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PubMed2002

Oral tolerisation to pyruvate dehydrogenase complex as a potential therapy for primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is characterised by immune-mediated damage to the intra-hepatic biliary epithelial cells (BEC). Immuno-modulatory/suppressive therapy represents, therefore, alogical approach to treatment in this disease. Conventional immuno-suppressive and immuno-modulatory agents suffer from the breadth of their action and/or excessive side effects. Autoreactive responses to pyruvate dehydrogenase complex (PDC) have been extensively characterised in PBC, and implicated in target cell damage. The aim of the current study was to study the potential efficacy of an antigen specific approach (oral tolerisation with autoantigen) to modulation of anti-PDC immune responses characteristic of PBC, utilising a mouse model of PDC immuno-reactivity. Groups of SJL/J mice were orally dosed with PDC alone, dosed with carrier only (saline) but systemically sensitised with PDC in adjuvant, or orally dosed with PDC at high (5 mg) or low (0.01 mg) dose and systemically sensitised with PDC. Oral dosing with PDC in isolation had no adverse effects on the animals and did not prime anti-PDC responses at doses below 1 mg. Pre-dosing with PDC at both high and low doses was effective at skewing the phenotype of the T-cell response to PDC induced by subsequent sensitisation away from the disease associated Th-1 phenotype (IL-2 and IFN-gamma secreting) and towards a theoretically protective Th-2 phenotype (IL-4 secreting) in a majority of, but not all, animals. No augmentation of Th-1 response was seen in any animal. Although the effects on liver histology remain to established oral tolerisation with PDC holds promise as a novel, antigen specific approach to therapy in PBC.

Animals↗
PubMed1992

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Acetyltransferases↗
PubMed1990

Ontogeny of pyruvate dehydrogenase complex and key enzymes involved in glycolysis and tricarboxylic acid cycle in rabbit fetal lung, heart, and liver.

The metabolic pathways by which the glycogen is utilized by fetal tissues is not well established. In the present study the ontogeny of seven key enzymes involved in glycolysis and the tricarboxylic acid cycle has been established for rabbit fetal lung, heart, and liver. In the fetal lung the activities of phosphofructokinase, pyruvate kinase, lactic dehydrogenase, citrate synthase, and malate dehydrogenase increase from day 21 to 25. Thereafter the levels either drop to day 19 levels or do not change. The isocitrate dehydrogenase activity continues to increase from day 19 of gestation to maximum level on day 31 of gestation. In fetal heart the pattern of activity is similar, but in fetal liver most of the enzymes reach maximum levels earlier and, with the exception of pyruvate kinase, do not show a significant fall in activity near term. The pattern of development of pyruvate dehydrogenase complex is different; maximum activity is observed on day 27 in fetal lung and heart and on day 21 in fetal liver. These results indicate that all three fetal tissues can oxidize glucose. Also, the accumulation of glycogen, particularly in fetal lung, appears to ensure that at specific times during gestation adequate quantities of energy (ATP) and substrates, required for surfactant phospholipid synthesis, are available independent of maternal supply of glucose or during brief episodes of hypoxia.

Animals↗
PubMed1992

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain.

The lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli have three tandemly repeated lipoyl domains, although net deletions of one or two has no apparent effect on the activity of the purified complexes. Plasmids containing IPTG-inducible aceEF-lpd operons, which encode PDH complexes bearing from one to nine lipoyl domains per E2p chain (24-216 per complex), were constructed. They were all capable of restoring the nutritional lesion of a strain lacking PDH complex and they all expressed active sedimentable multienzyme complexes having a relatively normal range of subunit stoichiometries. The extra domains are presumed to protrude from the E2p core (24-mer) without significantly affecting the assembly of the E1p and E3 subunits on the respective edges and faces of the cubic core. However, the catalytic activities of the overproduced complexes containing four to nine lipoyl domains per E2p chain were lower than those with fewer lipoyl domains. This could be due to under-lipoylation of the domains participating in catalysis and interference from unlipoylated domains.

Acetyltransferases↗
PubMed2000

Probable identification of a membrane-associated repressor of Bacillus subtilis DNA replication as the E2 subunit of the pyruvate dehydrogenase complex.

Two Bacillus subtilis lysogenic libraries were probed by an antibody specific for a previously described membrane-associated inhibitor of B. subtilis DNA replication (J. Laffan and W. Firshein, Proc. Natl. Acad. Sci. USA 85:7452-7456, 1988). Three clones that reacted strongly with the antibody contained an entire open reading frame. Sequencing identified one of the clones (R1-2) as containing the E2 subunit of the pyruvate dehydrogenase complex, dihydrolipoamide acetyltransferase. An AT-rich sequence in the origin region was identified initially as the site to which extracts from the R1-2 clone were bound. This sequence was almost identical to one detected in Bacillus thuringiensis that also bound the E2 subunit but which was involved in activating the Cry1 protoxin gene of the organism, not in inhibiting DNA replication (T. Walter and A. Aronson, J. Biol. Chem., 274:7901-7906, 1999). However, the exact sequence was not as important in B. subtilis as the AT-rich core region. Binding would occur as long as most of the AT character of the core remained. Purified E2 protein obtained by use of PCR and an expression vector reacted strongly with antibody prepared against the repressor protein and the protein in the R1-2 clone, but its specificity for the AT-rich region was altered. The purified E2 protein was capable of inhibiting membrane-associated DNA replication in vitro, but anti-E2 antibody was variable in its ability to rescue repression when added to the assay.

Acetyltransferases↗
PubMed2002

Exercise training increases the activity of pyruvate dehydrogenase complex in skeletal muscle of diabetic rats.

The effects of diabetes and exercise training on the activity of pyruvate dehydrogenase (PDH) complex in skeletal muscle were examined in rats. Male Sprague-Dawley rats were divided into four groups as follows: non-diabetic sedentary, non-diabetic trained, diabetic sedentary, and diabetic trained groups. Diabetic rats were prepared by a bolus injection of intravenous streptozotocin (50 mg/kg body weight). Exercise training was performed by having rats run on a treadmill at a speed of 25 m/min for 45 min/day, 6 days/wk for 4 wks. Exercise training decreased serum concentrations of glucose and non-esterified fatty acid in diabetic rats. GLUT4 content in skeletal muscle in sedentary rats was significantly decreased by diabetes; however, exercise training significantly increased the GLUT4 content in diabetic rats. The total and actual activities and the proportion of actual activity of the PDH complex were decreased in diabetic sedentary rats. Exercise training did not affect the total activity of the PDH complex in non-diabetic rats, whereas it increased the total activity in diabetic rats to the same level as that in non-diabetic rats. In diabetic rats, exercise training tended to increase the proportion of actual activity of the PDH complex from 2.7 +/- 0.4% to 4.7 +/- 0.8%, although the proportion of actual activity in non-diabetic rats was decreased by exercise training. The present study suggests that exercise training may improve glucose metabolism in the skeletal muscle of streptozotocin-induced diabetic rats probably through the mechanisms of increasing both GLUT4 content and the activity of the PDH complex.

Animals↗
PubMed2002

Bacterial motif DNA as an adjuvant for the breakdown of immune self-tolerance to pyruvate dehydrogenase complex.

Bacterial DNA containing unmethylated CpG dinucleotide motifs is immunostimulatory to mammals, skewing CD4(+) T-cell responses toward the Th1 phenotype. Autoreactive T-cell responses seen in primary biliary cirrhosis (PBC) are typically of the Th1 phenotype, raising the possibility that bacterial DNA might play a role in the generation of pathologic autoimmunity. We therefore studied the effects of CpG motif-containing oligodeoxynucleotides (ODN) on responses to pyruvate dehydrogenase complex (PDC, the autoantigen in PBC) in a murine model. Sensitization of SJL/J mice with non-self-PDC has been shown to result in induction of autoreactive T-cell responses to PDC sharing characteristics with those seen in patients with PBC. Administration of CpG ODN to SJL/J mice at the time of sensitization with PDC resulted in a significant skewing of splenic T-cell response to self-PDC, with significant augmentation of the Th1 cytokine response (interleukin [IL] 2 and interferon [IFN] gamma) and reduction of the Th2 response (IL-4 and IL-10). In fact, CpG ODN seemed to be more effective at biasing the response phenotype and as effective at inducing liver histologic change as complete Freund's adjuvant (CFA), the standard adjuvant used for induction of Th1 responses in murine autoimmune and infectious immunity models. In conclusion, our findings raise the possibility that bacteria play a role in the development of autoimmunity (in PBC at least) through the potential of their DNA to shift the T-cell responses toward the phenotype associated with autoimmune damage. Moreover, this study suggests caution in the therapeutic use of CpG ODN as vaccine adjuvants.

Adjuvants, Immunologic↗
PubMed1999

Insulin activation of pyruvate dehydrogenase complex is enhanced by exercise training.

We studied the effects of exercise training on the activity of the pyruvate dehydrogenase (PDH) complex in rat gastrocnemius muscle (experiment 1) and the response of the complex to glucose and insulin infusion (euglycemic clamp) in trained and sedentary rats (experiment 2). In experiment 1, half of the rats were randomly allocated as sedentary animals and the other half were trained by voluntary running exercise for 8 weeks. The total activity of the PDH complex was not affected by exercise training, and the activity state (proportion of the active form) of the PDH complex was decreased from 15.0%+/-2.4% to 7.5%+/-1.1% by exercise training. The activity of 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase ([3-HADH] an enzyme in beta-oxidation) was significantly higher in trained versus sedentary rats. In experiment 2, sedentary and trained rats were starved for 24 hours before performing the euglycemic clamp. Glucose and insulin infusion was performed by a euglycemic clamp (insulin infusion rate, 6 mU/kg/min) for 90 minutes. The PDH complex was inactivated to less than 1% in both sedentary and trained rats after 24 hours of starvation. The glucose infusion rate (GIR) during the euglycemic clamp was higher in trained versus sedentary rats. The euglycemic clamp resulted in activation of the PDH complex in both sedentary and trained rats, but the response of the PDH complex to the euglycemic clamp was significantly higher in trained rats (5.8%+/-0.5%) than in sedentary rats (2.9%+/-0.5%). These results suggest that exercise training promotes fatty acid oxidation in association with suppression of glucose oxidation in skeletal muscle under resting conditions, but increases the rate of carbohydrate oxidation when glucose flux into muscle cells is stimulated by insulin.

Animals↗
PubMed2005

Oxidative formation of thiolesters in a model system of the pyruvate dehydrogenase complex.

In the presence of a catalytic amount of 3-butyl-4-methylthiazolium bromide, the reaction of benzaldehydes with azobenzene in dichloromethane containing octanethiol and Et(3)N gave the corresponding S-octyl thiobenzoates in good yields. The thiolesters were produced by trapping of the 2-benzoylthiazolium salts with the thiol, which were generated through the azobenzene oxidation of the active aldehydes. This is the first example for the thiolester formation mimicking the function of the pyruvate dehydrogenase complex. An electron-withdrawing substituent at the 4-position of benzaldehyde enhanced the reaction rate. The effect of benzaldehyde substituents on the reaction rate was examined quantitatively on the basis of kinetic measurements, leading to a nonlinear correlation of log(k(obs)) with Hammett's substituent constants (sigma). The origin of the nonlinear Hammett plot was interpreted in terms of a shift in the rate-determining step of the multistep reaction with change of the electronic nature of substituent. Further support for this assumption was given by the observation that the reaction constant (rho) of the Hammett plot for the azobenzene substituent effect on the oxidation rate of 4-bromobenzaldehyde was much smaller than that of 4-cyanobenzaldehyde.

Catalysis↗
PubMed1991

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.

Antibodies↗
PubMed2002

The E1beta and E2 subunits of the Bacillus subtilis pyruvate dehydrogenase complex are involved in regulation of sporulation.

The pdhABCD operon of Bacillus subtilis encodes the pyruvate decarboxylase (E1alpha and E1beta), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH). There are two promoters: one for the entire operon and an internal one in front of the pdhC gene. The latter may serve to ensure adequate quantities of the E2 and E3 subunits, which are needed in greater amounts than E1alpha and E1beta. Disruptions of the pdhB, pdhC, and pdhD genes were isolated, but attempts to construct a pdhA mutant were unsuccessful, suggesting that E1alpha is essential. The three mutants lacked PDH activity, were unable to grow on glucose and grew poorly in an enriched medium. The pdhB and pdhC mutants sporulated to only 5% of the wild-type level, whereas the pdhD mutant strain sporulated to 55% of the wild-type level. This difference indicated that the sporulation defect of the pdhB and pdhC mutant strains was due to a function(s) of these subunits independent of enzymatic activity. Growth, but not low sporulation, was enhanced by the addition of acetate, glutamate, succinate, and divalent cations. Results from the expression of various spo-lacZ fusions revealed that the pdhB mutant was defective in the late stages of engulfment or membrane fusion (stage II), whereas the pdhC mutant was blocked after the completion of engulfment (stage III). This analysis was confirmed by fluorescent membrane staining. The E1beta and E2 subunits which are present in the soluble fraction of sporulating cells appear to function independently of enzymatic activity as checkpoints for stage II-III of sporulation.

Bacillus subtilis↗
PubMed2004

Apoptosis as a mechanism for cell surface expression of the autoantigen pyruvate dehydrogenase complex.

A number of antigens implicated in the pathogenesis of autoimmune diseases including Sjogren's syndrome and systemic lupus erythematosus (SLE) are expressed aberrantly by apoptotic cells. It is also known that apoptogenic proteins are released from the mitochondrial intermembrane space at an early stage during the induction and development of apoptosis. Combination of this evidence led us to test the hypothesis that apoptotic mechanisms provide an explanation for the abnormal expression of the inner mitochondrial enzyme, pyruvate dehydrogenase complex (PDC), observed on the surface of some cells in patients with the autoimmune liver disease primary biliary cirrhosis (PBC). Using one murine and two human cell lines it was found that the induction of apoptosis led to early detection of PDC within the cytoplasm. However, cytochrome c oxidase subunit 4 (COX 4), which is also present on the inner surface of the inner mitochondrial membrane, remained within the mitochondria. Immunoreactive PDC was also detected on the outer surface of the intact plasma membrane of cells sampled after the induction of apoptosis. Serial release of PDC to the cytoplasm and then onto the external surface of the plasma membrane provides direct evidence that the antigen on the cell surface is of mitochondrial origin. Immunoreactivity specific for PDC is strongly implicated in the pathogenesis of PBC, but this autoantigen is normally concealed from the immune system by three membrane systems. Release of PDC onto the cell surface during apoptosis provides a possible route for recognition of this antigen by the immune system which could contribute to both afferent and efferent phases of the disease process.

Animals↗
PubMed1994

Effects of L-carnitine on the pyruvate dehydrogenase complex and carnitine palmitoyl transferase activities in muscle of endurance athletes.

The effects of L-carnitine on the pyruvate dehydrogenase (PDH) complex and carnitine palmitoyl transferase (CPT) were studied in muscle of 16 long-distance runners (LDR). These subjects received placebo or L-carnitine (2 g orally) during a 4-week period of training. Athletes receiving L-carnitine showed a dramatic increase (P < 0.001) in the PDH complex activities. By contrast, the levels of CPT, both 1 and 2, were unchanged. No significant changes were observed after placebo administration. We previously reported [Huertas R. et al., Biochem. Biophys. Res. Commun. 188 (1992) 102-107] that L-carnitine induces an increase in the activities of complexes I, III and IV of the respiratory chain in muscle of LDR. Taken together, our data suggest that the improvement in (maximal oxygen consumption) VO2max observed in LDR after L-carnitine administration is based on these biochemical findings.

Adult↗
PubMed1994

Promoter analysis of the PDA1 gene encoding the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The location and sequence of the PDA1 gene, encoding the E1 alpha subunit of the pyruvate dehydrogenase (PDH) complex from Saccharomyces cerevisiae, were determined. The PDA1 gene was located on a 6.2 kb fragment of chromosome V, approximately 18 kb centromere distal to RAD3. Consistent with this, the PDA1 gene was genetically mapped at 4 cM from RAD3. A part of the 6.2 kb fragment of chromosome V was sequenced. The nucleotide sequence contained the PDA1 open reading frame and the entire putative promoter. Computer analysis revealed a putative GCN4 binding motif in the PDA1 promoter. The presence of transcriptional elements was experimentally determined by deletion analysis. To this end, ExoIII deletions were constructed in the 5' to 3' direction of the PDA1 promoter and effects on transcription were determined by Northern analysis. Transcription was unaffected upon deletion to position -190 relative to the ATG start codon. Deletions from position -148 and beyond, however, reduced promoter activity at least 40-fold. Apparently the 42 bp between nucleotides -190 and -148 contain an element essential for transcription. Inactivation of the PDA1 promoter could not be attributed to deletions of a recognizable TATA element or any known yeast regulatory motifs. The possible role of the CCCTT sequence present in the 42 bp region and also in the promoters of the other genes encoding subunits of the PDH complex is discussed.

Amino Acid Sequence↗
PubMed1994

Expression in Escherichia coli of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro.

The E1 alpha and E1 beta subunits of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus were produced from two genes overexpressed separately in Escherichia coli. A functional E1 enzyme was generated from disrupted mixtures of cells containing the separately overexpressed E1 alpha and E1 beta genes. The purified E1 enzyme exhibited an apparent molecular mass of 150,000 Da, consistent with an alpha 2 beta 2 structure. The Km for pyruvate and kcat (30 degrees C) were found to be 0.9 +/- 0.2 microM and 0.47 +/- 0.03 s-1, respectively. The purified E1 alpha subunit existed as a monomer (42,000 Da), whereas the E1 beta subunit existed mainly (95%) in a tetrameric form (145,000 Da). Mixing equimolar amounts of the pure recombinant E1 alpha and E1 beta subunits in vitro generated a functional E1 enzyme with a molecular mass and an E1 activity similar to those of the E1(alpha 2 beta 2) enzyme purified from disrupted mixtures of cells containing individually expressed subunits. Mixing individual subunits in vitro with one of the subunits in excess resulted in complete assembly of the lesser subunit into the intact E1 (alpha 2 beta 2) enzyme. Thus, no chaperonin is needed in vitro to promote the assembly of the separate subunits to form the E1 component of the pyruvate dehydrogenase multienzyme complex of B. stearothermophilus.

Base Sequence↗
PubMed2006

Transgenic mice aberrantly expressing pyruvate dehydrogenase complex E2 component on biliary epithelial cells do not show primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disorder that specifically destroys biliary epithelial cells (BECs). In patients with PBC, the immunodominant pyruvate dehydrogenase complex E2 component (PDC-E2), identified as an antigen for disease-specific anti-mitochondrial antibody, is expressed aberrantly in the BEC cytoplasm. The present study focused on the pathophysiological role of aberrant PDC-E2 in the development of PBC. The BEC-specific cytokeratin-19 promoter and PDC-E2 gene were cloned from a mouse cDNA library. The constructed transgene was microinjected into fertilized eggs of mice, and the offspring were identified by Southern blotting and reverse transcriptase-polymerase chain reaction. The protein expression was confirmed by immunoprecipitation, immunoblotting and immunohistochemical staining. Five founder lines were identified as carrying the PDC-E2 gene, and one of these lines expressed PDC-E2 mRNA. The protein expression of exogenous PDC-E2 was detected in the liver. The transgenic mouse line showed diffuse expression of PDC-E2 in the BEC cytoplasm. Biochemical, serological and histological features of PBC were not detected. We established transgenic mice that constitutively express PDC-E2. The results indicated that aberrant PDC-E2 expression in the cytoplasm of BECs is not sufficient for the initiation of autoimmunity. Additional factors may be required to establish a model of PBC.

Alanine Transaminase↗