Redox-Regulated, Targeted Affinity Isolation of NADH-Dependent Protein Interactions with the Branched Chain...

Isolation and identification of protein targets for redox-active proteins is challenging. The human branched chain aminotransferase (hBCAT) proteins are redox active transaminases that can be regulated through oxidation, S-nitrosation and S-glutathionylation. This metabolic protein was shown to associate with the E1 decarboxylase component of the branched-chain α-keto acid dehydrogenase complex in a NADH-dependent manner, where mutation of the CXXC center was shown to prevent complex formation. To determine if the redox state of the CXXC motif can influence other NADH-dependent protein-protein interactions, proteins were extracted from neuronal cells treated under reduced and oxidized conditions and then isolated using targeted affinity chromatography, resolved using 2D electrophoresis. Select proteins spots were excised and identified using a quadrupole time of flight mass spectrometer (Thermo) with a precursor tolerance of 10 ppm and subsequently analyzed using Proteome Discoverer 2.1 with Swiss-Prot human DB. Mass tolerances for precursor/product were set to 10 ppm/0.6 Da and data were filtered by peptide confidence with PD2.1. It was determined that the protein profile considerably altered in both number and abundance dependent on the redox state of the cell and also on the availability of the redox active thiol groups. The biological relevance of the newly identified partners was determined using DAVID, the bioinformatics database, which indicated that proteins important to cytoskeletal function, protein transport, protein synthesis, chaperone activity, and cell signaling.