Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies

Abstract

Protein chaperones play a critical role in proteostasis. The activity of the major endoplasmic reticulum chaperone BiP (GRP78) is regulated by Fic-mediated AMPylation during resting states. By contrast, during times of stress, BiP is deAMPylated. Here, we show that excessive AMPylation by a constitutively active Fic E247G mutant is lethal in Drosophila. This lethality is cell-autonomous, as directed expression of the mutant Fic E247G to the fly eye does not kill the fly but rather results in a rough and reduced eye. Lethality and eye phenotypes are rescued by the deAMPylation activity of wild-type Fic. Consistent with Fic acting as a deAMPylation enzyme, its activity was both time-and concentration-dependent. Furthermore, Fic deAMPylation activity was sufficient to suppress the AMPylation activity mediated by the constitutively active Fic E247G mutant in Drosophila S2 lysates. Further, we show that the dual enzymatic activity of Fic is, in part, regulated by Fic dimerization, as loss of this dimerization increases AMPylation and reduces deAMPylation of BiP.

Kinetic And Structural Parameters Governing Fic-Mediated Adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78

“…We hypothesize that depending on specific cellular signals, the rate of adenylylation versus de-AMPylation reaction differs, leading to elevated levels of either adenylylated or de-AMPylated BiP. Indeed, it was recently reported that while the HYPE dimer functions primarily as an adenylytransferase, HYPE in its monomeric form functions as a de-AMPylase (30). This finding supports our data showing de-AMPylation of BiP and HYPE by WT-HYPE even in the presence of an equal concentration of E234G-HYPE.…”

Regulation of the Unfolded Protein Response by BiP AMPylation protects photoreceptors from light-dependent degeneration

“…In contrast, a BiP transgene mutated at a second reported AMPylation site (Casey et 97 al., 2017;Preissler et al, 2015), BiP T518A , did not rescue BiP -/lethality. As fic null mutants that 98 lack BiP AMPylation are viable, the lethality of the BiP T518A mutant is not likely to be due to the 99 loss of AMPylation (Casey et al, 2017). Instead, these observations indicate an essential role for 100…”

“…Previously, we reported that over-expression of the constitutively active AMPylating 104 Fic E247G was lethal in a fic-null fly background (fic 30C ) because it lacks the essential 105 deAMPylation activity (Casey et al, 2017). We tested whether flies expressing the AMPylation-106 resistant BiP T366A could survive this lethality.…”

“…AMPylating UAS-Fic E247G transgene using a LongGMR-Gal4 driver in otherwise fic-null 117 animals results in a severe rough-eye defect (Casey et al, 2017). However, in a fic heterozygous 118 background, eye-specific expression of constitutively active AMPylating Fic E247G yields only a 119 mildly rough eye (Figure 2A).…”

FICD activity and AMPylation remodelling modulate human neurogenesis

“…Recent data show that FICD regulates the ATPase activity of HSPA5 and its interactions with unfolded proteins, but the exact function is not yet clear 13 . However, it was found that the HSPA5 AMPylation associates with changes in neuronal fitness in drosophila 3, [14][15][16] .…”