Folding and assembly defects of pyruvate dehydrogenase deficiency-related variants in the E1α subunit of the pyruvate...

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Pyruvate dehydrogenase complex (PDC) is crucial for linking glycolysis to the citric acid cycle.
• PDC deficiency in humans causes severe neurodevelopmental delay and neurodegeneration, often due to variants in the E1α subunit.
• The molecular mechanisms underlying these pathogenic variants remain largely unknown.

Purpose of the Study:

• To investigate the structural and functional consequences of specific amino acid substitutions in the E1α subunit of PDC.
• To elucidate how these variants affect PDC assembly, activity, and cofactor dependency using a yeast model system.

Main Methods:

• Site-directed mutagenesis was used to introduce specific substitutions (A189V, M230V, R322C) into yeast E1α.
• Analysis of PDC structure, subunit composition, enzymatic activity, and cofactor (thiamin pyrophosphate) sensitivity.
• Investigation of E1α variant interactions with chaperonins (Hsp60) and ATP.

Main Results:

• The A189V substitution led to a more compact PDC conformation, reduced E1α representation, and impaired activity.
• The M230V substitution resulted in a more open conformation, with heightened sensitivity to thiamin pyrophosphate.
• The R322C substitution abolished PDC activity and led to the loss of E3 subunits.
• The A189V variant E1α was found to accumulate in Hsp60 chaperonin, with release dependent on ATP.

Conclusions:

• Pathogenic E1α variants can induce significant structural alterations in the PDC.
• Impaired folding and assembly of E1α, potentially involving chaperonin interactions, are associated with these variants.
• Understanding these molecular defects provides insights into PDC deficiency-related neurodegeneration.