TAT-mediated Delivery of LAD Restores Pyruvate Dehydrogenase Complex Activity in the Mitochondria of Patients with LAD Deficiency

Abstract

Modern medicine offers no cure for mitochondrial disorders such as lipoamide dehydrogenase (LAD) deficiency. LAD is the E3 subunit shared by alpha-ketoacid dehydrogenase complexes in the mitochondrial matrix, and these complexes are crucial for the metabolism of carbohydrates and amino acids. We propose a novel concept for restoring the activity of an immense multicomponent enzymatic complex by replacing one mutated component, the LAD subunit. Our approach entails the fusing of LAD with the transactivator of transcription (TAT) peptide, which is capable of rapidly crossing biological membranes, thereby allowing TAT-LAD to be delivered into cells and their mitochondria where it can replace the mutated endogenous enzyme. Our results show that TAT-LAD is indeed delivered into the cells and their mitochondria, where it is processed, restoring LAD activity to normal values and, most importantly, increasing the activity of pyruvate dehydrogenase complex. We report here, for the first time, that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multicomponent enzymatic complex in cells of patients with enzyme deficiencies. We believe that this approach involving TAT-mediated enzyme replacement therapy (ERT) can be applied to the treatment of LAD deficiency as well as to other mitochondrial and metabolic disorders.

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

“…In 2008, the same protein was identified by Saada and her coworkers (15), in collaboration with our laboratory as an assembly factor.…”

“…Inside the mitochondria, TAT-LAD is processed normally and restores LAD activity. Most importantly, we showed that TAT-LAD is able to almost completely restore the activity of the mitochondrial multienzyme pyruvate dehydrogenase complex (PDHC) within the treated patients' cells (15). Moreover, we demonstrated the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of PDHC within the liver, heart and mainly the brain of TAT-LAD-treated E3-deficient mice (27).…”

“…Since then, genetic, bioinformatic and proteomic analyses have led to the identification of no less than 10 assembly factors (7). A number of complex I assembly factors are linked to human diseases (4,(8)(9)(10)(11)(12)(13)(14); among them is the chromosome 6, open reading frame 66 (C6ORF66) assembly factor (15).…”

“…The exact role of C6ORF66-later termed "NDUFAF4" (NADH dehydrogenase [ubiquinone] complex I assembly factor 4)-in complex I biogenesis is not yet fully understood; however, it was reported that together with the assembly factor NDUFAF3, these two proteins participate at early stages of complex I assembly (9). Pathogenic mutations in the gene encoding C6ORF66 result in fatal neonatal mitochondrial disease with severe complex I enzymatic deficiency (15). Isolated muscle mitochondria from a patient with a homozygous mutation retain only 30% residual mature complex I, with the accumulation of stalled assembly intermediates (15).…”

“…Pathogenic mutations in the gene encoding C6ORF66 result in fatal neonatal mitochondrial disease with severe complex I enzymatic deficiency (15). Isolated muscle mitochondria from a patient with a homozygous mutation retain only 30% residual mature complex I, with the accumulation of stalled assembly intermediates (15).…”