G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
pyruvate dehydrogenase (lipoamide) alpha 1
G00000468 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000021224 (Vega human gene)
ENSG00000131828 (Ensembl human gene)
5160 (Entrez Gene)
836 (G2Cdb plasticity & disease)
PDHA1 (GeneCards)
300502 (OMIM)
Marker Symbol
HGNC:8806 (HGNC)
Protein Sequence
P08559 (UniProt)

Literature (60)

Pubmed - other

  • AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells.

    Glushakova LG, Lisankie MJ, Eruslanov EB, Ojano-Dirain C, Zolotukhin I, Liu C, Srivastava A and Stacpoole PW

    Department of Medicine, Division of Endocrinology and Metabolism, College of Medicine, University of Florida, FL, USA.

    Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1alpha subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1alpha, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

    Funded by: NCRR NIH HHS: U54RR025208, UL1 RR029890, UL1 RR029890-01; NHLBI NIH HHS: R01 HL-07691, R01 HL076901, R01 HL076901-06; NIDDK NIH HHS: P01 DK-058327, P01 DK058327, P01 DK058327-060004

    Molecular genetics and metabolism 2009;98;3;289-99

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Pyruvate dehydrogenase deficiency: identification of a novel mutation in the PDHA1 gene which responds to amino acid supplementation.

    João Silva M, Pinheiro A, Eusébio F, Gaspar A, Tavares de Almeida I and Rivera I

    Unidade de Biologia Molecular e Biopatologia Experimental, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003, Lisboa, Portugal.

    Unlabelled: The pyruvate dehydrogenase complex (PDHc) is an intramitochondrial multienzyme system, which plays a key role in aerobic glucose metabolism by catalysing the oxidative decarboxylation of pyruvate to acetyl-CoA. Genetic defects in the PDHc lead to lactic acidemia and neurological abnormalities. In the majority of the cases, the defect appears to reside in the E(1)alpha subunit, the first catalytic component of the complex. The report is on a 6-year-old Portuguese boy with mild neurological involvement and low PDHc activity with absence of E1alpha on immunoblotting analysis. Molecular studies showed a novel and "de novo" mutation in the PDHA1 gene, R253G. Treatment with arginine aspartate showed complete clinical and biochemical recovery. We hypothesise that arginine aspartate acts as a chemical or pharmacological chaperone, and suggest amino acid supplementation as a possible therapy in PDHA1 mutations with mild phenotypes.

    Conclusion: our results encourage the use of amino acid supplementation to overcome the metabolic/biochemical changes induced by PDHA1 gene specific mutations associated with mild PDHc phenotypes.

    European journal of pediatrics 2009;168;1;17-22

  • Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.

    Boichard A, Venet L, Naas T, Boutron A, Chevret L, de Baulny HO, De Lonlay P, Legrand A, Nordman P and Brivet M

    Biochemistry laboratory, AP-HP hôpital de Bicêtre, Le Kremlin-Bicêtre, France.

    Background: Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts.

    One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5'ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5' splice site by site-directed mutagenesis.

    Conclusion: These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.

    Molecular genetics and metabolism 2008;93;3;323-30

  • Regulation of PDH in human arm and leg muscles at rest and during intense exercise.

    Kiilerich K, Birk JB, Damsgaard R, Wojtaszewski JF and Pilegaard H

    Copenhagen Muscle Research Center, August Krogh Bldg., University of Copenhagen, 2100 Copenhagen, Denmark. kkiilerich@aki.ku.dk

    To test the hypothesis that pyruvate dehydrogenase (PDH) is differentially regulated in specific human muscles, regulation of PDH was examined in triceps, deltoid, and vastus lateralis at rest and during intense exercise. To elicit considerable glycogen use, subjects performed 30 min of exhaustive arm cycling on two occasions and leg cycling exercise on a third day. Muscle biopsies were obtained from deltoid or triceps on the arm exercise days and from vastus lateralis on the leg cycling day. Resting PDH protein content and phosphorylation on PDH-E1 alpha sites 1 and 2 were higher (P < or = 0.05) in vastus lateralis than in triceps and deltoid as was the activity of oxidative enzymes. Net muscle glycogen utilization was similar in vastus lateralis and triceps ( approximately 50%) but less in deltoid (likely reflecting less recruitment of deltoid), while muscle lactate accumulation was approximately 55% higher (P < or = 0.05) in triceps than vastus lateralis. Exercise induced (P < or = 0.05) dephosphorylation of both PDH-E1 alpha site 1 and site 2 in all three muscles, but it was more pronounced at PDH-E1 alpha site 1 in triceps than in vastus lateralis (P < or = 0.05). The increase in activity of the active form of PDH (PDHa) after 10 min of exercise was more marked in vastus lateralis ( approximately 246%) than in triceps ( approximately 160%), but when it was related to total PDH-E1 alpha protein content, no difference was evident. In conclusion, PDH protein content seems to be related to metabolic enzyme profile, rather than myosin heavy chain composition, and less PDH capacity in triceps is a likely contributing factor to higher lactate accumulation in triceps than in vastus lateralis.

    American journal of physiology. Endocrinology and metabolism 2008;294;1;E36-42

  • The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity.

    Bradley NS, Heigenhauser GJ, Roy BD, Staples EM, Inglis JG, LeBlanc PJ and Peters SJ

    Faculty of Applied Health Sciences, Brock Univ., St. Catharines, Ontario, Canada.

    Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise, and its activity can be downregulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of the PDH complex (PDHa activity) at rest and at the onset of moderate-intensity exercise. University-aged male subjects (n = 7) underwent two fat-loading trials spaced at least 2 wk apart. Subjects consumed approximately 300 g saturated (SFA) or n-6 polyunsaturated fatty acid (PUFA) fat over the course of 5 h. Following this, participants cycled at 65% of their maximum oxygen uptake for 15 min. Muscle biopsies were taken before and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 +/- 0.07 to 0.54 +/- 0.19 mM over 5 h with SFA and from 0.11 +/- 0.04 to 0.35 +/- 0.13 mM with n-6 PUFA and were significantly lower throughout the n-6 PUFA trial. PDHa activity was unchanged following fat loading but increased at the onset of exercise in the SFA trial, from 1.18 +/- 0.27 to 2.16 +/- 0.37 mmol x min(-1) x kg wet wt(-1). This effect was negated in the n-6 PUFA trial (1.04 +/- 0.20 to 1.28 +/- 0.36 mmol x min(-1) x kg wet wt(-1)). PDH kinase was unchanged in both trials, suggesting that the attenuation of PDHa activity with n-6 PUFA was a result of changes in the concentrations of intramitochondrial effectors, potentially intramitochondrial NADH or Ca(2+). Our findings suggest that attenuated PDHa activity contributes to the preferential oxidation of n-6 PUFA during moderate-intensity exercise.

    Journal of applied physiology (Bethesda, Md. : 1985) 2008;104;1;1-9

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • PDH-E1alpha dephosphorylation and activation in human skeletal muscle during exercise: effect of intralipid infusion.

    Pilegaard H, Birk JB, Sacchetti M, Mourtzakis M, Hardie DG, Stewart G, Neufer PD, Saltin B, van Hall G and Wojtaszewski JF

    August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark. hpilegaard@aki.ku.dk

    To investigate pyruvate dehydrogenase (PDH)-E1alpha subunit phosphorylation and whether free fatty acids (FFAs) regulate PDH activity, seven subjects completed two trials: saline (control) and intralipid/heparin (intralipid). Each infusion trial consisted of a 4-h rest followed by a 3-h two-legged knee extensor exercise at moderate intensity. During the 4-h resting period, activity of PDH in the active form (PDHa) did not change in either trial, yet phosphorylation of PDH-E1alpha site 1 (PDH-P1) and site 2 (PDH-P2) was elevated in the intralipid compared with the control trial. PDHa activity increased during exercise similarly in the two trials. After 3 h of exercise, PDHa activity remained elevated in the intralipid trial but returned to resting levels in the control trial. Accordingly, in both trials PDH-P1 and PDH-P2 decreased during exercise, and the decrease was more marked during intralipid infusion. Phosphorylation had returned to resting levels at 3 h of exercise only in the control trial. Thus, an inverse association between PDH-E1alpha phosphorylation and PDHa activity exists. Short-term elevation in plasma FFA at rest increases PDH-E1alpha phosphorylation, but exercise overrules this effect of FFA on PDH-E1alpha phosphorylation leading to even greater dephosphorylation during exercise with intralipid infusion than with saline.

    Diabetes 2006;55;11;3020-7

  • Acute flaccid paralysis as initial symptom in 4 patients with novel E1alpha mutations of the pyruvate dehydrogenase complex.

    Strassburg HM, Koch J, Mayr J, Sperl W and Boltshauser E

    University Children's Hospital Würzburg, Germany.

    We report on 4 boys from 3 families presenting initially in infancy with an acute onset of flaccid tetraparesis and areflexia, resembling Guillain-Barré syndrome (GBS). However, the cerebrospinal fluid (CSF) protein was normal, while serum and CSF lactate were elevated. All patients had recurrent similar episodes, usually associated with infections. Brain MRI showed T (2) hyperintensities in the basal ganglia in two boys, in one of them at the first clinical presentation; the other one had a normal brain MRI during the first episode. A third boy had a normal MRI twice but an increased lactate peak in the basal ganglia in (1)H-MR spectroscopy. Motor nerve conduction velocities (NCV) were normal in all patients. Biochemical analyses of muscle tissue, performed in two patients, revealed a deficiency of the pyruvate dehydrogenase (PDH). Molecular genetic analysis of the X-chromosomal E1alpha subunit of PDH showed three new mutations in phylogenetically conserved areas of the protein: Glu358Lys in patient 1; Arg88Lys in patient 2 and 3 (brothers); and Leu216Ser in patient 4. In conclusion, children with "atypical GBS" should be evaluated for a mitochondrial disorder, including pyruvate dehydrogenase deficiency, even after a first episode.

    Neuropediatrics 2006;37;3;137-41

  • Females with PDHA1 gene mutations: a diagnostic challenge.

    Willemsen M, Rodenburg RJ, Teszas A, van den Heuvel L, Kosztolanyi G and Morava E

    Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 Nijmegen, HB, The Netherlands.

    Biochemical analysis was performed in muscle tissue and in fibroblasts of four unrelated females consecutively diagnosed with a 'de novo' point mutation in the PDHA1 gene. Pyruvate dehydrogenase E1 subunit deficiency was confirmed in the muscle sample of all patients, however, in three out of four cases the activity of the pyruvate dehydrogenase complex in fibroblasts showed a normal activity. A skewed inactivation was confirmed of the maternal X chromosome in fibroblasts in all children. Due to the possibility of a skewed X inactivation pattern enzyme measurements in fibroblasts are not always reliable for the diagnosis of a PDHc defect in females. Based on the overlapping features of PDHc deficiency with those of the disorders of the oxidative phosphorylation we suggest performing a fresh muscle biopsy for detailed biochemical analysis in females with a suspected pyruvate dehydrogenase deficiency, followed by molecular genetic analysis of the PDHA1 gene.

    Mitochondrion 2006;6;3;155-9

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex.

    Kato M, Chuang JL, Tso SC, Wynn RM and Chuang DT

    Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

    The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino-acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60-meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3-L2 complex with and without bound ADP or ATP. These structures disclose that the C-terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl-binding pocket in the N-terminal domain of the opposing subunit. The two swapped C-terminal tails promote conformational changes in active-site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP-bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p-augmented PDK3 activity.

    Funded by: NIDDK NIH HHS: DK 26758, DK 62308, R01 DK026758, R01 DK062306, R56 DK062306

    The EMBO journal 2005;24;10;1763-74

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components.

    Hiromasa Y, Fujisawa T, Aso Y and Roche TE

    Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.

    The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.

    Funded by: NIDDK NIH HHS: DK18320

    The Journal of biological chemistry 2004;279;8;6921-33

  • Epitope mapping on E1alpha subunit of pyruvate dehydrogenase complex with autoantibodies of patients with primary biliary cirrhosis.

    Mori T, Ono K, Hakozaki M and Kochi H

    Department of Biochemistry, Fukushima Medical University, Fukushima, Japan.

    Background: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The alpha subunit of pyruvate decarboxylase (E1alpha) of PDH is also recognized in some E2-reactive PBC sera, suggesting that the occurrence of autoimmunity against Elalpha is subsequent to that against E2. METHODS. To investigate the mechanism inducing autoimmunity against E1alpha, we surveyed immunoreactive sequences of E1alpha by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant.

    Results: The major determinants of E1alpha appeared to reside in its N-terminal region, apparently forming 'nested epitopes', and all E1alpha-reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1alpha, and many of them were out of the known functional domains (TPP-binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans.

    Conclusion: These observations suggest that determinant spreading might underlie the autoimmunity against Elalpha.

    Liver international : official journal of the International Association for the Study of the Liver 2003;23;5;355-62

  • Structural basis for flip-flop action of thiamin pyrophosphate-dependent enzymes revealed by human pyruvate dehydrogenase.

    Ciszak EM, Korotchkina LG, Dominiak PM, Sidhu S and Patel MS

    Biological and Physical Space Research Laboratory, National Aeronautics and Space Administration/Marshall Space Flight Center and Universities Space Research Association, Huntsville, Alabama 35812, USA. Ewa.M.Ciszak@nasa.gov

    The derivative of vitamin B1, thiamin pyrophosphate, is a cofactor of enzymes performing catalysis in pathways of energy production. In alpha2beta2-heterotetrameric human pyruvate dehydrogenase, this cofactor is used to cleave the Calpha-C(=O) bond of pyruvate followed by reductive acetyl transfer to lipoyl-dihydrolipoamide acetyltransferase. The dynamic nonequivalence of two, otherwise chemically equivalent, catalytic sites has not yet been understood. To understand the mechanism of action of this enzyme, we determined the crystal structure of the holo-form of human pyruvate dehydrogenase at 1.95-A resolution. We propose a model for the flip-flop action of this enzyme through a concerted approximately 2-A shuttle-like motion of its heterodimers. Similarity of thiamin pyrophosphate binding in human pyruvate dehydrogenase with functionally related enzymes suggests that this newly defined shuttle-like motion of domains is common to the family of thiamin pyrophosphate-dependent enzymes.

    Funded by: NIDDK NIH HHS: DK20478

    The Journal of biological chemistry 2003;278;23;21240-6

  • Recent advances in mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs.

    Sugden MC and Holness MJ

    Department of Diabetes and Metabolic Medicine, Division of General and Developmental Medicine, Bart's and the London, Queen Mary's School of Medicine and Dentistry, University of London, Mile End Road, London E1 4NS, United Kingdom. m.c.sugden@qmul.ac.uk

    The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate, linking glycolysis to the tricarboxylic acid cycle and fatty acid (FA) synthesis. Knowledge of the mechanisms that regulate PDC activity is important, because PDC inactivation is crucial for glucose conservation when glucose is scarce, whereas adequate PDC activity is required to allow both ATP and FA production from glucose. The mechanisms that control mammalian PDC activity include its phosphorylation (inactivation) by a family of pyruvate dehydrogenase kinases (PDKs 1-4) and its dephosphorylation (activation, reactivation) by the pyruvate dehydrogenase phosphate phosphatases (PDPs 1 and 2). Isoform-specific differences in kinetic parameters, regulation, and phosphorylation site specificity of the PDKs introduce variations in the regulation of PDC activity in differing endocrine and metabolic states. In this review, we summarize recent significant advances in our knowledge of the mechanisms regulating PDC with emphasis on the PDKs, in particular PDK4, whose expression is linked with sustained changes in tissue lipid handling and which may represent an attractive target for pharmacological interventions aimed at modulating whole body glucose, lipid, and lactate homeostasis in disease states.

    American journal of physiology. Endocrinology and metabolism 2003;284;5;E855-62

  • Site specificity of four pyruvate dehydrogenase kinase isoenzymes toward the three phosphorylation sites of human pyruvate dehydrogenase.

    Korotchkina LG and Patel MS

    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214, USA.

    Activity of the mammalian pyruvate dehydrogenase complex is regulated by phosphorylation-dephosphorylation of three specific serine residues (site 1, Ser-264; site 2, Ser-271; site 3, Ser-203) of the alpha subunit of the pyruvate dehydrogenase (E1) component. Phosphorylation is carried out by four pyruvate dehydrogenase kinase (PDK) isoenzymes. Specificity of the four mammalian PDKs toward the three phosphorylation sites of E1 was investigated using the recombinant E1 mutant proteins with only one functional phosphorylation site present. All four PDKs phosphorylated site 1 and site 2, however, with different rates in phosphate buffer (for site 1, PDK2 > PDK4 approximately PDK1 > PDK3; for site 2, PDK3 > PDK4 > PDK2 > PDK1). Site 3 was phosphorylated by PDK1 only. The maximum activation by dihydrolipoamide acetyltransferase was demonstrated by PDK3. In the free form, all PDKs phosphorylated site 1, and PDK4 had the highest activity toward site 2. The activity of the four PDKs was stimulated to a different extent by the reduction and acetylation state of the lipoyl moieties of dihydrolipoamide acetyltransferase with the maximum stimulation of PDK2. Substitution of the site 1 serine with glutamate, which mimics phosphorylation-dependent inactivation of E1, did not affect phosphorylation of site 2 by four PDKs and of site 3 by PDK1. Site specificity for phosphorylation of four PDKs with unique tissue distribution could contribute to the tissue-specific regulation of the pyruvate dehydrogenase complex in normal and pathophysiological states.

    Funded by: NIDDK NIH HHS: DK20478

    The Journal of biological chemistry 2001;276;40;37223-9

  • Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites.

    Kolobova E, Tuganova A, Boulatnikov I and Popov KM

    Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110-2499, USA.

    The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3>or=PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4>or=PDK3>PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

    Funded by: NIDDK NIH HHS: DK 56898; NIGMS NIH HHS: GM 51262, R01 GM051262, R01 GM051262-09

    The Biochemical journal 2001;358;Pt 1;69-77

  • X chromosome evidence for ancient human histories.

    Harris EE and Hey J

    Department of Genetics, Rutgers University, Nelson Biological Labs, 604 Allison Road, Piscataway, NJ 08854-8082, USA.

    Diverse African and non-African samples of the X-linked PDHA1 (pyruvate dehydrogenase E1 alpha subunit) locus revealed a fixed DNA sequence difference between the two sample groups. The age of onset of population subdivision appears to be about 200 thousand years ago. This predates the earliest modern human fossils, suggesting the transformation to modern humans occurred in a subdivided population. The base of the PDHA1 gene tree is relatively ancient, with an estimated age of 1.86 million years, a late Pliocene time associated with early species of Homo. PDHA1 revealed very low variation among non-Africans, but in other respects the data are consistent with reports from other X-linked and autosomal haplotype data sets. Like these other genes, but in conflict with microsatellite and mitochondrial data, PDHA1 does not show evidence of human population expansion.

    Funded by: NIGMS NIH HHS: R55GM54684

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;6;3320-4

  • DNA variability and recombination rates at X-linked loci in humans.

    Nachman MW, Bauer VL, Crowell SL and Aquadro CF

    Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721, USA. nachman@u.arizona.edu

    We sequenced 11,365 bp from introns of seven X-linked genes in 10 humans, one chimpanzee, and one orangutan to (i) provide an average estimate of nucleotide diversity (pi) in humans, (ii) investigate whether there is variation in pi among loci, (iii) compare ratios of polymorphism to divergence among loci, and (iv) provide a preliminary test of the hypothesis that heterozygosity is positively correlated with the local rate of recombination. The average value for pi was low 0.063%, SE = 0.036%, about one order of magnitude smaller than for Drosophila melanogaster, the species for which the best data are available. Among loci, pi varied by over one order of magnitude. Statistical tests of neutrality based on ratios of polymorphism to divergence or based on the frequency spectrum of variation within humans failed to reject a neutral, equilibrium model. However, there was a positive correlation between heterozygosity and rate of recombination, suggesting that the joint effects of selection and linkage are important in shaping patterns of nucleotide variation in humans.

    Funded by: NIGMS NIH HHS: R01 GM036431

    Genetics 1998;150;3;1133-41

  • Arginine 302 mutations in the pyruvate dehydrogenase E1alpha subunit gene: identification of further patients and in vitro demonstration of pathogenicity.

    Otero LJ, Brown RM and Brown GK

    Department of Biochemistry, University of Oxford, United Kingdom.

    Three further patients with mutations in the codon for arginine 302 of the E1alpha subunit of the pyruvate dehydrogenase complex have been identified. Mutations in this codon have now been found in nine patients with pyruvate dehydrogenase deficiency in seven unrelated families, in sharp contrast to the great majority of other PDH E1alpha mutations which have been described in single individuals only. Because of the relatively high frequency of this mutation and because very few PDH E1alpha mutations have been demonstrated to be causative, we have established a system for analysing the consequences of defined mutations using transfection of normal and mutant PDH E1alpha cDNA into transformed human fibroblasts which have no endogenous E1alpha mRNA or protein. Using this test system, we have demonstrated that the R302C mutation results in the production of PDH E1alpha protein which is devoid of enzymic activity.

    Funded by: Wellcome Trust

    Human mutation 1998;12;2;114-21

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • Mitochondrial and nuclear genes present conflicting portraits of human origins.

    Hey J

    Department of Ecology, Evolution, and Natural Resources, Rutgers University, Piscataway 08855-1059, USA. bey@mbel.rutgers.edu

    Human mitochondrial DNA (mtDNA) sequences reveal an abundance of polymorphic sites in which the frequencies of the segregating bases are very different. A typical polymorphism involves one base at low frequency and the other base at high frequency. In contrast, nuclear gene data sets tend to show an excess of polymorphisms in which both segregating bases are at intermediate frequencies. A new statistical test of this difference finds significant differences between mtDNA and nuclear gene data sets reported in the literature. However, differences in the polymorphism patterns could be caused by different sample origins for the different data sets. To examine the mtDNA-nuclear difference more closely, DNA sequences were generated from a portion of the X-linked pyruvate dehydrogenase E1 alpha subunit (PDHA1) locus and from a portion of mitochondrial control region I (CRI) from each of eight individuals, four from sub-Saharan Africa. The two genes revealed a significant difference in the site frequency distribution of polymorphic sites. PDHA1 revealed an excess of intermediate-frequency polymorphisms, while CRI showed an excess of sites with the low-high frequency pattern. The discrepancy suggests that mitochondrial variation has been shaped by natural selection, and may not be ideal for some questions on human origins.

    Molecular biology and evolution 1997;14;2;166-72

  • Refined localization of the pyruvate dehydrogenase E1 alpha gene (PDHA1) by linkage analysis.

    Børglum AD, Flint T, Hansen LL and Kruse TA

    Institute of Human Genetics, Aarhus University, Denmark.

    Pyruvate dehydrogenase (PDH) E1 alpha is a key component in the PDH complex which catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. Defects in the gene coding for PDH E1 alpha (PDHA1) are associated with a variety of clinical symptoms, often of a severe character. In the present study, the segregation of three polymorphic CA repeats located in PDHA1 was followed in the 40 CEPH reference pedigrees. Using these data, multipoint linkage analysis was carried out, refining the genetic location of PDHA1. The 16-point map presented locates PDHA1 in an approximately 3-cM interval between DXS999 and DXS365 with odds of more than 1000:1. From known physical localizations of the flanking marker loci, PDHA1 could be regionally assigned to Xp22.1-p22.2. The information provided should be of value in clinical settings.

    Human genetics 1997;99;1;80-2

  • Three new mutations of the pyruvate dehydrogenase alpha subunit: a point mutation (M181V), 3 bp deletion (-R282), and 16 bp insertion/frameshift (K358SVS-->TVDQS).

    Tripatara A, Kerr DS, Lusk MM, Kolli M, Tan J and Patel MS

    Department of Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.

    Three novel mutations in the coding region of E1 alpha gene were found in three PDC-deficient male patients, including a missense mutation (M181V), a 3 bp deletion (AGA, corresponding to R282), and a 16 bp insertion (CAGTGGATCAAGTTTA), causing a frameshift starting with lysine 358 and resulting in decrease of both E1 subunits.

    Funded by: NIDDK NIH HHS: DK-20478; PHS HHS: MCJ-009122

    Human mutation 1996;8;2;180-2

  • Mutation analysis of the pyruvate dehydrogenase E1 alpha gene in eight patients with a pyruvate dehydrogenase complex deficiency.

    Lissens W, De Meirleir L, Seneca S, Benelli C, Marsac C, Poll-The BT, Briones P, Ruitenbeek W, van Diggelen O, Chaigne D, Ramaekers V and Liebaers I

    Department of Medical Genetics, University Hospital, Brussels, Belgium.

    Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X-linked E1 alpha gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single-strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA. The 4 female patients were shown to carry both a normal and a mutated E1 alpha gene.

    Human mutation 1996;7;1;46-51

  • An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

    Takakubo F, Cartwright P, Hoogenraad N, Thorburn DR, Collins F, Lithgow T and Dahl HH

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia.

    A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.

    American journal of human genetics 1995;57;4;772-80

  • Mutagenesis studies of the phosphorylation sites of recombinant human pyruvate dehydrogenase. Site-specific regulation.

    Korotchkina LG and Patel MS

    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.

    Mammalian pyruvate dehydrogenase (alpha 2 beta 2) (E1) is regulated by phosphorylation-dephosphorylation, catalyzed by the E1-kinase and the phospho-E1-phosphatase. Using site-directed mutagenesis of the three phosphorylation sites (sites 1, 2, and 3) on E1 alpha, several human E1 mutants were made with single, double, and triple mutations by changing Ser to Ala. Mutation at site 1 but not at sites 2 and/or 3 decreased E1 specific activity and also increased Km values for thiamin pyrophosphate and pyruvate. Sites 1, 2, and 3 in the E1 mutants were phosphorylated either individually or in the presence of the other sites by the dihydrolipoamide acetyltransferase-protein X-E1 kinase indicating a site-independent mechanism of phosphorylation. Phosphorylation of each site resulted in complete inactivation of the E1. However, the rates of phosphorylation and inactivation were site-specific. Sites 1, 2, and 3 were dephosphorylated either individually or in the presence of the other sites by the phospho-E1-phosphatase resulting in complete reactivation of the E1. The rates of dephosphorylation and reactivation were similar for sites 1, 2, and 3, indicating a random dephosphorylation mechanism.

    Funded by: NIDDK NIH HHS: DK20478

    The Journal of biological chemistry 1995;270;24;14297-304

  • Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase: exon skipping, insertion of duplicate sequence, and missense mutations leading to the deficiency of the pyruvate dehydrogenase complex.

    Chun K, MacKay N, Petrova-Benedict R, Federico A, Fois A, Cole DE, Robertson E and Robinson BH

    Department of Paediatrics, University of Toronto, Ontario, Canada.

    Human pyruvate dehydrogenase (PDH)-complex deficiency is an inborn error of metabolism that is extremely heterogeneous in its presentation and clinical course. In a study of 14 patients (7 females and 7 males), we have found a mutation in the coding region of the E1 alpha gene in all 14 patients. Two female patients had the same 7-bp deletion at nt 927; another female patient had a 3-bp deletion at nt 931. Another female patient was found to have a deletion of exon 6 in her cDNA. Two other female patients were found to have insertions, one of 13 bp at nt 981 and one of 46 bp at nucleotide 1078. Two male patients were found to have a 4-bp insertion at nucleotide 1163. The remaining six patients all had missense mutations. A male patient and a female patient both had an A1133G mutation. The other missense mutations were C214T, C615A, and C787G (two patients). Five of these mutations are novel mutations, five have been previously reported in other patients, and two were published observations in other patients in an E1 alpha-mutation summary. In the four cases where parent DNA was available, only one mother was found to be a carrier of the same mutation as her child.

    American journal of human genetics 1995;56;3;558-69

  • Recombinant expression and evaluation of the lipoyl domains of the dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex.

    Liu S, Baker JC, Andrews PC and Roche TE

    Department of Biochemistry, Kansas State University, Manhattan 66406.

    The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.

    Funded by: NIDDK NIH HHS: DK18320

    Archives of biochemistry and biophysics 1995;316;2;926-40

  • Pyruvate dehydrogenase complex deficiency due to a point mutation (P188L) within the thiamine pyrophosphate binding loop of the E1 alpha subunit.

    Hemalatha SG, Kerr DS, Wexler ID, Lusk MM, Kaung M, Du Y, Kolli M, Schelper RL and Patel MS

    Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

    Funded by: NICHD NIH HHS: HD 00878; NIDDK NIH HHS: DK-20478; PHS HHS: MCJ-009122

    Human molecular genetics 1995;4;2;315-8

  • Pyruvate dehydrogenase deficiency.

    Brown GK, Otero LJ, LeGris M and Brown RM

    Department of Biochemistry, University of Oxford, UK.

    Funded by: Wellcome Trust

    Journal of medical genetics 1994;31;11;875-9

  • Pyruvate dehydrogenase deficiency caused by a 33 base pair duplication in the PDH E1 alpha subunit.

    Hansen LL, Horn N, Dahl HH and Kruse TA

    Institute of Human Genetics, University of Aarhus, Denmark.

    Human molecular genetics 1994;3;6;1021-2

  • Pyruvate dehydrogenase deficiency. Clinical presentation and molecular genetic characterization of five new patients.

    Matthews PM, Brown RM, Otero LJ, Marchington DR, LeGris M, Howes R, Meadows LS, Shevell M, Scriver CR and Brown GK

    Department of Biochemistry, University of Oxford, UK.

    Fibroblast cultures from five patients with early onset severe encephalopathy and lactic acidosis were studied for evidence of pyruvate dehydrogenase (PDH) deficiency. Three males had significantly reduced activity (0.29-0.45 nmol/mg protein/min versus normal controls 0.7-1.1 nmol/mg protein/min); two females had PDH activity within the normal range. However, as the majority of cases of PDH deficiency result from defects in the X-linked E1 alpha subunit and both females had biased patterns of X-inactivation (making it impossible to rule out the possibility that they were heterozygous for an E1 alpha gene defect) molecular genetic studies were performed. cDNA from the male patients was sequenced and mis-sense mutations found: Y243N (T-->A) in exon 7, D315A (G-->A) in exon 10 and R378H (G-->A) in exon 11. Single-strand conformation polymorphism analysis of amplified genomic DNA fragments and sequencing revealed a mis-sense mutation M282L (A-->C) in one female and a frameshift mutation caused by insertion of T (R288ins) in the other. Adding to recent descriptions of new mutations, this report emphasizes the allelic heterogeneity of the condition. The identification of mutations in females with a suggestive clinical phenotype, even when peripheral fibroblasts do not show deficient PDH activity, illustrates the importance of molecular analysis of this disease.

    Funded by: Wellcome Trust

    Brain : a journal of neurology 1994;117 ( Pt 3);435-43

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Characterization of a point mutation in the pyruvate dehydrogenase E1 alpha gene from two boys with primary lactic acidaemia.

    Awata H, Endo F, Tanoue A, Kitano A and Matsuda I

    Department of Pediatrics, Kumamoto University Medical School, Japan.

    We report here a novel mutation in the codon for amino acid 263 resulting in the change from arginine to glutamine in the pyruvate dehydrogenase (PDH) E1 alpha gene, in two boys with primary lactic acidaemia, from independent families. The mutation changes an amino acid located between the two serine residues which are the sites of phosphorylation of the subunit protein. In one family, the mutation was de novo and in the other it was transmitted from mother to son. The amino acid substitution may affect function of the PDH complex via phosphorylation and dephosphorylation of the E1 alpha subunit. Derangement in the regulation of activity of the PDH complex may explain the primary lactic acidaemia in the patients.

    Journal of inherited metabolic disease 1994;17;2;189-95

  • Pyruvate dehydrogenase deficiency in a male caused by a point mutation (F205L) in the E1 alpha subunit.

    Dahl HH and Brown GK

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Parkville, Melbourne, Victoria, Australia.

    Human mutation 1994;3;2;152-5

  • Molecular genetic characterization of an X-linked form of Leigh's syndrome.

    Matthews PM, Marchington DR, Squier M, Land J, Brown RM and Brown GK

    Department of Biochemistry, University of Oxford, UK.

    We report a patient with necrotizing encephalomyelopathy (Leigh's syndrome) associated with a deficiency of pyruvate dehydrogenase complex activity. The underlying mutation is an A to C transversion in the pyruvate dehydrogenase complex E1 alpha subunit gene. As the E1 alpha subunit is encoded on the X chromosome, this observation confirms that some patients with Leigh's syndrome may potentially exhibit X-linked inheritance.

    Annals of neurology 1993;33;6;652-5

  • Mutations in the X-linked E1 alpha subunit of pyruvate dehydrogenase leading to deficiency of the pyruvate dehydrogenase complex.

    Chun K, MacKay N, Petrova-Benedict R and Robinson BH

    Department of Paediatrics, University of Toronto, Ontario, Canada.

    Human PDH complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. In an investigation at the level of the gene into ten cases of PDH complex (E1) deficiency, we found that all had mutations in the coding sequence of the X-linked E1 alpha gene while the E1 beta coding sequence was normal. Six of these patients (three males, three females) had missense mutations resulting in a changed amino acid residue in the E1 alpha subunit at positions amino acid 148 (in two siblings), 170, 202, 234 and 263 of the mature protein. Two of the females had one normal E1 alpha gene and one with a deletion at the sites of tandem repeats of AGTAAGA and TAT respectively. The two remaining females also had one normal E1 alpha gene and one with an insertion. Both insertions, one of 2 bp and one of 4 bp, occurred in DNA hotspots normally associated with deletions. Only two of these ten mutations have been reported in other patients previously. In the five cases (including the two siblings) where parent DNA was available, only in one case could the same mutation be found in the patient as well as the maternal genomic DNA.

    Human molecular genetics 1993;2;4;449-54

  • Pyruvate dehydrogenase (PDH) deficiency caused by a 21-base pair insertion mutation in the E1 alpha subunit.

    De Meirleir L, Lissens W, Vamos E and Liebaers I

    Laboratory of Medical Genetics, Vrije Universiteit Brussel (VUB), Belgium.

    We report the molecular characterization of a case of a functional PDH-E1 (E1 subunit of pyruvate dehydrogenase) deficiency, a cause of severe congenital lactic acidosis. Residual PDH-E1 activity was reduced to 10% of normal values, although the subunit appeared to be quantitatively and qualitatively normal at the protein level as determined by Western blotting. The sequence of PDH-E1 alpha mRNA and the corresponding genomic DNA revealed an in-frame 21-bp insertion between codons 305 and 306 of the normal E1 alpha cDNA. The mutational insert commences with a novel GAT codon and is a nearly perfect tandem duplication of the wild type DNA sequence. A serine phosphorylation site regulating the activity of the PDH complex is altered by this insertion, which in all likelihood is responsible for the functional enzymatic deficiency leading to lactic acidosis.

    Human genetics 1992;88;6;649-52

  • Mutation of E1 alpha gene in a female patient with pyruvate dehydrogenase deficiency due to rapid degradation of E1 protein.

    Ito M, Huq AH, Naito E, Saijo T, Takeda E and Kuroda Y

    Department of Pediatrics School of Medicine, University of Tokushima, Japan.

    A mutation of an insertion of 4 bp in the gene for the alpha subunit of pyruvate dehydrogenase (E1 alpha) was found in a female with pyruvate dehydrogenase deficiency due to the rapid degradation of alpha and beta subunit proteins of pyruvate dehydrogenase. This mutation caused a frameshift that altered the amino acid sequence and created a premature stop codon. This 4-bp insertion has been found in an unrelated female patient with E1 alpha deficiency. It is rare that the same mutation is found in unrelated patients with this rare inborn error of metabolism. Furthermore, short deletions or duplications in the E1 alpha gene of patients with E1 alpha deficiency have been found only in exons 10 and 11. These exons may be hot spots for the mutations by the recombinational processes. This patient was heterozygous for the normal and a mutant allele. However, in most of the cultured skin fibroblasts from this patient, the mutant allele was expressed. These observations suggest that the X chromosome containing the normal allele was predominantly inactivated so that she developed lactic acidaemia and neurological abnormalities despite being heterozygous. The mutant alpha subunit protein failed to form a stable structure of pyruvate dehydrogenase, so that both alpha and beta subunit proteins were degraded rapidly.

    Journal of inherited metabolic disease 1992;15;6;848-56

  • Mutations and polymorphisms in the pyruvate dehydrogenase E1 alpha gene.

    Dahl HH, Brown GK, Brown RM, Hansen LL, Kerr DS, Wexler ID, Patel MS, De Meirleir L, Lissens W, Chun K et al.

    Murdoch Institute for Research Into Birth Defects, Royal Children's Hospital, Parkville, Melbourne, Australia.

    We present an update on mutations and polymorphisms in the human X chromosome located pyruvate dehydrogenase E1 alpha gene. A total of 20 different mutations are tabulated. The mutations include deletions, insertions, and point mutations. Certain sequences seem particularly prone to mutation. Most of the mutations are found in exons 10 and 11. Furthermore, four of the mutations are seen in unrelated patients. Little is known about how the mutations affect the structure or function of the pyruvate dehydrogenase complex.

    Funded by: NIADDK NIH HHS: AM07319; NIDDK NIH HHS: DK20478

    Human mutation 1992;1;2;97-102

  • X-linked pyruvate dehydrogenase E1 alpha subunit deficiency in heterozygous females: variable manifestation of the same mutation.

    Dahl HH, Hansen LL, Brown RM, Danks DM, Rogers JG and Brown GK

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia.

    Three female patients are described with pyruvate dehydrogenase (PDH) deficiency as a result of mutation in the X-linked gene for the E1 alpha subunit of the complex. Two of these patients illustrate typical presentations of PDH E1 alpha deficiency, with severe neurological dysfunction, degenerative changes and developmental anomalies in the brain, together with variable lactic acidosis. The third patient extends the known spectrum of the condition to include mild to moderate mental retardation and seizures in an adult. All three patients have the same mutation in the PDH E1 alpha gene. This mutation, a C-to-T substitution in a CpG dinucleotide in amino acid codon 302 (designated R302C), results in the replacement of arginine by cysteine at this position. The mildly affected adult was the mother of one of the other patient, making this the first described instance of mother-to-daughter transmission of a mutation causing PDH E1 alpha deficiency. The genetic basis of the variable expression of X-linked PDH E1 alpha deficiency in heterozygous females is discussed.

    Journal of inherited metabolic disease 1992;15;6;835-47

  • Characterization of the mutations in three patients with pyruvate dehydrogenase E1 alpha deficiency.

    Hansen LL, Brown GK, Kirby DM and Dahl HH

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Victoria, Australia.

    The human pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. Defects in several of the seven subunits have been reported, but the majority of mutations affect the E1 component and especially the E1 alpha subunit. However, the clinical presentation of patients with pyruvate dehydrogenase E1 alpha deficiency is extremely variable. Dependency of the brain on pyruvate dehydrogenase activity and localization of the gene for the somatic form of the pyruvate dehydrogenase E1 alpha subunit to the X chromosome provide the basis for a better understanding of the variation in the clinical manifestations. Further understanding of the function and interaction of subunits and the pathophysiology of pyruvate dehydrogenase deficiency necessitates the characterization of mutations in the pyruvate dehydrogenase complex. We report the analysis of three patients with pyruvate dehydrogenase E1 alpha deficiency. One female has a three base pair deletion which affects dephosphorylation of the subunit. Of two males analysed, one has a two base pair deletion causing a shift in the reading frame. The other has a base change, resulting in an Arg to His substitution. All three mutations are located near the carboxyl terminus of the subunit.

    Journal of inherited metabolic disease 1991;14;2;140-51

  • Characterization and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase alpha-subunit.

    Koike K, Urata Y, Matsuo S and Koike M

    Department of Pathological Biochemistry, Nagasaki University School of Medicine, Japan.

    Genomic clones encompassing the entire gene (PDH alpha) encoding the human pyruvate dehydrogenase alpha-subunit (PDH alpha) have been isolated by screening a leukocyte genomic library in the cloning vector, lambda EMBL4. The PDH alpha gene spans 17082 bp and is composed of eleven exons and ten introns. All intron/exon splice junctions follow the GT/AG rule. A total of seven Alu repeats were found in five introns. The entire nucleotide (nt) sequence of the PDH alpha gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. The results of primer extension analysis imply that the PDH alpha gene transcription start point (tsp) is a thymine residue 124 bp upstream from the ATG start codon in exon 1. The structural organization and the tsp were compared with the recent report [Maragos et al., J. Biol. Chem. 264 (1989) 12294-12298]. Analysis of the PDH alpha gene resolves existing discrepancies among four published sequences of PDH alpha cDNAs. A 93-bp sequence that was missing in our sequence of cultured foreskin fibroblast PDH alpha cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45] was identified in the gene as exon 6.

    Gene 1990;93;2;307-11

  • Molecular cloning and characterization of human pyruvate dehydrogenase beta subunit gene.

    Koike K, Urata Y and Koike M

    Department of Pathological Biochemistry, Nagasaki University School of Medicine, Japan.

    A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase beta subunit (PDH beta) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH beta cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH beta structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5' flanking region of the PDH beta gene contains a "CAAT" consensus promoter sequence but no "TATA" sequence. Primer-extension analysis indicated that the PDH beta gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

    Proceedings of the National Academy of Sciences of the United States of America 1990;87;15;5594-7

  • Structural organization of the gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex.

    Maragos C, Hutchison WM, Hayasaka K, Brown GK and Dahl HH

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia.

    The structural organization of the X-linked gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been determined by restriction endonuclease mapping and DNA sequence analysis of overlapping genomic clones. The gene is approximately 17 kilobase pairs long. It contains 11 exons ranging from 61 to 174 base pairs and introns ranging from 600 base pairs to 5.7 kilobase pairs. All the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by S1 nuclease mapping. The DNA sequence around this site is very GC-rich. A "TATA box"-like sequence and a "CAAT box"-like sequence are present 24 and 113 bases upstream from the cap site, respectively. Also upstream from the cap site are several sets of inverted repeats, direct repeats, several sequences resembling the transcription factor Sp1 binding site, a glucocorticoid-responsive element, and two cAMP receptor binding sites.

    The Journal of biological chemistry 1989;264;21;12294-8

  • Characterization of cDNAs encoding human pyruvate dehydrogenase alpha subunit.

    Ho L, Wexler ID, Liu TC, Thekkumkara TJ and Patel MS

    Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, OH 44106.

    A cDNA clone (1423 base pairs) comprising the entire coding region of the precursor form of the alpha subunit of pyruvate dehydrogenase (E1 alpha) has been isolated from a human liver cDNA library in phage lambda gt11. The first 29 amino acids deduced from the open reading frame correspond to a typical mitochondrial targeting leader sequence. The remaining 361 amino acids, starting at the N terminus with phenylalanine, represent the mature mitochondrial E1 alpha peptide. The cDNA has 43 base pairs in the 5' untranslated region and 210 base pairs in the 3' untranslated region, including a polyadenylylation signal and a short poly(A) tract. The nucleotide sequence of human liver E1 alpha cDNA was confirmed by the nucleotide sequences of three overlapping fragments generated from human liver and fibroblast RNA by reverse transcription and DNA amplification by the polymerase chain reaction. This consensus nucleotide sequence of human liver E1 alpha cDNA resolves existing discrepancies among three previously reported human E1 alpha cDNAs and provides the unambiguous reference sequence needed for the characterization of genetic mutations in pyruvate dehydrogenase-deficient patients.

    Funded by: NIADDK NIH HHS: AM07319; NIDDK NIH HHS: DK20478

    Proceedings of the National Academy of Sciences of the United States of America 1989;86;14;5330-4

  • Defective gene in lactic acidosis: abnormal pyruvate dehydrogenase E1 alpha-subunit caused by a frame shift.

    Endo H, Hasegawa K, Narisawa K, Tada K, Kagawa Y and Ohta S

    Department of Biochemistry, Jichi Medical School, Tochigi-ken, Japan.

    A patient with lactic acidosis showed a lowered pyruvate dehydrogenase E1 activity and fatigued on slight exercise. The cDNA encoding the pyruvate dehydrogenase E1 alpha-subunit from his lymphocytes, transformed by infection of Epstein-Barr virus, was cloned and sequenced. The nucleotide sequence determination revealed that the gene had a deletion of four nucleotides at the second codon upstream from the termination codon. This deletion would lead to a reading-frame shift and make a new termination codon at the 33d codon downstream from the "normal" termination codon. An S1 nuclease-protection experiment confirmed the presence of mRNA with its deletion in the patient. Amplification, by the polymerase chain reaction method, of the genomic-DNA region from his peripheral blood cells showed that the deletion was localized in an exon and that it was not caused by an abnormal splicing at the intron/exon junction. This is the first report on cloning a defective gene of the pyruvate dehydrogenase complex.

    American journal of human genetics 1989;44;3;358-64

  • X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex.

    Brown RM, Dahl HH and Brown GK

    Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne, Australia.

    The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.

    Genomics 1989;4;2;174-81

  • Isolation of a full-length complementary DNA coding for human E1 alpha subunit of the pyruvate dehydrogenase complex.

    De Meirleir L, MacKay N, Lam Hon Wah AM and Robinson BH

    Department of Pediatrics and Biochemistry, University of Toronto, Ontario, Canada.

    A 1.5-kilobase cDNA clone for human pyruvate dehydrogenase E1 was isolated from a lambda gt11 expression library by screening with polyclonal antiserum to the E1 alpha subunit of the porcine pyruvate dehydrogenase complex, a polyclonal antibody against bovine pyruvate dehydrogenase complex and a synthetic oligonucleotide based on the known amino acid sequence of the amino-terminal of the bovine pyruvate dehydrogenase-E1 alpha subunit. Nucleotide sequence analysis of the cDNA revealed a 5'-untranslated sequence of 72 nucleotides, a translated sequence of 1170 nucleotides, and a 3'-untranslated sequence of 223 nucleotides with a poly(A) tail. The cDNA structure predicts a leader sequence of 29 amino acids and a mature protein of 362 amino acids comprising an amino-terminal peptide identical to that of the bovine E1 alpha subunit and three serine phosphorylation sites whose sequence was also identical to those in the bovine E1 alpha subunit. The translated sequence for the mature protein differs substantially from that described by Dahl et al. (Dahl, H. H., Hunt, S. M., Hutchison, W. M., and Brown, G. K. (1987) J. Biol. Chem. 262, 7398-7403) by virtue of a frameslip between bases 390 and 594. This amended sequence is confirmed by the presence of additional restriction sites for the enzymes NaeI and HaeII at the beginning and end, respectively, of this section. The leader sequence is typical for mitochondrial enzymes being composed of a combination of neutral and basic residues. The amino acid composition is strikingly similar to that of the bovine protein. This cDNA clone hybridizes with a 1.8-kilobase mRNA on a Northern blot analysis of human fibroblasts, and a second minor band of 4.4 kilobases is also detected.

    The Journal of biological chemistry 1988;263;4;1991-5

  • Cloning and sequencing of cDNAs encoding alpha and beta subunits of human pyruvate dehydrogenase.

    Koike K, Ohta S, Urata Y, Kagawa Y and Koike M

    Department of Pathological Biochemistry, Nagasaki University School of Medicine, Japan.

    The cDNAs encoding fragments of the alpha and beta subunits (PDH alpha and PDH beta) of human pyruvate dehydrogenase (PDH, EC were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening. Phage cDNA fragments were subsequently used to screen a human foreskin fibroblast cDNA library by colony hybridization. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH alpha and one of 1.69 kb for PDH beta, respectively, allowing us to predict the complete amino acid sequences of the precursor and mature proteins of these two subunits. A putative leader sequence of 29 amino acid residues was identified in pHPDA, resulting in a precursor protein of 392 amino acid residues (Mr 43,414) and a mature protein of 363 residues (Mr 40,334). A similar leader sequence of 30 amino acid residues in pHPDB was also identified, resulting in a precursor protein of 359 amino acid residues (Mr 39,046) and a mature protein of 329 residues (Mr 35,911). The amino acid sequences of NH2-terminal regions of the two subunits of human PDH were highly homologous with those of mature porcine PDH. The amino acid sequences of phosphorylation sites determined in PDH alpha of bovine and porcine enzymes were also conserved in the human PDH alpha. Blot analysis of HeLa cell poly(A)+ RNA showed a single mRNA of 1.8 kb for PDH alpha and 1.7 kb for PDH beta, respectively. The precursor proteins of PDH alpha and PDH beta were detected by immunoprecipitation from an 35S-labeled cell-free translation system.

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;1;41-5

  • The human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA.

    Dahl HH, Hunt SM, Hutchison WM and Brown GK

    cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC, the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.

    The Journal of biological chemistry 1987;262;15;7398-403

  • The glucose-lactic acid cycle and gluconeogenesis.

    Cori CF

    Current topics in cellular regulation 1981;18;377-87

Gene lists (10)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000010 G2C Homo sapiens Human mitochondria Human orthologues of mouse mitochondria adapted from Collins et al (2006) 91
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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