Protein Replacement Therapy for a mitochondrial protein: The Pyruvate Dehydrogenase E1 alpha (PDHE1a) subunit

Pyruvate dehydrogenase complex (PDC) is a nuclear-encoded mitochondrial matrix multienzyme complex of three enzymes that converts pyruvate into acetyl-CoA. The E1 component is a heterotetramer of 2 alpha and 2 beta subunits.

Pyruvate dehydrogenase deficiency is one of the most common genetic defects of mitochondrial energy metabolism. The clinical spectrum ranging from fatal lactic acidosis in the newborn period to a chronic neurodegenerative condition often categorized as Leigh syndrome. Medicine nowadays offers no cure for mitochondrial disorders.

Our goal is to test if protein replacement therapy for PDH E1a deficiency can rescue a normal metabolism of the cells/organism. Therefore, we designed, constructed and produced a fusion protein containing three parts: (1) Transactivating transcriptional activator (TAT) that is used for the delivery of proteins across biological membranes; (2) The natural N-terminus mitochondrial targeting sequence (MTS) of PDH E1a, thus enabling its import and processing into the mitochondria; and (3) The mature human active PDHE1a protein.

We developed a purification protocol for producing a biologically active fusion protein. When added to cells in culture, TAT-MTS- PDHE1a fusion protein reached the mitochondrial matrix. Treatment of PDH E1a siRNA knockdown cells with TAT-MTS- PDHE1a fusion protein improves bio-energetic capabilities as measured by oxygen consumption rate and ATP production. Most importantly, treatment with TAT-MTS- PDHE1a fusion protein restores the whole Pyruvate Dehydrogenase Complex activity.

Studies are now being performed to test the potential therapeutic effect of TAT-MTS- PDHE1a fusion protein in vivo in a mouse model.