Phenylbutyrate increases pyruvate dehydrogenase complex activity in cells harboring a variety of defects

Abstract

Objective: Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. PDHC deficiency is genetically heterogenous and most patients have defects in the X-linked E1-α gene but defects in the other components of the complex encoded by PDHB, PDHX, DLAT, DLD genes or in the regulatory enzyme encoded by PDP1 have also been found. Phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of pyruvate dehydrogenase kinases and thus, has potential for therapy of patients with PDHC deficiency. In the present study, we investigated response to phenylbutyrate of multiple cell lines harboring all known gene defects resulting in PDHC deficiency.

Methods: Fibroblasts of patients with PDHC deficiency were studied for their enzyme activity at baseline and following phenylbutyrate incubation. Drug responses were correlated with genotypes and protein levels by Western blotting.

Results: Large deletions affecting PDHA1 that result in lack of detectable protein were unresponsive to phenylbutyrate, whereas increased PDHC activity was detected in most fibroblasts harboring PDHA1 missense mutations. Mutations affecting the R349-α residue were directed to proteasome degradation and were consistently unresponsive to short-time drug incubation but longer incubation resulted in increased levels of enzyme activity and protein that may be due to an additional effect of phenylbutyrate as a molecular chaperone.

Interpretation: PDHC enzyme activity was enhanced by phenylbutyrate in cells harboring missense mutations in PDHB, PDHX, DLAT, DLD, and PDP1 genes. In the prospect of a clinical trial, the results of this study may allow prediction of in vivo response in patients with PDHC deficiency harboring a wide spectrum of molecular defects.

Figures

Figure 1

(A) PDHC activity in fibroblasts with PDHA1 mutations. PDHC activity is shown as fold increase in baseline activity after incubation with phenylbutyrate. Bars indicate average ± standard error of the mean; *P < 0.05. PDHC activity was increased in four of the nine fibroblast cell lines from male patients shown as black bars and in 10 of the 14 fibroblast cell lines from female patients shown as white bars. (B) Western blotting of skin fibroblasts with PDHA1 mutations with a cocktail of antibodies recognizing E1-α (43 KDa), E1-β (39 kDa), E2 (69 kDa), and E3BP (54 kDa) proteins. Western blotting for cytochrome c oxidase (COX; 17 kDa) was performed as mitochondrial marker. The PDHA1 mutations of the corresponding patient cell lines are shown in Table1. wt, wild-type control fibroblasts.

Figure 2

(A) PDHC activity in fibroblasts with mutations affecting R349 residue of E1-α protein following shorter (1 day) and longer (5 days) phenylbutyrate incubations. (B) Western blotting for E1-α (43 kDa), E1-β (39 kDa), and E2 (69 kDa) protein of cell line 5 harboring p.R349C mutation incubated with either vehicle, phenylbutyrate, MG132, or a combination of phenylbutyrate and MG132. Cytochrome c oxidase (COX; 17 kDa) was used as mitochondrial control marker.

Figure 3

(A) PDHC activity in fibroblasts with PDHB, PDHX, DLD, or PDP1 mutations. PDHC activity is shown as fold increase in baseline activity following incubation with phenylbutyrate. Bars indicate average ± standard error of the mean; *P < 0.05. Western blotting for E1-α (43 kDa), E1-β (39 kDa), E2 (69 kDa), and E3BP (54 kDa) proteins of skin fibroblasts with PDHB (B), PDHX (C), or DLAT (D) mutations. (E) Western blotting for E3 protein of cell lines 34 and 35 harboring DLD mutations. PDHC phosphorylation was evaluated with an antibody against the phosphorylated E1-α (E1-α-Ser264). Cytochrome c oxidase (COX; 17 kDa) was used as mitochondrial marker. (F) Western blotting with antibodies against the three phosphorylated sites of E1-α or total E1-α in fibroblast cell line 36 harboring a homozygous PDP1 mutation. wt1, control 1; wt2, control 2. The PDHB, PDHX, DLD, or PDP1 mutations for each cell lines are shown in Table1.