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We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42°C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.
Indomethacin, a potent anti-inflammatory drug, activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1), but this is insufficient to elevate heat shock gene expression. However, indomethacin pretreatment leads to a complete heat shock response at temperatures that are by themselves insufficient. Here, we showed that the heat-induced loss of enzymatic activity of a nuclear or a cytoplasmic luciferase expressed in murine cells was enhanced when cells had been pretreated with indomethacin. Additionally, in these cells the 70-kDa constitutive heat shock protein exhibited an enhanced aggregation in the presence of indomethacin. Similarly an increase in the aggregation of β-galactosidase was observed. These data suggest that indomethacin at moderate temperatures accelerates the presence of denatured proteins in the cell, thus lowering the temperature threshold for a heat shock response.
Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.
The complete dnaK operon of Listeria monocytogenes was isolated by chromosome walking using the previously cloned dnaK gene as a probe. Molecular analysis of the locus identified 6 genes in the order hrcA, grpE, dnaK, dnaJ, orf35, and orf29. Primer extension analysis revealed 3 transcription start sites—S1, S2, and S3—upstream of the hrcA, grpE, and dnaJ, respectively. The transcription from S1 was heat inducible. Analysis of the sequences revealed the consensus promoter sequences of gram-positive bacteria, P1 and P2 upstream of the hrcA and dnaJ, respectively. The hrcA gene and a regulatory sequence, designated CIRCE (controlling inverted repeat of chaperone expression), play a role in the regulation of expression of the dnaK locus in response to heat shock in several gram-positive bacteria. Their presence upstream of the dnaK locus in L monocytogenes suggested a similar regulatory mechanism for the transcription initiated at the promoter, P1. Northern blot analysis led to the detection of 4 mRNA species of 4.9 kb, 3.6 kb, 3.6 kb, and 1.2 kb; the first 2 species were heat inducible. The current results indicate that 4 distinct transcripts directed by 3 promoters are involved in the expression of the dnaK operon of L. monocytogenes.
We describe a reverse transcriptase–polymerase chain reaction method for the semiquantitative detection of mRNAs encoding the human heat shock proteins αB-crystallin, Hsp27, and Hsp60. The method involves the coamplification of cellular mRNA-derived cDNA with a dilution series of a competitor fragment (internal standard), using 1 primer pair common to both templates. Internal standards were based on cellular-derived cDNA engineered to be slightly smaller to differentiate between the target and the standard on electrophoretic separation. Initial cDNA quantitations can be corrected for possible variations during cDNA synthesis by standardizing to the levels of β-actin–encoding cDNA. We show that the coamplified templates accumulate in a parallel manner with the cellular-derived cDNA throughout both the exponential and the nonexponential phase of amplification. Furthermore, we illustrate the utility of this technique by quantifying increased expression of αB-crystallin, Hsp27, and Hsp60 mRNA in astroglioma cells on heat shock.
In this study, we have employed whole-mount, in situ hybridization to study the spatial pattern of hsc70 and hsp70 mRNA accumulation in normal and heat shocked embryos during Xenopus laevis development. Our findings revealed that hsc70 mRNA was constitutively present in a global fashion throughout the embryo and was not heat inducible. Accumulation of hsp70 mRNA, however, was detected only in heat shocked embryos. Furthermore, hsp70 mRNA accumulation was enriched in a tissue-specific manner in X laevis tailbud embryos within 15 minutes of a 33°C heat shock. Abundant levels of heat shock–induced hsp70 mRNA were detected in the head region, including the lens placode, the cement gland, and in the somitic region and proctodeum. Preferential heat-induced accumulation of hsp70 mRNA was first detected at a heat shock temperature of 30°C. Placement of embryos at 22°C after a 1-hour, 33°C heat shock resulted in decreased hsp70 mRNA with time, but the message persisted in selected tissues, including the lens placode and somites. Treatment of tailbud embryos with either sodium arsenite or zinc chloride induced a tissue-specific enrichment of hsp70 mRNA in the lens placode and somitic region. These studies reveal the complex nature of the heat shock response in different embryonic tissues and suggest the presence of regulatory mechanisms that lead to a stressor-induced, tissue-specific enrichment of hsp70 mRNA.
Induction of the protective heat shock proteins (Hsps), and of Hsp72 in particular, has been reported to be decreased in certain tissues from aged animals. To determine if both fast and slow skeletal muscles from aged animals demonstrate an altered ability to induce and accumulate Hsp72, adult (age, 6 months) and aged (age, 20 months) Fischer 344 rats were subjected to heat stress. At selected times (0, 1, 3, and 24 hours) after a 10-minute, 41°C heat stress, fast (white gastrocnemius [WG]) and slow (soleus) skeletal muscles were examined for either heat shock transcription factor (HSF) activation (trimerization and DNA-binding activity) or Hsp72 content using electrophoretic gel mobility shift assays and Western blotting, respectively. Immediately after heat stress, the level of HSF activation between aged and adult animals was similar for both muscles. HSF activation was undetectable at 1 and 3 hours after heat stress in all cases. Twenty-four hours after heat stress, Hsp72 content in the WG muscles from both aged and adult animals was significantly increased compared with unstressed, age-matched controls (P < 0.05). In contrast, perhaps because of their high constitutive Hsp72 levels, soleus muscles from both aged and adult animals did not demonstrate a significant increase in Hsp72 content after heat shock, but there was a trend toward increased levels. Hsp72 content in both the soleus and WG muscles demonstrated no significant differences between adult and aged animals in either the unstressed state (controls) or after heat shock. These results suggest that skeletal muscles from aged animals are capable of inducing the heat shock response and accumulating Hsp72.
The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of eukaryotic cells. Its chaperone function in folding nascent proteins seems to be restricted to a subset of proteins including major components of signal transduction pathways (eg, nuclear hormone receptors, transcription factors, and protein kinases). Improper function of these proteins can be induced by selective disruption of their complexes with Hsp90 using the benzoquinonoid ansamycin geldanamycin. In this study, we demonstrate that geldanamycin treatment blocks interleukin (IL)-2 secretion, IL-2 receptor expression, and proliferation of stimulated T-lymphocytes. Moreover, geldanamycin decreases the amount and phosphorylation of Lck and Raf-1 kinases and prevents activation of the extracellular signal regulated kinase (ERK)-2 kinase. Geldanamycin also disrupts the T-cell receptor–mediated activation of nuclear factor of activated T-cells (NF-AT). Treatment with geldanamycin, however, does not affect the activation of lysophosphatide acyltransferase, which is a plasma membrane enzyme coupled to the T-cell receptor after T-cell stimulation. Through demonstrating the selective inhibition of kinase-related T-lymphocyte responses by geldanamycin, our results emphasize the substantial role of Hsp90–kinase complexes in T-cell activation.
Chloroplast transit peptides have been proposed to function as substrates for Hsp70 molecular chaperones. Many models of chloroplast protein import depict Hsp70s as the translocation motors that drive protein import into the organelle, but to our knowledge, no direct evidence has demonstrated that transit peptides function either in vivo or in vitro as substrates for the chaperone. In this report, we demonstrate that DnaK binds SStp (the full-length transit peptide for the precursor to the small subunit of Rubisco) in vivo when fused to either glutathione-S-transferase (GST) or to an His6-S-peptide tag (His-S) via an ATP-dependent mechanism. Three independent biophysical and biochemical assays confirm the ability of DnaK and SStp to interact in vitro. The cochaperones, DnaJ and GrpE, were also associated with the DnaK/SStp complex. Therefore, both GST-SStp and His-S–SStp can be used as affinity-tagged substrates to study prokaryotic chaperone/transit peptide interactions as well as to provide a novel functional probe to study the dynamics of DnaK/DnaJ/GrpE interactions in vivo. The combination of these results provides the first experimental support for a transit peptide–dependent interaction between a chloroplast precursor and Hsp70. These results are discussed in light of a general mechanism for protein translocation into chloroplasts and mitochondria.