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.     

Folding of the glucocorticoid receptor by the heat shock protein (hsp) 90-based chaperone machinery. The role of p23 is to stabilize receptor.hsp90 heterocomplexes formed by hsp90.p60.hsp70

In cytosols from animal and plant cells, the abundant heat shock protein hsp90 is associated with several proteins that act together to assemble steroid receptors into receptor.hsp90 heterocomplexes. We have reconstituted a minimal receptor.hsp90 assembly system containing four required components, hsp90, hsp70, p60, and p23 (Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and Pratt, W. B. (1996) J. Biol. Chem. 271, 12833-12839). We have shown that hsp90, p60, and hsp70 are sufficient for carrying out the folding change that converts the glucocorticoid receptor (GR) hormone binding domain (HBD) from a non-steroid binding to a steroid binding conformation, but to form stable GR.hsp90 heterocomplexes, p23 must also be present in the incubation mix (Dittmar, K. D., and Pratt, W. B. (1997) J. Biol. Chem. 272, 13047-13054). In this work, we show that addition of p23 to native GR.hsp90 heterocomplexes immunoadsorbed from L cell cytosol or to GR.hsp90 heterocomplexes prepared with the minimal (hsp90.p60.hsp70) assembly system inhibits both receptor heterocomplex disassembly and loss of steroid binding activity. p23 stabilizes the GR.hsp90 heterocomplex in a dynamic and ATP-independent manner. In contrast to hsp90 that is bound to the GR, free hsp90 binds p23 in an ATP-dependent manner, and hsp90 in the hsp90.p60.hsp70 heterocomplex is in a conformation that does not bind p23 at all. The effect of p23 in the minimal GR heterocomplex assembly system is to stabilize GR.hsp90 heterocomplexes once they are formed and it does not appear to affect the rate of heterocomplex assembly. Molybdate has the same ability as p23 to stabilize GR heterocomplexes with mammalian hsp90, but GR heterocomplexes with plant hsp90 are stabilized by p23 and not by molybdate. We propose that incubation of the GR with hsp90.p60.hsp70 forms a GR.hsp90 heterocomplex in which hsp90 is in an ATP-dependent conformation. The ATP-dependent conformation of hsp90 is required for the hormone binding domain to have a steroid binding site, and binding of p23 to that state of hsp90 stabilizes the GR.hsp90 heterocomplex to inactivation and disassembly.     

ATPase activity and molecular chaperone function of the stress70 proteins

The codons for the amino acid residues making up the proposed ATP-binding sites of the maize (Zea mays L.) endoplasmic reticulum and tomato (Lycopersicon esculentum) cytoplasmic Stress70 proteins were deleted from their respective cDNAs. The deletions had little effect on the predicted secondary structure characteristics of the encoded proteins. Both wild-type and mutant proteins were expressed in Escherichia coli and purified to electrophoretic homogeneity. The mutant recombinant proteins did not bind to immobilized ATP columns, had no detectable ATPase activity, and were unable to function in vitro as molecular chaperones. Additionally, the inability to bind ATP was associated with changes in the oligomerization state of the Stress70 proteins.     

Isolation and characterization of a DnaJ-like protein in rats: the C-terminal 10-kDa domain of hsc70 is not essential for stimulating the ATP-hydrolytic activity of hsc70 by a DnaJ-like protein

A DnaJ-like protein, RDJ1, was isolated from a rat brain cDNA library. The protein is predicted to have 397 amino acid residues and shares 99% identity to that of HDJ2, a human DnaJ-like protein. RDJ1 was also shown to rescue the temperature-sensitive lethality of a strain containing a mutated cytosolic DnaJ in yeast, ydj1-151. Fragments containing the J-domain of RDJ1 either with or without the G/F motif were expressed in Escherichia coli. The purified proteins stimulated the ATPase activity of hsc70 and of the 60-kDa N-terminal fragment of hsc70. These results imply that RDJ1 can interact with the N-terminal 60-kDa fragment of hsc70 to activate ATP hydrolysis by hsc70.     

Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated

OBJECTIVES: We sought to evaluate the immediate angiographic results and intermediate-term follow-up after percutaneous treatment of left main coronary stenoses in the new device era. BACKGROUND: Historically, balloon angioplasty of left main coronary stenoses has been associated with high procedural morbidity and poor long-term results. It is not clear whether new devices are more effective in this anatomic setting. METHODS: Between July 1993 and July 1995, we performed initial left main coronary interventions on 46 patients (mean age 67 +/- 12 years, 26% women). Quantitative angiography was available for 42 of 46 interventions, and clinical follow-up was obtained for all patients at 1 month, 6 months and 1 year after initial revascularization. RESULTS: Most interventions (42 of 46) were performed in patients with "protected" coronary stenoses to the left coronary system owing to the presence of one or more patent left main coronary grafts. Seventy-seven percent of screened patients were deemed unsuitable for repeat coronary artery bypass surgery. Procedures performed included stenting in 73% of patients (alone in 30% and after rotational atherectomy in 43%), rotational atherectomy in 58% (alone in 15% and before stenting in 43%), directional atherectomy in 4% and angioplasty alone in 7%. Initial procedural success was achieved in all interventions, with no deaths, myocardial infarctions (creatine kinase, MB fraction > 50 IU/liter) or emergent bypass surgery. Follow-up data to date (median duration 9 months, range 6 to 19) demonstrate a 98% overall survival rate and a 6-month event-free survival rate of 78% (six target vessel revascularizations [TVRs], four non-TVRs). CONCLUSIONS: Treatment of protected left main coronary artery stenoses can be accomplished safely and effectively with new device technology. Intermediate-term follow-up demonstrates an acceptably low rate of death, myocardial infarction or repeat revascularization at 6 months and 1 year.     

Plasma leptin is directly related to body adiposity in subjects with spinal cord injury

We addressed the relationship between plasma leptin and body mass index in 48 able-bodied male controls and 34 male subjects with spinal cord injury, as well as the association between plasma leptin with body fat by dual energy x-ray absorptiometry in those with spinal cord injury. In subjects with spinal cord injury, the effect of an oral glucose tolerance test and the relationship of the serum lipid profile with plasma leptin levels were determined. Body mass index was not significantly different between the spinal cord injury and control groups. Plasma leptin was significantly higher in the group with spinal cord injury than in the control group (12.7 +/- 1.7 vs. 7.6 +/- 0.9 ng/ml, p < 0.005). A linear relationship was found between plasma leptin and body mass index in both groups separately (spinal cord injury: r = 0.59, p < 0.0002; control: r = 0.67, p < 0.0001). In those with SCI, a polynomial relationship was evident between plasma leptin and percent fat (r = 0.82, p < 0.0001). After an oral glucose load, plasma insulin levels and serum glucose concentrations were not related to plasma leptin levels. Serum triglycerides were found to be weakly correlated with plasma leptin levels (r = 0.35, p < 0.05). The higher plasma leptin levels in the group with spinal cord injury compared with the control group was probably due to a relatively increased percentage of adiposity in those with spinal cord injury.     

Effect of herbimycin A on hsp30 and hsp70 heat shock protein gene expression in Xenopus cultured cells

Glucose intolerance and diabetes mellitus are both prevalent in patients with chronic liver diseases. We examined the efficacy and systemic safety of therapy with an alpha-glucosidase inhibitor, acarbose, in diabetes mellitus associated with chronic liver diseases. Twenty patients with chronic hepatitis or liver cirrhosis and overt diabetes mellitus received acarbose (taken orally) for 8 weeks. The initial dosage of acarbose was 50 mg three times daily, taken before meals; this was increased to 100 mg three times daily after 2 weeks. The mean fasting plasma glucose level was 173.7 +/- 18.6 mg/dl (mean +/- SE) at entry, and was significantly decreased to 132.9 +/- 7.5 mg/dl (P < 0.05) after 8 weeks of acarbose treatment. The improved glycemic control was reflected by a significant decrease in glycosylated hemoglobin (HbA1c) from 7.2 +/- 0.3% at entry to 6.3 +/- 0.2% (P < 0.05) after 8 weeks. Serum levels of both aspartate and alanine aminotransferases fluctuated during acarbose treatment, probably due to the natural course of chronic liver diseases, but the mean values had decreased after 8 weeks of treatment. Plasma ammonia levels increased, from 61.3 +/- 10.7 micrograms/dl to 71.1 +/- 9.6 micrograms/dl after 8 weeks of acarbose treatment but the increase was not significant. Clinically significant elevation of plasma ammonia concentration was seen in 2 cirrhotic patients (121 and 124 micrograms/dl); this was asymptomatic and gradually returned to the normal range despite continuous acarbose treatment in one patient, and was reversed after the withdrawal of acarbose with the concomitant administration of lactulose in the other patient. No other blood tests results, including albumin, cholinesterase, and prothrombin time, or lipid profile and nutritional status, in terms of rapid turnover proteins, prealbumin, retinol binding protein, and transferin, were altered throughout the study period. These results indicate that diabetes mellitus associated with chronic liver diseases may be safely and effectively treated with acarbose. However, clinicians must be aware of the possibility of hyperammonemia when they prescribe acarbose for patients with diabetes mellitus and advanced liver cirrhosis.     

Protein folding activity of Hsp70 is modified differentially by the hsp40 co-chaperones Sis1 and Ydj1

Specification of Hsp70 action in cellular protein metabolism may occur through the formation of specialized Hsp70:Hsp40 pairs. To test this model, we compared the ability of purified Sis1 and Ydj1 to regulate the ATPase and protein-folding activity of Hsp70 Ssa1 and Ssb1/2 proteins. Ydj1 and Sis1 could both functionally interact with Ssa1, but not the Ssb1/2 proteins, to refold luciferase. Interestingly, Ydj1:Ssa1 could promote up to four times more luciferase folding than Sis1:Ssa1. This functional difference was explored and could not be accounted for by differences in the ability of Sis1 and Ydj1 to regulate Ssa1 ATPase activity. Instead, differences in the chaperone function of Ydj1 and Sis1 were observed. Ydj1 was dramatically more effective than Sis1 at suppressing the thermally induced aggregation of luciferase. Paradoxically, Sis1 and Ydj1 could bind similar quantities of chemically denatured luciferase. The polypeptide binding domain of Sis1 was found to lie between residues 171-352 and correspond to its conserved carboxyl terminus. The conserved carboxyl terminus of Ydj1 is also known to participate in the binding of nonnative polypeptides. Thus, Ydj1 appears more efficient at assisting Ssa1 in folding luciferase because its contains a zinc finger-like region that is absent from Sis1. Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins that can specify Ssa1 action by generating Hsp70:Hsp40 pairs that exhibit different chaperone activities.     

Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10

The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins. In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed. We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10. The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60. The majority of the identified substrates are group I proteins. For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10. We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.     

Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70

Two different tetrapeptides, AlaTrpTrpPro and AlaIleIlePro, were inserted near the C-terminus of the protein ZZT0. The Trp-rich peptide unit strongly increased both the partitioning of ZZT0 into the polyethylene glycol (PEG)-rich phase in a PEG-potassium phosphate aqueous two-phase system and its retention on PEG and propyl hydrophobic interaction chromatographic columns with potassium phosphate as eluent. Both the partitioning and the retention increased with increasing number of Trp-rich peptide units inserted into ZZT0. Insertion of Ile-rich tetrapeptide units affected the partitioning and retention to a much lesser extent. Partition data also indicated a folding of inserted Trp tetrapeptides units, probably to minimize their water contact.     

The role of G-CSF in mature neutrophil function is not related to GM-CSF-type cell priming

Because of uncertainties regarding the comparability of granulocyte-macrophage and granulocyte colony-stimulating factors with regard to their effects on mature neutrophils (PMNs), we compared the actions of the two cytokines on reactive oxidant production and granular secretion by these cells. We found that chemiluminescence (CL) stimulated by formylmethionyl-leucyl-phenylalanine (fMLP) was not influenced by G-CSF (0.1-100 ng/ml), whereas GM-CSF priming (10 ng/ml) caused a nearly twofold increase in this PMN response. Moreover, the reactivity of PMNs treated with GM-CSF and G-CSF in combination was not different from that of PMNs treated with GM-CSF alone. GM-CSF (10 ng/ml) increased the rate of O2- production by 79%, caused a fivefold increase in fMLP-induced myeloperoxidase (MPO) secretion, and strongly enhanced CD11b expression. In contrast, G-CSF (50 ng/ml) only slightly increased O2- production (by 15%), and MPO secretion and CD11b expression remained unchanged. Both cytokines together gave results similar to those obtained with GM-CSF alone. In the presence of platelets (which by themselves enhanced PMN reactivity), the differences in the effects of the two cytokines persisted. We conclude that the priming effect of G-CSF on mature PMNs is negligible compared with that of GM-CSF. Our results are in conflict with previous reports of much more pronounced G-CSF effects but in accord with recent work showing the failure of this cytokine to induce a range of effects produced by GM-CSF. We therefore suggest that the primary role of G-CSF in mature PMN function is still unclear but may be related to the control of PMN distribution in view of the mobilizing and marginating effects of the cytokine in vivo.     

Oxidative susceptibility of low density lipoprotein subfractions is related to their ubiquinol-10 and alpha-tocopherol content

The conjugated polyene fatty acid parinaric acid (PnA) undergoes a stoichiometric loss in fluorescence upon oxidation and can be used to directly monitor peroxidative stress within lipid environments. We evaluated the course of potentially atherogenic oxidative changes in low density lipoproteins (LDL) by monitoring the oxidation of PnA following its incorporation into buoyant (p = 1.026-1.032 g/ml) and dense (p = 1.040-1.054 g/ml) LDL subfractions. Copper-induced oxidation of LDL-associated PnA exhibited an initial lag phase followed by an increased rate of loss until depletion. Increased PnA oxidation occurred immediately after the antioxidants ubiquinol-10 and alpha-tocopherol were consumed but before there were marked elevations in conjugated dienes. Despite differences in sensitivity to early oxidation events, PnA oxidation and conjugated diene lag times were correlated (r = 0.582; P = 0.03), and both indicated a greater susceptibility of dense than buoyant LDL in accordance with previous reports. The greater susceptibility of PnA in dense LDL was attributed to reduced levels of ubiquinol-10 and alpha-tocopherol, which were approximately 50% lower than in buoyant LDL (mol of antioxidant/mol of LDL) and together accounted for 80% of the variation in PnA oxidation lag times. These results suggest that PnA is a useful probe of LDL oxidative susceptibility and may be superior to conjugated dienes for monitoring the initial stages of LDL lipid peroxidation. Differences in oxidative susceptibility among LDL density subfractions are detected by the PnA assay and are due in large part to differences in their antioxidant content.     

Heat shock proteins hsp27 and hsp70: lack of correlation with response to tamoxifen and clinical course of disease in estrogen receptor-positive metastatic breast cancer (a Southwest Oncology Group Study)

In this study, we tested the hypothesis that heat shock proteins (hsps) 27 and 70 are associated with clinical resistance to tamoxifen. hsp27 is, like progesterone receptor, an estrogen-regulated protein. hsp70 is also of interest because of its interaction with estrogen receptors and because hsp70 is a component of the molecular chaperone machinery functioning in the assembly and trafficking of steroid receptors. In addition, hsps in general help protect cells against noxious stimuli and stress, and their expression has been linked to drug resistance. The study involved 205 tumors from estrogen receptor-positive tamoxifen-treated breast cancer patients with metastatic disease. All patients received daily tamoxifen as initial therapy for metastatic disease. The study began in 1982, and follow-up is now 9 years. hsp27 and hsp70 were detected by immunohistochemistry and scored according to the nuclear and/or cytoplasmic content. Expression of hsp27 or hsp70 was unrelated to estrogen receptor content, progesterone receptor content, menopausal status, age, and presence of visceral disease. Cytoplasmic and nuclear hsp27 positivities were weakly and inversely related to each other (P = 0.05). There was a significant association between cytoplasmic hsp27 and cytoplasmic hsp70 content (P < 0.001), as well as between nuclear hsp70 and nuclear hsp27 content (P = 0.001). Cytoplasmic and nuclear hsp70 were also associated (P = 0.02). However, increased hsp27 and hsp70 expression (nuclear or cytoplasmic) was not significantly associated with response to tamoxifen, time to treatment failure, or survival. Thus, this study clarifies the lack of clinical utility of hsp27 and hsp70 in predicting the response to tamoxifen in an estrogen receptor-positive breast cancer population.     

24-h blood pressure and autonomic function is related to albumin excretion within the normoalbuminuric range in IDDM patients

A retrospective review was performed to determine "crankshaft" prevalence in 86 immature patients who underwent posterior spinal fusion for idiopathic scoliosis. Tanner stage, chronologic age, bone age, and epiphyseal status were used as maturity indicators. Overall, 62 (72%) patients progressed < or = 10 degrees, 18 (21%) patients progressed 11-15 degrees, and six (7%) patients progressed > or = 16 degrees in the coronal plane. Tanner I patients with open triradiate cartilage had the highest rate of crankshaft occurrence; nine (75%) of 12 patients progressed >10 degrees (p < 0.05). Fifty-two percent of Tanner I, 26% of Tanner II, 11% of Tanner III, and no Tanner IV patients progressed >10 degrees (p < 0.05). Cobb angle increases of >10 degrees degrees occurred in 54% of patients with open triradiate cartilage (p < 0.05) and in 48% of patients with open capital femoral epiphyses (p < 0.05). Anterior and posterior spinal fusion should be considered in prepubertal (Tanner I) patients with open triradiate cartilage.