Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The acyltransferase components (E2) from the family of 2-oxo acid dehydrogenase multienzyme complexes form large protein scaffolds, to which multiple copies of peripheral enzymes bind tightly but non-covalently. Sixty copies of the E2 polypeptide from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus assemble to form a pentagonal dodecahedral scaffold with icosahedral symmetry. This protein scaffold can be modified to present foreign peptides and proteins on its surface. We show that it is possible to display two epitopes (MAL1 and MAL2) from the circumsporozoite CS proteins of Plasmodium falciparum and Plasmodium berghei, respectively, and a green fluorescent protein (EGFP), on the E2 surface. Immunization with an E2 scaffold displaying the MAL1 epitope elicited MAL1-specific antibodies in rabbits. EGFP (25 kDa) displayed as an N-terminal fusion in each of the 60 copies of the E2 chain folded into its active form, as judged by its fluorescence and detection in localized foci in Escherichia coli cells in vivo. Simultaneous display of a hexahistidine affinity tag, the MAL1 epitope and the green fluorescent protein, all on the same E2 scaffold, could be achieved by reversible denaturation and reassembly of mixtures of appropriately modified E2 chains. This new methodology offers several important advantages over other current display technologies, not least in the size of insert that can be accommodated and the multiplicity of display that can be achieved.
J Mol Biol 2001 Jan 12
PMID:Multiple display of peptides and proteins on a macromolecular scaffold derived from a multienzyme complex. 1112 4

One cause of congenital lactic acidosis is a mutation in the E1 alpha-subunit of the pyruvate dehydrogenase multienzyme complex. Little is known about the consequences of these mutations at the enzymatic level. Here we study the A199T mutation by expressing the protein in Escherichia coli. The specific activity is 25% of normal and the K(m) for pyruvate is elevated by 10-fold. Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations.
Mol Genet Metab 2001 Mar
PMID:Congenital lactic acidosis: evaluation of the properties of the a199t natural variant of human pyruvate dehydrogenase e1alpha by in vitro mutation. 1124 35

Most eukaryotes perform the oxidative decarboxylation of pyruvate in mitochondria using pyruvate dehydrogenase (PDH). Eukaryotes that lack mitochondria also lack PDH, using instead the O(2)-sensitive enzyme pyruvate : ferredoxin oxidoreductase (PFO), which is localized either in the cytosol or in hydrogenosomes. The facultatively anaerobic mitochondria of the photosynthetic protist Euglena gracilis constitute a hitherto unique exception in that these mitochondria oxidize pyruvate with the O(2)-sensitive enzyme pyruvate : NADP oxidoreductase (PNO). Cloning and analysis of Euglena PNO revealed that the cDNA encodes a mitochondrial transit peptide followed by an N-terminal PFO domain that is fused to a C-terminal NADPH-cytochrome P450 reductase (CPR) domain. Two independent 5.8-kb full-size cDNAs for Euglena mitochondrial PNO were isolated; the gene was expressed in cultures supplied with 2% CO(2) in air and with 2% CO(2) in N(2). The apicomplexan Cryptosporidium parvum was also shown to encode and express the same PFO-CPR fusion, except that, unlike E. gracilis, no mitochondrial transit peptide for C. parvum PNO was found. Recombination-derived remnants of PNO are conserved in the genomes of Saccharomyces cerevisiae and Schizosaccharomyces pombe as proteins involved in sulfite reduction. Notably, Trypanosoma brucei was found to encode homologs of both PFO and all four PDH subunits. Gene organization and phylogeny revealed that eukaryotic nuclear genes for mitochondrial, hydrogenosomal, and cytosolic PFO trace to a single eubacterial acquisition. These findings suggest a common ancestry of PFO in amitochondriate protists with Euglena mitochondrial PNO and Cryptosporidium PNO. They are also consistent with the view that eukaryotic PFO domains are biochemical relics inherited from a facultatively anaerobic, eubacterial ancestor of mitochondria and hydrogenosomes.
Mol Biol Evol 2001 May
PMID:Pyruvate : NADP+ oxidoreductase from the mitochondrion of Euglena gracilis and from the apicomplexan Cryptosporidium parvum: a biochemical relic linking pyruvate metabolism in mitochondriate and amitochondriate protists. 1131 55

Nitrogen-fixing Sinorhizobium meliloti cells depend upon dicarboxylic acids as carbon and energy sources. The metabolism of these intermediate compounds of the trichloroacetic acid cycle is dependent upon the availability of acetyl-coenzyme A (CoA). In bacteroids, the combined activities of malic enzymes and pyruvate dehydrogenase (PDH) have been proposed to be responsible for the anaplerotic synthesis of acetyl-CoA. We obtained a S. meliloti mutant strain, PD3, in which a Tn5 insertion led to a significant decrease in the overall PDH activity. The genetic characterization of this mutant revealed that the transposon is located at the 3' end of a gene (ada) encoding a putative arylesterase. The mutant PD3 is deficient in nitrogen fixation, which strengthens the physiological importance of PDH activity in the symbiosis of S. meliloti with alfalfa plants.
Mol Plant Microbe Interact 2001 Jun
PMID:The disruption of a gene encoding a putative arylesterase impairs pyruvate dehydrogenase complex activity and nitrogen fixation in Sinorhizobium meliloti. 1138 77

We have previously shown that infection with Plasmodium yoelii malaria or injection of extracts from malaria-parasitized red cells induces hypoglycemia in normal mice and normalizes the hyperglycemia in mice made moderately diabetic with streptozotocin. Inositol phosphoglycans (IPGs) are released outside cells by hydrolysis of membrane-bound glycosylphosphatidylinositols (GPIs), and act as second messengers mediating insulin action. The C57BL/Ks-db/db and C57BL/6J-ob/ob mice offer good models for studies on human obesity and Type 2 diabetes. In the present study, we show that a single iv injection of IPG-A or IPG-P extracted from P. yoelii significantly (P < 0.02) lowers the blood glucose in STZ-diabetic, db/db, and in ob/ob mice for at least 4--6 h. Using rat white adipocytes, IPG-P increased lipogenesis by 20--30% in the presence and absence of maximal concentrations of insulin (10(-8) M) (P < 0.01) and stimulated pyruvate dehydrogenase (PDH) phosphatase in a dose-related manner. Both IPG-A and IPG-P inhibited c-AMP-dependent protein kinase (PKA) in a dose-related manner. Compositional analysis of IPGs after 24 h hydrolysis revealed the presence of myo-inositol, phosphorus, galactosamine, glucosamine, and glucose in both IPG-A and IPG-P. However, hydrolysis of IPGs for 4 h highlighted differences between IPG-A and IPG-P. There are some functional similarities between P. yoelii IPGs and those previously described for mammalian liver. However, this is the first report of the hypoglycemic effect of IPGs in murine models of Type 2 diabetes. We suggest that IPGs isolated from P. yoelii, when fully characterized, may provide structural information for the synthesis of new drugs for the management of diabetes mellitus.
Mol Genet Metab 2001 Jul
PMID:Reversal of type 2 diabetes in mice by products of malaria parasites. II. Role of inositol phosphoglycans (IPGs). 1146 Nov 92

The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids. The activity of this mitochondrial complex is regulated by the continuous operation of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) reactions. The resulting interconversion cycle determines the fraction of active (nonphosphorylated) pyruvate dehydrogenase (E1) component. Tissue-specific and metabolic state-specific control is achieved by the selective expression and distinct regulatory properties of at least four PDK isozymes and two PDP isozymes. The PDK isoforms are members of a family of serine kinases that are not structurally related to cytoplasmic Ser/Thr/Tyr kinases. The catalytic subunits of the PDP isoforms are Mg2+-dependent members of the phosphatase 2C family that has binuclear metal-binding sites within the active site. The dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex. One or more of their three lipoyl domains (two in E2) selectively bind each PDK and PDP1. These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes. When fatty acids are the preferred source of acetyl-CoA and NADH, feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group. PDC activity is increased in Ca2+-sensitive tissues by elevating PDP1 activity via the Ca2+-dependent binding of PDP1 to a lipoyl domain of E2. During starvation, the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms. Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes.
Prog Nucleic Acid Res Mol Biol 2001
PMID:Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms. 1164 66

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the alpha subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1(flox8) allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1(flox8) females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1(Deltaex8). The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1(Deltaex8) allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1(Deltaex8) allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.
Mol Genet Metab 2001 Nov
PMID:Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic development. 1170 58

This review summarizes the recent developments on the regulation of human pyruvate dehydrogenase complex (PDC) by site-specific phosphorylation by four kinases. Mutagenic analysis of the three phosphorylation sites of human pyruvate dehydrogenase (E1) showed the site-independent mechanism of phosphorylation as well as site-independent dephosphorylation of the three phosphorylation sites and the importance of each phosphorylation site for the inactivation of E1. Both the negative charge and size of the group introduced at site 1 were involved in human E1 inactivation. Mechanism of inactivation of E1 was suggested to be site-specific. Phosphorylation of site 1 affected E1 interaction with the lipoyl domain of dihydrolipoamide acetyltransferase, whereas phosphorylation site 3 appeared to be closer to the thiamine pyrophosphate (TPP)-binding region affecting coenzyme interaction with human E1. Four isoenzymes of pyruvate dehydrogenase kinase (PDK) showed different specificity for the three phosphorylation sites of E1. All four PDKs phosphorylated sites 1 and 2 in PDC with different rates, and only PDK1 phosphorylated site 3. PDK2 was maximally stimulated by the reduction/acetylation of the lipoyl groups of E2. Presence of the multiple phosphorylation sites and isoenzymes of PDK is important for the tissue-specific regulation of PDC under different physiological conditions.
Exp Mol Med 2001 Dec 31
PMID:Regulation of mammalian pyruvate dehydrogenase complex by phosphorylation: complexity of multiple phosphorylation sites and kinases. 1179 79

To determine the feasibility of gene transfer to correct defects in the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDC), we constructed rAAV vectors that expressed PDH E1alpha, either alone or with a green fluorescent protein tag, from a hybrid cytomegalovirus (CMV) enhancer/chicken beta-actin (CB) promoter. These vectors were functional in vitro, as judged by increased expression of mRNA in vector-transduced deficient cell lines and correction of the biochemical defect in PDH activity in these cells. Approximately 30% of wild-type levels of PDH activity were restored under conditions with which only about 15% of cells were transduced. These same vectors were then used in vivo to transduce neurons within the rat striatum. Gene transfer, expression, and translocation into mitochondria were observed, without any obvious untoward effects. In vivo vector-mediated PDH expression persisted for at least 1 year after injection, indicating the stability of gene transfer. These studies provide the basis for future efforts to develop a recombinant AAV (rAAV)-based gene therapy approach for the correction of PDC deficiency.
Mol Ther 2002 Sep
PMID:Gene therapy for pyruvate dehydrogenase E1alpha deficiency using recombinant adeno-associated virus 2 (rAAV2) vectors. 1223 Nov 76

The interactions between pathogenic bacteria and extracellular matrix (ECM) components markedly influence the initiation and establishment of infection. We have identified two surface proteins of virulent Mycoplasma pneumoniae with molecular masses of 45 and 30 kDa that bind to the ECM constituent, fibronectin (Fn). These Fn-binding proteins (FnBPs) were purified to near homogeneity using Fn-coupled Sepharose 4B-affinity column chromatography, and amino acid sequence analysis of the 45 and the 30 kDa proteins identified them as elongation factor Tu (EF-Tu) and pyruvate dehydrogenase E1 beta subunit (PDH-B) respectively. The genes for EF-Tu and PDH-B were cloned, and the entire EF-Tu gene and NH2-terminus of PDH-B (NPDH (pyruvate dehydrogenase E1 beta subunit from amino acid 1-244)-B) gene were overexpressed in Escherichia coli. The recombinant proteins, rEF-Tu and rNPDH-B, were purified to homogeneity by His-tag affinity column chromatography and used to immunize rabbits. Purified rEF-Tu and rNPDH-B bound to Fn using a ligand immunoblot assay and ELISA. Immunogold electron microscopy with polyclonal antibodies reactive against rEF-Tu (antirEF-Tu) and rNPDH-B (antirNPDH-B) and whole cell radioimmunoprecipitation (WCRIP) revealed the surface location of these proteins. Adherence of viable M. pneumoniae to immobilized Fn was inhibited by antirEF-Tu and antirNPDH-B antisera in a dose-dependent and cumulative manner. These results demonstrate that M. pneumoniae EF-Tu and PDH-B, in addition to their major cytoplasmic biosynthetic and metabolic roles, can be surface translocated, which confers additional important biological functions.
Mol Microbiol 2002 Nov
PMID:Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae. 1242 10


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