Redox Modification of PKA-Cα Differentially Affects Its Substrate Selection.

The cyclic AMP-dependent protein kinase (PKA) plays an essential role in the regulation of many important cellular processes and is dysregulated in several pervasive diseases, including diabetes, cardiovascular disease, and various neurodegenerative disorders. Previous studies suggest that the alpha isoform of the catalytic subunit of PKA (PKA-Cα) is oxidized on C199, both in vitro and in situ. However, the molecular consequences of these modifications on PKA-Cα's substrate selection remain largely unexplored. C199 is located on the P + 1 loop within PKA-Cα's active site, suggesting that redox modification may affect its kinase activity. Given the proximity of C199 to the substrate binding pocket, we hypothesized that oxidation could differentially alter PKA-Cα's activity toward its substrates. To this end, we examined the effects of diamide- and H2O2-dependent oxidation on PKA-Cα's activity toward select peptide and protein substrates using a combination of biochemical (i.e., trans-phosphorylation assays and steady-state kinetics analysis) and biophysical (i.e., surface plasmon resonance and fluorescence polarization assays) strategies. These studies suggest that redox modification of PKA-Cα differentially affects its activity toward different substrates. For instance, we found that diamide-mediated oxidation caused a marked decrease in PKA-Cα's activity toward some substrates (e.g., Kemptide and CREBtide) while having little effect on others (e.g., Crosstide). In contrast, H2O2-dependent oxidation of PKA-Cα led to an increase in its activity toward each of the substrates at relatively low H2O2 concentrations, with differential effects at higher peroxide concentrations. Together, these studies offer novel insights into crosstalk between redox- and phosphorylation-dependent signaling pathways mediated by PKA. Likewise, since C199 is highly conserved among AGC kinase family members, they also lay the foundation for future studies designed to elucidate the role of redox-dependent modification of kinase substrate selection in physiological and pathological states.

Computational Analysis of the Binding Mechanism of GenX and HSA.

One PFOS alternative, ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoate, known as GenX, was created to replace one of the original PFAS. This small and tough molecule has been found in surface water, groundwater, drinking water, rainwater, and air emissions in some areas in the United States. Recently, GenX has been shown to have an impact on several disease-related proteins in humans, and just like PFOS, it binds to human protein human serum albumin (HSA). In this paper, we reported four binding sites of GenX on HSA protein via docking and molecular dynamics simulation.