse oocytes constitutively synthesized 70- and 89-kDa polypeptides resembling hsc70 and hsp89 of granulosa cells.     

The heat-shock response in Drosophila KC 161 cells. mRNA competition is the main explanation for reduction of normal protein synthesis

The effect of heat shock on protein synthesis in the Drosophila melanogaster KC 161 tissue culture cell line was examined with a view to investigating the mechanism underlying the acute reduction in normal cellular protein synthesis typical of heat-shocked Drosophila cells. However, at 36-37 degrees C, the optimum temperature for induction of the 70-kDa heat-shock protein, this cell line did not show such a response. The synthesis of a very limited number of proteins was abruptly turned off following heat shock in the presence or absence of actinomycin, but the rate of synthesis of the majority of normal cellular proteins declined slowly over a three-hour period. Incubation of heat-shocked cells in hypertonic media increased the relative proportion of protein synthesis directed towards heat-shock proteins (as opposed to normal cellular proteins). Incubation with low concentrations of cycloheximide had the converse effect and resulted in a preferential increase in the size of polysomes translating normal cellular mRNAs, greater than the increase in size of polysomes synthesising heat-shock proteins. Heat shock also resulted in some mRNAs being almost completely displaced from polysomes into the postribosomal supernatant. These observations suggest that competition between normal cellular mRNAs and increasing amounts of heat-shock mRNAs with a higher affinity for the translation machinery was the main explanation for the gradual reduction in the synthesis of normal cellular proteins, although a slight reduction in overall translation initiation rates cannot be excluded as a subsidiary cause. The results demonstrate that the acute reduction in normal cellular protein synthesis seen in other Drosophila cell lines is not an integral and necessary feature of the heat-shock response in this organism, which makes it unlikely that the mechanism of this acute shut-off is intimately connected with the mechanism of induction of heat-shock mRNAs.     

Elevated levels of stress proteins associated with bacterial symbiosis in Amoeba proteus and soybean root nodule cells

Obligatory bacterial endosymbionts of Amoeba proteus and symbiotic Bradyrhizobium japonicum bacteroids in soybean-root nodules contained large amounts of 67-kDa and 65-kDa proteins, respectively, antigenically related to groEL of E. coli and the 58-kDa heat-shock protein of Tetrahymena. Monoclonal antibodies against the 67-kDa protein recognized groEL analogs from several different organisms. The quantity of the stress protein in symbiotic B. japonicum bacteroids was augmented seven times that in the free-living counterparts. The increase in these proteins in endosymbionts, as determined by immunoblot techniques, indicated that intracellular symbiosis is a stress condition even when the symbiotic relationship is considered to be mutually beneficial. Mitochondria and chloroplasts may also be under a stressed condition like endosymbionts in view of the presence of heat-shock proteins in these cell organelles.