Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β 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 E1␤ 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 E1␣ (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 E1␤ could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyrphostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1␤ 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 El␣-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 E1␤ 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.The nuclear encoded mitochondrial pyruvate dehydrogenase (PDH) 2 complex plays major roles in regulating cellular fuel metabolism, acid-base equilibrium and energetics (1, 2). The complex consists of three catalytic components: pyruvate dehydrogenase (E1; EC 1.2.4.1), dihydrolipoamide transacetylase (E2; EC 2.3.1.12), and dihydro-lipoamide dehydrogenase (E3; EC 1.8.1.4), and an additional protein known as the E3-binding protein (E3BP). Rapid post-translational regulation of the complex is mediated in large part by reversible phosphorylation of the ␣ subunit of E1 that is catalyzed by isoforms of PDH kinase and PDH phosphatase.The E1 enzyme (EC 1.2.4.1) is a heterotetrameric (␣ 2 ␤ 2 ) ␣-ketoacid decarboxylase that irreversibly oxidizes pyruvate to acetyl-CoA in the presence of thiamine pyrophosphate (TPP) and also catalyzes the subsequent reductive acetylation of the lipoyl moiety of E2. Most reported mutations of the PDH complex are located in the X-linked gene for the ␣ subunit of the E1 component (MIM 312170). Only a small number of patients have been described with mutations in the E2, E3, E3BP, or PDH phosphatase g...

Phenylbutyrate Therapy for Pyruvate Dehydrogenase Complex Deficiency and Lactic Acidosis

“…The amino acid positions of the mutations are reported considering the mature NCBI protein sequence (NP_000275.1 for PDHA1 and NP_003468.2 for PDHX ) after signal peptide cleavage. # This patient was found to have PDH deficiency due to abnormal ubiquitination and proteasome degradation of E1β encoded by PDHB1 gene, as previously described (66). N.D.= not detected.…”

“…The molecular defects of the corresponding patient cell lines are shown in Table 1. Patients 1-12 (dark gray bars) have mutations in PDHA1 , patient 13 (light gray bar) has PDHC deficiency due to abnormal ubiquitination and degradation of E1β (66), and patients 14 and 15 (black bars) have mutations in PDHX . *p<0.05.…”

MuRF1-dependent Regulation of Systemic Carbohydrate Metabolism as Revealed from Transgenic Mouse Studies

“…24 While PDH activities can be reversibly regulated by PDK2-and PDK4-dependent phosphorylation, PDH has been identified recently as an ubiquitination target. 31 Thus, MuRF1 is an excellent candidate for PDH inhibition control in muscle via its E3 ubiquitin ligase activity. Since PDH is a multienzyme complex, further studies are required to dissect the interaction of MuRF1 and other MuRF family members with PDH and its subunits in more detail: While we identified the interaction of MuRF1 with the β (lipoamide) subunit of PDH β, our proteome studies identified the PDH α-E1 subunit as being downregulated in MuRF1-TG mice.…”

NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation

“…Inhibition of PDH-E1α phosphorylation by dichloroacetate, a PDK inhibitor, decreases the E1α degradation and maximizes the PDH activity (35). In contrast, phosphorylation of PDH-E1β at tyrosine residues is associated with enhanced E1β degradation via ubiquitination and decreased PDH activity (57). Indeed, the most notable change in M1 Macs is NOTCH-dependent expression of the PDH-E1α protein.…”