Grubraw, a chemogenetic mitochondrial activator, reveals new mechanisms underlying the Warburg effect

Here we created a chemogenetic instrument, Grubraw, that generates pyruvate directly in the mitochondrial matrix bypassing restricted pyruvate influx. Grubraw, chemogenetic molecular tool is based on Pseudomonas aeruginosa FAD-dependent D-amino acid dehydrogenase DadA that allows bypass of the pyruvate transport block and activation of mitochondrial respiration by generating pyruvate from D-alanine directly in the mitochondria of eukaryotic cells and donating electrons downstream of Complex I.To control the unspecific effects of heterologous protein expression, an inactive mutant was created by changing the conservative Tyr17 and Tyr18 residues in the predicted FAD-binding domain to Ala residuesExperiment was made on Lu451 cancer cell line. Four conditions were analyzed: Grubraw + D-Ala (pyruvate generation) and three controls (Grubraw without D-Ala; Inactive mutant Grubraw with D-Ala; Inactive mutant Grubraw without D-Ala). Each in three biological replicates.