CbpA, a DnaJ homolog, is a DnaK co-chaperone, and its activity is modulated by CbpM

The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogs in Escherichia coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of Thermus thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. The E. coli YccD was isolated because of its potential functional relationship to CbpA. Purified YccD specifically inhibited both the co-chaperone activity and the DNA binding activity of CbpA, suggesting that YccD modulates the activity of CbpA. We named the product of the yccD gene CbpM for CbpA modulator.     

Functional analysis of CbpA, a DnaJ homolog and nucleoid-associated DNA-binding protein

DnaK/Hsp70 proteins are universally conserved ATP-dependent molecular chaperones that help proteins adopt and maintain their native conformations. DnaJ/Hsp40 and GrpE are co-chaperones that assist DnaK. CbpA is an Escherichia coli DnaJ homolog. It acts as a multicopy suppressor for dnaJ mutations and functions in vitro in combination with DnaK and GrpE in protein remodeling reactions. CbpA binds nonspecifically to DNA with preference for curved DNA and is a nucleoid-associated protein. The DNA binding and co-chaperone activities of CbpA are modulated by CbpM, a small protein that binds specifically to CbpA. To identify the regions of CbpA involved in the interaction of CbpA with CbpM and those involved in DNA binding, we constructed and characterized deletion and substitution mutants of CbpA. We discovered that CbpA interacted with CbpM through its N-terminal J-domain. We found that the region C-terminal to the J-domain was required for DNA binding. Moreover, we found that the CbpM interaction, DNA binding, and co-chaperone activities were separable; some mutants were proficient in some functions and defective in others.     

Structural Basis of the Regulation of the CbpA Co-chaperone by its Specific Modulator CbpM

CbpA, one of the Escherichia coli DnaJ homologues, acts as a co-chaperone in the DnaK chaperone system. Despite its extensive similarity in domain structure and function to DnaJ, CbpA has a unique and specific regulatory mechanism mediated through the small protein CbpM. Both CbpA and CbpM are highly conserved in bacteria. Earlier studies showed that CbpM interacts with the N-terminal J-domain of CbpA inhibiting its co-chaperone activity but the structural basis of this interaction is not known. Here, we have combined NMR spectroscopy, site-directed mutagenesis and surface plasmon resonance to characterize the CbpA/CbpM interaction at the molecular level. We have determined the solution structure of the CbpA J-domain and mapped the residues that are perturbed upon CbpM binding. The NMR data defined a broad region on helices α2 and α3 as involved in the interactions. Site-directed mutagenesis has been used to further delineate the CbpA J-domain/CbpM interface. We show that the binding sites of CbpM and DnaK on CbpA J-domain overlap, which suggests a competition between DnaK and CbpM for binding to CbpA as a mechanism for CbpA regulation. This study also provides the explanation for the specificity of CbpM for CbpA versus DnaJ, by identifying the key residues for differential binding.     

Regulation of Hsp70 function by a eukaryotic DnaJ homolog.

We report that a purified cytoplasmic Hsp70 homolog from Saccharomyces cerevisiae, Hsp70SSA1, exhibits a weak ATPase activity, which is stimulated by a purified eukaryotic dnaJp homolog (YDJ1p). Stable complex formation between Hsp70SSA1 and the permanently unfolded protein carboxymethylated alpha-lactalbumin (CMLA) was assayed by native gel electrophoresis. The affinity of Hsp70SSA1 for CMLA appeared to be regulated by YDJ1p. Significant reduction in both CMLA-Hsp70SSA1 complex formation and the release of CMLA pre-bound to Hsp70SSA1 was observed only in the presence of both YDJ1p and ATP. Thus, Hsp70SSA1 and YDJ1p interact functionally in the execution of Hsp70SSA1 chaperone activities in the eukaryotic cell.     

In vivo modulation of N-myristoyltransferase activity by orthovanadate

N-Myristoyltransferase (NMT) catalyses the transfer of myristate from myristoyl-CoA to the NH₂-terminal glycine residue of several proteins and are important in signal transduction. STZ-induced diabetes (an animal model for insulin-dependent diabetes mellitus, IDDM) resulted in a 2-fold increase in rat liver NMT activity as compared with control animals. In obese Zucker (fa/fa) rats (an animal model for non-insulin dependent diabetes mellitus, NIDDM) there was a4.7-fold lower liver particulate NMT activity as compared with the control lean rat livers. Administration of sodium orthovanadate to the diabetic rats normalised liver NMT activity. These results would indicate that the rat liver particulate N-myristoyltransferase activity appears to be inversely proportional to the level of plasma insulin, implicating insulin in the control of N-myristoylation.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - IDDM insulin-dependent diabetes mellitus - NIDDM non-insulin-dependent diabetes mellitus - NIP₇₁ 71 kDa N-myristoyltransferase inhibitor protein - NAF₄₅ 45 kDa N-myristoyltransferase activating factor     

MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import.

The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.     

The rpoD gene functions as a multicopy suppressor for mutations in the chaperones, CbpA, DnaJ and DnaK, in Escherichia coli

Abstract The CbpA protein is an analog of the DnaJ molecular chaperone of Escherichia coli . The dnaJ ⁻ cbpA ⁻ double-null mutant exhibits severe defects in cell growth, namely, a very narrow temperature range for growth. To gain insight into the functions of CbpA as well as DnaJ, we isolated a multicopy suppressor gene that permits this dnaJ ⁻ cbpA ⁻~ mutant to grow normally at low temperatures. The suppressor gene was identified as rpoD , the gene that encodes the major σ ⁷⁰. The biological implications of this finding are examined and discussed.     

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.     

In vitro and in vivo TNFalpha synthesis modulation by methylguanidine, an uremic catabolyte.

This study was performed in order to examine whether the uraemic toxin, methylguanidine (MG), can modulate tumor necrosis factor alpha (TNF alpha) release by activated macrophages. In this study we have evaluated the ability of MG to influence TNF alpha release in vitro, in Escherichia coli lypopolysaccharide- (LPS)-stimulated J774 cells preincubated overnight with MG, and in vivo in rats treated with MG before and after LPS challenge. Parallel experiments employing N(G)-nitro-L-arginine methyl esther (L-NAME) were also carried out for comparison. The effect of LPS (6 x 10(3) u/ml) on TNF alpha release by J774, following overnight incubation with MG or L-NAME (1 mM), was examined 3 hours after LPS challenge. LPS-stimulated J774 released 287.83+/-88 u/ml TNF alpha into the culture medium. MG (1 mM) significantly inhibited TNF alpha release by 73% (P<0.05). L-NAME (1 mM) significantly inhibited TNF alpha release too by 72.88% (P<0.05). The effect of MG and L-NAME have been also studied in vivo. Serum TNF alpha levels in LPS treated rats 2 h after LPS challenge were 88.33+/-31.7 u/ml as compared to the serum TNF alpha levels of control rats (undetectable). Treatment of rats with MG (30 mg/kg, i.p.) strongly and significantly reduced TNF alpha release (98.71% inhibition; with P<0.001); in the same experimental setting L-NAME (10 mg/kg, i.p.) also significantly reduced TNF alpha serum levels (76.47% inhibition; with P<0.01). These results could indicate that immune disfunction related to uremia may be related to the inhibitory capability of uremic catabolyte, MG, on TNF alpha synthesis and release.     

Modulation of chaperone activities of Hsp70 and Hsp70-2 by a mammalian DnaJ/Hsp40 homolog, DjA4

Type I DnaJs comprise one type of Hsp70 cochaperones. Previously, we showed that two type I DnaJ cochaperones, DjA1 (HSDJ/Hdj-2/Rdj-1/dj2) and DjA2 (cpr3/DNAJ3/Rdj-2/dj3), are important for mitochondrial protein import and luciferase refolding. Another type I DnaJ homolog, DjA4 (mmDjA4/dj4), is highly expressed in heart and testis, and the coexpression of Hsp70 and DjA4 protects against heat stress-induced cell death. Here, we have studied the chaperone functions of DjA4 by assaying the refolding of chemically or thermally denatured luciferase, suppression of luciferase aggregation, and the ATPase of Hsp70s, and compared these activities with those of DjA2. DjA4 stimulates the hydrolysis of ATP by Hsp70. DjA2, but not DjA4, together with Hsp70 caused denatured luciferase to refold efficiently. Together with Hsp70, both DjA2 and DjA4 are efficient in suppressing luciferase aggregation. bag-1 further stimulates ATP hydrolysis and protein refolding by Hsp70 plus DjA2 but not by Hsp70 plus DjA4. Hsp70-2, a testis-specific Hsp70 family member, behaves very similarly to Hsp70 in all these assays. Thus, Hsp70 and Hsp70-2 have similar activities in vitro, and DjA2 and DjA4 can function as partner cochaperones of Hsp70 and Hsp70-2. However, DjA4 is not functionally equivalent in modulating Hsp70s.     

Mutation of host DnaJ homolog inhibits brome mosaic virus negative-strand RNA synthesis

The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.     

Characterization and functional analysis of a heart-enriched DnaJ/ Hsp40 homolog dj4/DjA4

DnaJ homologs are cochaperones of the heat shock protein 70 (hsp70) family. Homologs dj1 (hsp40/hdj-1/ DjB1), dj2 (HSDJ/hdj-2/rdj-1/DjA1), and dj3 (cpr3/DNAJ3/HIRIP4/rdj2/DjA2) have been identified in the mammalian cytosol and characterized. In this paper we characterized newly found dj4 (DjA4) and compared it with other chaperones. The dj4 messenger ribonucleic acid (mRNA) and protein were expressed strongly in heart and testis, moderately in brain and ovary, and weakly in other tissues in mice. Dj4 constituted about 1% of the total protein in heart. Testis gave extraspecies of dj4 mRNA and protein in addition to those seen in other tissues. On subcellular fractionation of the mouse heart, dj4 was recovered mostly in the cytosol fraction. In immunocytochemical analysis of the H9c2 heart muscle cells, dj4 and heat shock cognate 70 (hsc70) colocalized in the cytoplasm under normal conditions, whereas they colocalized in the nucleus after heat shock. When H9c2 cells were differentiated by culturing for up to 28 days with a lowered serum concentration, dj4 was increased markedly, dj3 was increased moderately, and dj1 and dj2 were little changed. The homolog dj4 as well as hsp70, dj1, and dj2 were induced in H9c2 cells by heat treatment at 43 degrees C for 30 minutes, whereas hsc70 and dj3 were not induced. Heat pretreatment promoted survival of cells after severe heat shock at 47 degrees C for 90 minutes or 120 minutes. H9c2 cells overexpressing hsp70 were more resistant to severe heat shock, and a better survival was obtained when dj4 or dj2 was co-overexpressed with hsp70. Taking a high concentration of dj4 in heart into consideration, these results suggest that the hsc70/hsp70-dj4 chaperone pair protects the heart muscle cells from various stresses.     

In vivo modulation of platelet deposition on human atherosclerotic lesions by various antiaggregatory prostaglandins

The influence of intravenous infusions of various prostaglandins on in vivo platelet function was studied after labelling of autologous platelets with 100 mu ci 111 indium-oxinesulfate in patients with peripheral vascular disease stage II according to FONTAINE. PGI2 (5 ng/kg/min) provoked a significant decrease of platelet deposition and a prolongation in platelet half-life time (74 +/- 6 vs 68 +/- 5 hours). PGE1 (25 ng/kg/min) failed to influence platelet deposition, but prolonged significantly platelet half-life time (82 +/- 6 vs 76 +/- 8 hours). CG 4203 (25 ng/kg/min) decreased significantly platelet deposition and prolonged significantly platelet half-life time (73 +/- 10 vs 67 +/- 11 hours). Iloprost (1 and 2 ng/kg/min) reduced significantly platelet deposition without dose relation. Half-life time was increased significantly after therapy compared to placebo (1 ng: 76 +/- 7 vs 69 +/- 7; 2 ng: 73 +/- 9 vs 67 +/- 9 hours).     

In vivo postirradiation protection by a vitamin E analog, alpha-TMG

The water-soluble vitamin E derivative alpha-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with alpha-TMG against a range of whole-body lethal (8.5-12 Gy) and sublethal (1-5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.     

In vivo modulation of T cell responses and protective immunity by TCR antagonism during infection

Infectious agents are known to express altered peptide ligands that antagonize T cells in vitro; however, direct evidence of TCR antagonism during infection is still lacking, and its importance in the context of infection remains to be established. In this study, we used a murine model of infection with recombinant Listeria monocytogenes and addressed three issues that are critical for assessing the role of TCR antagonism in the modulation of the immune response. First, we demonstrated that the antagonist peptide efficiently inhibited the ability of the agonist to prime naive TCR-transgenic T cells in vivo. Second, we showed clonal memory T cells were antagonized during recall responses, resulting in loss of protective immunity. Lastly, we observed that even in the context of a polyclonal response, TCR antagonism greatly inhibits the agonist-specific response, leading to altered hierarchy of immunodominance and reduced T cell memory and protective immunity. These results provide direct evidence of clonal TCR antagonism of naive and memory CD8 T cells during infection and demonstrate the effect of TCR antagonism on protective immunity. Thus, agonist/antagonist interactions may play an important role in determining the immunodominance and repertoire of T cell targets, and evaluation of immune responses and vaccine strategies may require examination of not only agonists but also antagonists and their interactions during an infection.     

In vivo modulation by alpha 2-adrenoceptors of adrenal catecholamine release in the anaesthetized dog

In this study, the reversal of the potentiating effect of idazoxan, a selective alpha 2-antagonist, on adrenal catecholamine release elicited by splanchnic nerve stimulation in anaesthetized and vagotomized dogs, was investigated with the use of oxymetazoline, a selective alpha 2-agonist. Stimulation of the left splanchnic nerve (5.0-V pulses of 2 ms duration for 3 min at a frequency of 2 Hz) was applied before and 20 min after the i.v. injection of each drug. Blood samples were collected in the adrenal vein before and at the end of each stimulation. The results show that the release of catecholamines induced by electrical stimulation was potentiated by 50% after idazoxan injection (0.1 mg/kg). This enhanced response was significantly antagonized by the subsequent injection of oxymetazoline (2 micrograms/kg). The alpha 2-modulating effect appears to be related to the amount of catecholamines released during the stimulation, since by subgrouping of the data on the basis of the degree of potentiation by idazoxan, it was observed that this drug was more efficient when catecholamine release was higher during control stimulation. In contrast, the reversing effect of oxymetazoline was found to be more pronounced when catecholamine release was lower. These results thus suggest that the sensitivity of the alpha 2-adrenoceptor mechanism may depend upon the in situ concentration of adrenal catecholamine release during electrical stimulation and that the potentiating effect of alpha 2-blockade can be reversed by activation of those receptors by a selective alpha 2-agonist.