title: Pyruvate dehydrogenase complex deficiency caused by ubiquitination and proteasome-mediated degradation of the E1 subunit.
abstract: Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.
authors.affiliation: Departments of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida 32610.
authors.forename: Zongchao
authors.initials: Z
authors.lastname: Han
authors.affiliation: Departments of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida 32610.
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.affiliation: Departments of Pediatrics, Division of Cellular and Molecular Therapy, Gainesville, Florida 32610; Molecular Genetics and Microbiology, Gainesville, Florida 32610; The General Clinical Research Center, Gainesville, Florida 32610.
authors.forename: Arun
authors.initials: A
authors.lastname: Srivastava
authors.affiliation: The General Clinical Research Center, Gainesville, Florida 32610; Medicine, Division of Endocrinology and Metabolism, Gainesville, Florida 32610; Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610. Electronic address: peter.stacpoole@medicine.ufl.edu.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Stacpoole
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007976:Leupeptins / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000235:genetics / Q000378:metabolism*; D015325:Pyruvate Dehydrogenase Complex Deficiency Disease / Q000378:metabolism* / Q000473:pathology; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D007976:Leupeptins; D061988:Proteasome Inhibitors; D030481:Pyruvate Dehydrogenase (Lipoamide); C111305:pyruvate dehydrogenase E1 beta subunit; C065640:pyruvate dehydrogenase E1alpha subunit; D066246:ErbB Receptors; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde