Exogenous heat shock cognate protein Hsc 70 prevents axotomy-induced death of spinal sensory neurons

The objective of this study was to evaluate the individual and combined effects of Nacystelyn (NAL) and rhDNase in vitro on the rheological properties of cystic fibrosis (CF) sputum. Sputum samples were collected from 11 CF patients and subjected to the following protocols: 1) negative control sample without any treatment; 2) positive control sample incubated with 0.02 ml of normal saline; 3) incubation of CF sputum with 0.02 mL DNase (25 micrograms/mL in normal saline) at 37 degrees C to achieve 2.5 micrograms/g final sputum concentration (approximately 100 nM); 4) incubation of CF sputum with 0.02 mL NAL (30.9 micrograms/mL in normal saline) at 37 degrees C to achieve 3.09 micrograms/g final sputum concentration (10 microM); and 5) combination of protocols 3 and 4 with half the concentration of each drug. The samples in protocols 2 through 5 were incubated for 30 minutes at 37 degrees C. For each protocol, spinnability by filancemeter and viscoelasticity (log G*) by magnetic microrheometer were measured at baseline and 30 minutes. Treatment of the sputum with rhDNase alone or NAL alone decreased spinnability more than control treatment with saline. Combining NAL with rhDNase at half the concentration of each drug significantly decreased spinnability more than either treatment by itself. There were no significant changes in log G* or the derivative parameters, mucociliary clearability index (MCI) and cough clearability index (CCI). The enhanced reduction in sputum spinnability by the combination of NAL and rhDNase indicates additative effects between these two mucolytic treatments. These results suggest that combined treatment with rhDNase and NAL should be considered as a potential therapy for CF patients.     

The hydroxyl of threonine 13 of the bovine 70-kDa heat shock cognate protein is essential for transducing the ATP-induced conformational change

The mechanism by which ATP binding transduces a conformational change in 70-kDa heat shock proteins that results in release of bound peptides remains obscure. Wei and Hendershot demonstrated that mutating Thr37 of hamster BiP to glycine impeded the ATP-induced conformational change, as monitored by proteolysis [(1995) J. Biol. Chem. 270, 26670-26676]. We have mutated the equivalent resitude of the bovine heat shock cognate protein (Hsc70), Thr13, to serine, valine, and glycine. Solution small-angle X-ray scattering experiments on a 60-kDa fragment of Hsc70 show that ATP binding induces a conformational change in the T13S mutant but not the T13V or T13G mutants. The kinetics of ATP-induced tryptophan fluorescence intensity changes in the 60-kDa proteins is biphasic for the T13S mutant but monophasic for T13V or T13G, consistent with a conformational change following initial ATP binding in the T13S mutant but not the other two. Crystallographic structures of the ATPase fragments of the T13S and T13G mutants at 1.7 A resolution show that the mutations do not disrupt the ATP binding site and that the serine hydroxyl mimics the threonine hydroxyl in the wild-type structure. We conclude that the hydroxyl of Thr13 is essential for coupling ATP binding to a conformational change in Hsc70. Molecular modeling suggests this may result from the threonine hydroxyl hydrogen-bonding to a gamma-phosphate oxygen of ATP, thereby inducing a structural shift within the ATPase domain that couples to its interactions with the peptide binding domain.     

Binding of heptapeptides or unfolded proteins to the chimeric C-terminal domains of 70-kDa heat shock cognate protein

Seventy-kDa heat shock cognate protein (hsc70) and its homologs in bacteria, yeast and vertebrates are known to form complexes with S-carboxymethyl-alpha-lactalbumin (CMLA), an unfolded protein; and, this activity has been attributed to its C-terminal 30-kDa domain. Herein, we show that hsc70s isolated from the seeds of mung bean and peas, however, are not effective in complexing with CMLA, and that the 30-kDa domain of Arabidopsis hsc70 (At30) cannot form stable complexes with CMLA either. Moreover, chimeric 30-kDa domains, either composed of rat 18-kDa and Arabidopsis 10-kDa subdomains (R18At10) or with Arabidopsis 18-kDa and rat 10-kDa subdomains (At18R10), were prepared and tested for their ability to complex with CMLA or a heptapeptide FYQLALT. At18R10 cannot complex with both CMLA and FYQLALT. On the other hand, R18At10 is capable of forming complexes with FYQLALT at a level similar to that of the rat 30-kDa domain (R30). R18At10 also forms complexes with CMLA, but the amount of the R18At10/CMLA complexes is much less than that of R30/CMLA. The results imply that the 18-kDa subdomain dictates the binding specificity for heptapeptide, and that the C-terminal 10-kDa subdomain may also provide some selection or restriction for unfolded proteins to form complexes with hsc70.     

Localization of the gene encoding the human heat shock cognate protein, HSP73, to chromosome 11

The heat shock cognate protein HSP73 (or HSC70) is a member of the HSP70 multigene family. This protein has several functions, including binding to nascent polypeptides to facilitate correct folding and the uncoating of clathrin-coated vesicles. Analysis of somatic cell hybrids by two-dimensional protein gel electrophoresis revealed the presence of a 73-kDa protein in two hybrids containing human chromosomes 5, 6, 9, and 11 in common. Using Western blot analysis, we demonstrate that this protein is a member of the HSP70 family and, by Southern blot analysis, that the HSP73 gene is located on human chromosome 11. Fluorescence in situ hybridization further localized HSP73 to the region 11q23.3-q25. This region is involved in a number of genetic rearrangements and is associated with several well-characterized tumours.     

Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells

The EphA3 receptor tyrosine kinase has been implicated in guiding the axons of retinal ganglion cells as they extend in the optic tectum. A repulsive mechanism involving opposing gradients of the EphA3 receptor on retinal axons and its ligands, ephrin-A2 and ephrin-A5, in the tectum influences topographic mapping of the retinotectal projection. To investigate the overall role of the Eph family in patterning of the visual system, we have used in situ hybridization to localize nine Eph receptors in the chicken retina and optic tectum at Embryonic Day 8. Three of the receptors examined correspond to the novel chicken homologs of EphA2, EphA6, and EphA7. Unexpectedly, we found that many Eph receptors are expressed not only in retinal ganglion cells, but also in tectal cells, In particular, EphA3 mRNA is prominently expressed in the anterior tectum, with a pattern reciprocal to that of ephrin-A2 and ephrin-A5. Similarly, ephrin-A5 is expressed not only in tectal cells but also in the nasal retina, with a pattern reciprocal to that of its receptor EphA3 and partially overlapping with that of its other receptor EphA4. Consistent with the even distribution of EphA4 and the polarized distribution of EphA4 ligands in the retina, probing EphA4 immunoprecipitates from different sectors of the retina with anti-phosphotyrosine antibodies revealed spatial differences in receptor phosphorylation. These complex patterns of expression and tyrosine phosphorylation suggest that Eph receptors and ephrins contribute to establishing topography of retinal axons through multiple mechanisms, in addition to playing a role in intraretinal and intratectal organization.     

Destabilization of peptide binding and interdomain communication by an E543K mutation in the bovine 70-kDa heat shock cognate protein, a molecular chaperone

We have compared 70-kDa heat shock cognate protein (Hsc70) isolated from bovine brain with recombinant wild type protein and mutant E543K protein (previously studied as wild type in our laboratory). Wild type bovine and recombinant protein differ by posttranslational modification of lysine 561 but interact similarly with a short peptide (fluorescein-labeled FYQLALT) and with denatured staphylococcal nuclease-(Delta135-149). Mutation E543K results in 4. 5-fold faster release of peptide and lower stability of complexes with staphylococcal nuclease-(Delta135-149). ATP hydrolysis rates of the wild type proteins are enhanced 6-10-fold by the addition of peptide. The E543K mutant has a peptide-stimulated hydrolytic rate similar to that of wild type protein but a higher unstimulated rate, yielding a mere 2-fold enhancement. All three versions of Hsc70 possess similar ATP-dependent conformational shifts, and all show potassium ion dependence. These data support the following model: (i) in the presence of K+, Mg2+, and ATP, the peptide binding domain inhibits the ATPase; (ii) binding of peptide relieves this inhibition; and (iii) the E543K mutation significantly attenuates the inhibition by the peptide binding domain and destabilizes Hsc70-peptide complexes.     

Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis

Insights have emerged concerning insulin function during development, from the finding that apoptosis during chicken embryo neurulation is prevented by prepancreatic (pro)insulin. While characterizing the molecules involved in this survival effect of insulin, we found insulin-dependent regulation of the molecular chaperone heat shock cognate 70 kDa (Hsc70), whose cloning in chicken is reported here. This chaperone, generally considered constitutively expressed, showed regulation of its mRNA and protein levels in unstressed embryos during early development. More important, Hsc70 levels were found to depend on endogenous (pro)insulin, as shown by using antisense oligodeoxynucleotides against (pro)insulin mRNA in cultured neurulating embryos. Further, in the cultured embryos, apoptosis affected mainly cells with the lowest level of Hsc70, as shown by simultaneous Hsc70 immunostaining and terminal deoxynucleotidyltransferase-mediated UTP nick end labeling. These results argue in favor of Hsc70 involvement, modulated by embryonic (pro)insulin, in the prevention of apoptosis during early development and suggest a role for a molecular chaperone in normal embryogenesis.     

Thermotolerance and heat shock protein (HSP 70) in vascular endothelial cells

Recently, it was determined that the endothelial cells of blood vessel play a very important physiological role in the regulation of blood coagulation and selective permeability. To study the thermotolerance of vascular endothelial cells, human umbilical vein endothelial cells (HUVEC) were heated at 40, 43, 45 or 50 degrees C for various lengths of time with or without preheating at 40 or 43 degrees C for 30 min. The cell viability (CV) of HUVEC decreased gradually according to heating time. However, the CV of preheated HUVEC decreased slightly or not at all. Heat shock protein (HSP) in HUVEC heated at 37, 40, 43, or 45 degrees C was examined by immunoblotting. A new HSP 70 band was detected in HUVEC by heating at 40, 43 or 45 degrees C. HUVEC revealed thermotolerance with induction of HSP by heat stress.     

The degradation of apolipoprotein B100 is mediated by the ubiquitin-proteasome pathway and involves heat shock protein 70

Apolipoprotein B (apoB) is the major protein component of atherogenic lipoproteins of hepatic origin. In HepG2 cells, the standard cell culture model of human hepatic lipoprotein metabolism, there is a limited availability of core lipids in the endoplasmic reticulum for association with nascent apoB. Under these conditions, apoB is partially translocated, interacts with cytosolic Hsp70, and undergoes rapid degradation. We show that increasing the expression of Hsp70 in HepG2 cells promotes apoB degradation. In addition, apoB is polyubiquitinated and its degradation both normally and after Hsp70 induction is blocked by inhibitors of the proteasome. The apoB that accumulates after proteasome inhibition is endoplasmic reticulum-associated and can be assembled into lipoproteins and secreted if new lipid synthesis is stimulated. Thus, apoB is the first example of a wild-type mammalian protein whose secretion is regulated by degradation in the cytosol via the ubiquitin-proteasome pathway. Furthermore, targeting of this secretory protein to the proteasome is regulated by the molecular chaperone Hsp70 and the availability of apoB's lipid-ligands.     

Hop as an adaptor in the heat shock protein 70 (Hsp70) and hsp90 chaperone machinery

Hop, an abundant and conserved protein of unresolved function, binds concomitantly with heat shock protein 70 (Hsp70) and Hsp90, participates with heat shock proteins at an intermediate stage of progesterone receptor assembly, and is required for efficient assembly of mature receptor complexes in vitro. A largely untested hypothesis is that Hop functions as an adaptor that targets Hsp90- to Hsp70-substrate complexes; if true, then loss of either Hsp70 binding or Hsp90 binding by Hop should equally disrupt its ability to promote assembly of mature receptor complexes. To generate Hop mutants that selectively disrupt heat shock protein interactions, highly conserved amino acids in the previously mapped Hsp70 and Hsp90 binding domains of Hop and in a conserved C-terminal domain were targeted for small substitutions and deletions. In co-precipitation assays, these mutants displayed selective loss of association with heat shock proteins. In assays using Hop-depleted rabbit reticulocyte lysate for the cell-free assembly of receptor complexes, none of the Hop mutants inhibited Hsp70 binding to receptor, but all mutants were defective in supporting Hsp90-receptor interactions. Thus, Hop has a novel role in the chaperone machinery as an adaptor that can integrate Hsp70 and Hsp90 interactions.     

Expression of foreign gene in mycobacterium regulated by human Mycobacterium tuberculosis heat shock protein 70 promoter

PURPOSE: Phagocytosis is a major mechanism of defense against bacterial infections. The ingestion of bacteria by phagocytes involves a variety of cell membrane recognition structures and, among them, immunoglobulin receptors. The aim of this study was to test the phagocytic activity of granulocytes and monocytes of intensive care unit (ICU) patients, and to evaluate the effects of intravenous polyvalent immunoglobulins (IVIG) used as adjunct treatment of nosocomial pneumonia on some phagocyte membrane receptors of these patients. MATERIALS AND METHODS: The phagocytic activity of granulocytes and monocytes of 41 mechanically ventilated patients with nosocomial bacterial pneumonia was studied during the acute phase of infection. These ICU patients were compared with 21 hospitalized, noninfected volunteer patients hospitalized in a medical ward. Peripheral blood granulocytes and monocytes were studied. Of the 41 ICU patients, after randomization, 21 received IVIG at a dose of 1 g/kg for 3 days. The 41 ICU patients were compared with the 21 non-ICU, noninfected hospitalized controls. The 21 ICU patients who received 3 days of IVIG were also compared with the 20 ICU patients not receiving IVIG. Cells were tested in standard immunoglobulin-free medium (fetal calf serum) and in the presence of patients' serum. Blood granulocytes and monocytes were purified and separately exposed to three types of particles: antibody-coated erythrocytes (to test immunoglobulin receptors), opsonized zymosan (to test C3 receptors), and glutaraldehyde-treated erythrocytes (to test lectinlike or other nonspecific binding sites). Phagocytosis and superoxide anion production (oxidative burst) were measured. RESULTS: Granulocytes of ICU patients compared with those of non-ICU, noninfected patients exhibited a substantial decrease of zymosan ingestion (P < .05), whereas phagocytosis of other particles was normal. Monocytes from the ICU patients, compared with those of the non-ICU, noninfected patients, displayed an unselective overall decrease of phagocytic ability for the three particle types (P < .05). The phagocytosis activity of the three membrane receptor species of blood monocytes and granulocytes of ICU patients was not significantly modified by the IVIG infusion. For both monocytes and granulocytes, no significant improvement was observed in the fraction of cells that ingested at least one foreign particle and the mean number of particles per cell having phagocytized at least one foreign particle. Granulocyte and monocyte functions were also tested by the production of reduced ferricytochrome and no significant improvement in the oxidative burst was observed after infusion of IVIG. CONCLUSION: Infected ICU patients display a deficiency of phagocytosis membrane receptors of blood granulocytes and monocytes. The addition of IVIG to standard therapy does not improve the phagocytic activity of ICU patients with nosocomial pneumonia.     

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of alpha-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.     

Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers

1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals.     

Characteristic expression of 105-kDa heat shock protein (HSP105) in various tissues of nonstressed and heat-stressed rats

BACKGROUND: Transient lower esophageal sphincter relaxations (TLESRs) are the major mechanism permitting gastroesophageal reflux (GER). Little information is available on how anti-reflux surgery affects reflux mechanisms, especially TLESRs. We evaluated the effects of partial fundoplication (Belsey Mark IV) on reflux mechanisms. METHODS: Sixteen patients were prospectively studied before and after Belsey Mark-IV operation by endoscopy, 24-h esophageal pH-metry, and simultaneous recording of pH and lower esophageal sphincter (LES) characteristics by sleeve manometry. RESULTS: The operation was successful in 14 of 16 patients (87%). Fasting and postprandial reflux decreased significantly (P < 0.01) after the operation. Partial fundoplication significantly (P < 0.05) decreased the number of TLESRs per hour in the fasting and postprandial period from 3.2+/-0.4 and 5.6+/-0.5 to 1.7+/-0.3 and 2.8+/-0.4, respectively. The percentage of TLESRs associated with reflux also decreased significantly (P < 0.05). Basal LES pressure increased from 14.7+/-2.1 mmHg to 17.9+/-2.6 mmHg (not significant). CONCLUSIONS: Partial fundoplication controls GER through a reduction in the number of TLESRs and by decreasing the number of relaxations associated with reflux.     

Expression of the heat shock protein HSP27 in human ovarian cancer

The relationship of the heat shock protein HSP27 in ovarian cancer to several biological and clinical parameters was investigated in a series of primary tumors and cell lines. Analysis of 72 primary tumors (54 malignant, 5 borderline, and 13 benign neoplasms) indicated that malignant tumors expressed higher HSP27 concentrations than benign tumors (median values, 0.56 versus 0.25 ng/microgram cytosolic protein; P = 0.032). Tumors from patients with advanced stage (stages II, III, or IV) disease contained significantly higher HSP27 concentrations than tumors from stage I patients (P = 0.018), and an HSP27 content >2.0 ng/microgram cytosolic protein was associated with reduced survival (P = 0.03). Tumors that had demonstrated progressive growth after chemotherapy had a significantly higher HSP27 content than tumors that were static or responsive (P = 0.022). These data indicate that HSP27 is associated with more aggressive malignant ovarian disease and with inherent resistance to chemotherapy. Concentrations of HSP27 were also correlated with indicators of estrogen sensitivity. Therefore, the HSP27 concentration correlated with the estrogen receptor (all tumors, P = 0.0014; malignant tumors only, P = 0.047) but not with the progesterone receptor concentration. Analysis of ovarian cancer cell lines in vitro and in vivo indicated that the HSP27 content was higher in cell lines that were estrogen receptor rich and whose growth was modulated by estrogen as compared with those that were not. Additionally, two estrogen receptor-rich ovarian carcinoma lines demonstrated a small but significant decrease in HSP27 levels in response to 17beta-estradiol in culture. These results suggest that HSP27 may help identify tumors responsive to estrogens.     

Identification and characterization of a heat shock protein 70 family member in channel catfish (Ictalurus punctatus)

The primary objective of this study was to evaluate the electrophysiologic effects of large-dose propofol, used as the sole anesthetic in patients with epilepsy. Nine patients with medically intractable complex partial epilepsy undergoing a three-stage approach to the surgical management of epilepsy were recruited. State I involved placement of the intracranial electrode array, while Stage II consisted of extraoperative localization of the seizure focus. The patients were studied during induction of anesthesia for Stage III (removal of electrodes and resection of seizure focus). Unpremedicated patients were induced with a propofol infusion (0.5 mg.kg-1.min-1) until one of the following occurred: 1) electrical seizure activity, 2) burst suppression, or 3) total dose of 10 mg/mg. Electrocorticography (ECoG) was recorded continuously during this period. Two patients were excluded from the study after experiencing delayed awakening after the Stage I procedure. Both had received propofol along with other anesthetics. No ECoG evidence of seizure activity was detected in the seven patients completing the study. Burst suppression was attained in six patients using a mean dose of 5.7 mg/kg +/- 2.6. We conclude that large dose propofol alone does not trigger electrical epileptiform activity on the ECoG of seizure patients.     

The different expression of proteins recognized by monoclonal anti-heat shock protein 70 (hsp70) antibody in human colonic diseases

Analysis of the expression of hsp70 (70 kDa heat-shock proteins) in normal and pathological tissues might prove their potential diagnostic and prognostic values. In the present study, we combined high-resolution two-dimensional polyacrylamide gel electrophoresis with immunoblotting to study hsp70 expression in normal, preneoplastic and neoplastic colonic mucosa. By monoclonal anti-hsp70 antibody, recognizing both the constitutive and inducible forms of hsp70, we have detected six charge isoforms localized in the pH 5.1-5.3 range and in the molecular mass range of 68-69 kDa in normal colonic mucosa. Immunostaining of hsp70 in polypous and malignant tissues revealed qualitative as well as quantitative changes in the expression of more acidic isoforms of hsp70 in comparison with normal tissue. Furthermore, the different basic isoforms of hsp70 were detected in chronically inflamed colonic mucosa from patients suffering from ulcerative colitis (UC) or Crohn's disease (CD). Besides the standard hsp70 protein pattern, additional proteins with molecular masses of about 39, 40, 74 and 75 kDa were variably immunostained in normal and pathological specimens. These observations suggest that hsp70 expression may be closely linked to disease etiology and/or pathophysiology.     

The cellular inhibitor of the PKR protein kinase, P58(IPK), is an influenza virus-activated co-chaperone that modulates heat shock protein 70 activity

P58(IPK), a member of the tetratricopeptide repeat and J-domain protein families, was first recognized for its ability to inhibit the double-stranded RNA-activated protein kinase, PKR. PKR is part of the interferon-induced host defense against viral infection, and down-regulates translation initiation via phosphorylation of eukaryotic initiation factor 2 on the alpha-subunit. P58(IPK) is activated in response to infection by influenza virus, and inhibits PKR through direct protein-protein interaction. Previously, we demonstrated that the molecular chaperone heat shock protein 40 (hsp40) was a negative regulator of P58(IPK). We could now report that influenza virus activates the P58(IPK) pathway by promoting the dissociation of hsp40 from P58(IPK) during infection. We also found that the P58(IPK)-hsp40 association was disrupted during recovery from heat shock, which suggested a regulatory role for P58(IPK) in the absence of virus infection. The PKR pathway is even more complex as we show in this report that the molecular chaperone, hsp/Hsc70, was a component of a trimeric complex with hsp40 and P58(IPK). Moreover, like other J-domain proteins, P58(IPK) stimulated the ATPase activity of Hsc70. Taken together, our data suggest that P58(IPK) is a co-chaperone, possibly directing hsp/Hsc70 to refold, and thus inhibit kinase function.