The liver is one of the largest and most important organs in the mammalian body with major functions in whole-body nutrient homeostasis. The response to nutrient deprivation (fasting/starvation) is a particularly important function for the liver. Regulation of hepatocyte signalling by circulating hormones, which can modulate protein-protein interactions, plays a critical role in this response.
To identify proteins that may regulate the liver fasting response by altering protein-protein interactions, we capitalised on the ability for size exclusion chromatography (SEC) separations to identify complex assembly and disassembly. We collected 48 SEC fractions per sample using two complementary SEC buffer systems and 3 biological replicates per condition (overnight fasted or fed). High sensitivity nanoLC-MS/MS analysis on a QExactive Plus and data analysis using the MaxQuant package, yielded >5,200 proteins identified and quantified across the biological replicates. After Pearson correlation analysis, we set thresholds for replicate profile variation (>0.75) and for inter-condition variation (<0.75). Less than 80 proteins showed a significant deviation in protein complex assembly/disassembly under these criteria that was not coupled to a simultaneous total protein abundance change.
One of the most interesting changes observed was for the protein phosphatase PP2A-B56 complex, containing the catalytic C subunit PPP2CA, the constant regulatory A subunit PPP2R1A and the variable regulatory B subunits of the B56-family. The PP2A-B56 complex displayed significant deviation in assembly of the main 300 kDa peak between fed and fasted animals. We have followed up these observed changes with immunoprecipitation of endogenous PP2A complexes from liver tissue for both proteome analysis and activity measurements in each condition. We hypothesise that this fasting-regulation of PP2A mediates control of the negative feedback this phosphatase provides directly on Akt, which forms a key element of the insulin signalling cascade in hepatocytes.