title: Sequential deletion of C-terminal amino acids of the E(1)alpha component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E(1)alpha(2)beta(2) tetramers: implications for patients with PDH deficiency.
abstract: Human pyruvate dehydrogenase (PDH) complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. We have characterized novel mutations that affect the C-terminal portion of the PDH-E(1)alpha-coding sequence. Although the molecular defects underlying these mutations are different, both effectively produce a stop codon prematurely three amino acids from the C-terminus. The clinical and biochemical consequences of these mutations are unusual in that the affected individuals are very long-term survivors with PDH complex deficiency despite having low (<20%) activity in skin fibroblasts. These findings prompted us to investigate the C-terminus of E(1)alpha in greater detail. We constructed and expressed a series of PDH-E(1)alpha deletion mutants in a cell line with zero PDH complex activity due to a null E(1)alpha allele. Sequential deletion of the C-terminus by one, two, three and four amino acids resulted in PDH complex activities of 100, 60, 36 and 14%, respectively, compared with wild-type E(1)alpha expressed in PDH complex-deficient cells. The immunodetectable protein was decreased by the same amount as the activity, suggesting that the stability and/or assembly of the E(1)alpha(2)beta(2)heterotetramer might depend on the intactness of the PDH-E(1)alpha C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E(1)alphaand concluded that they are biochemically equivalent.
authors.affiliation: Department of Biochemistry, University of Toronto, and Metabolism Research Programme, Research Institute, The Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Seyda
authors.forename: G
authors.initials: G
authors.lastname: McEachern
authors.forename: R
authors.initials: R
authors.lastname: Haas
authors.forename: B H
authors.initials: BH
authors.lastname: Robinson
mesh_terms: D000328:Adult; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D030481:Pyruvate Dehydrogenase (Lipoamide); D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013737:Testis / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D011768:Pyruvate Dehydrogenase Complex; D012333:RNA, Messenger; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit