Abstract 1710 rAMPing up the stress: Investigating the regulation of bifunctional FicD activity

FicD is an Endoplasmic Reticulum (ER) transmembrane protein that reversibly AMPylates and deAMPylates BiP, an HSP70 chaperone and master regulator of the Unfolded Protein Response (UPR). Previous work in cultured cells has shown that the bifunctional activity of FicD is highly dependent on levels of ER stress. During low ER stress, FicD generates an inactive pool of AMPylated BiP. Conversely, FicD deAMPylates this pool upon rising ER stress. Consistent with these observations, we show that BiP is AMPylated in the pancreases of fasted WT mice and deAMPylated upon refeeding. Furthermore, FicD-/- mice engage amplified UPR signaling and sustained more severe pancreatic damage upon pharmacological induction of pancreatic ER stress. These results suggest that FicD activity acts as a rheostat for the UPR, restraining its activation until ER chaperone capacity reaches a critical minimum. However, the mechanisms through which FicD's bifunctional activity is regulated remain unclear. Here, we describe in-vitro biochemical analyses that elucidate the parameters that govern BiP's AMPylation and deAMPylation. Using in-vitro activity assays with recombinantly purified proteins, we show that physiologic concentrations of divalent cations potently inhibit FicD-mediated AMPylation of BiP. Furthermore, using in-vitro thermal shift assays, pulldown assays, and analytical chromatography, we show that divalent cations exert an inhibitory effect by disrupting the FicD-BiP enzyme/substrate complex. We hypothesize that the fluctuating environment in the ER alters the kinetics of BiP AMPylation and deAMPylation to tune UPR signaling. This work provides insight into how cells sense and respond to dynamic and idiosyncratic stressors. Our ongoing work seeks to further understand the conditions which delineate FicD's enzymatic switch from AMPylator to deAMPylator. This work was funded by Welch Foundation grant I-1561, the Once Upon a TimeFoundation, NIH R35GM134945, the Life Sciences Research Foundation Fellowship, NIH EY10199, and NIH T32GM127216.