Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Zongchao Han,Li Zhong,Arun Srivastava,Peter W Stacpoole

doi:10.1074/jbc.m704748200

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.

Highlights

The nuclear encoded mitochondrial pyruvate dehydrogenase (PDH)2 complex plays major roles in regulating cellular fuel metabolism, acid-base equilibrium and energetics [1, 2]
Analysis of the PDH E1␤ subunit (PDHB) cDNA revealed an A 3 G sense mutation at nucleotide 438 in exon 6 that results in a new codon that still codes for the same amino acid glycine (G146G; data not shown)
These data corroborate our hypothesis that in cells that overexpress EGFR-PTK, the E1␤ subunit becomes phosphorylated at tyrosine residues, which leads to ubiquitination, and subsequent degradation by the proteasome

Summary

EXPERIMENTAL PROCEDURES

This study was approved by the Institutional Review Board of Shands Hospital, University of Florida. Cells were cultured as monolayers in T-flasks in complete Dulbecco’s modified Eagle’s medium (Sigma). Lysates were precleared by adding 1.0 ␮g of mouse IgG (Santa Cruz Biotechnology), together with 20 ␮l of agarose conjugate (protein A/G-agarose, Santa Cruz Biotechnology) at 4 °C for 30 min. Samples were boiled for 2–3 min prior to adding 10 ␮l of complexes per well for Western analysis using tyrosine phosphor-specific antibody, PY99 (1:300), and secondary antibody, goat anti-mouse IgG2b-HRP (1:10,000, Santa Cruz Biotechnology), as described above. Patient and healthy control fibroblast cDNAs, each synthesized from 50 ng of total RNA, were used for each qPCR reaction. Plasmid Construction—Patient and control cells were cultured and total cellular RNAs were isolated by a RiboPure kit (Ambion), followed by reverse transcription using Omniscript RT kit (Qiagen), according to the manufacturers’ instructions. Synthesized proteins were analyzed by SDS-PAGE with anti-E1␣ and anti-E1␤ mAbs

RESULTS

PDH activitya

DISCUSSION

PTKs catalyze the phosphorylation