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De-AMPylation
De-AMPylation is the reverse reaction in which an AMP molecule is detached from the amino acid side of a chain protein.

GS-ATase (GlnE) is an AMPylator that has been shown to catalyze de-AMPylation of glutamine synthetase by removing the covalent linkage between AMP and a hydroxyl residue of a protein. It contains two adenylyl transferase domains regulated by PII and its associated posttranslational modifications. The de-AMPylation of glutamine synthetase is caused by the phosphorylation of the adenyl-tyrosine bond using orthophosphate, leading to the creation of ADP and unmodified glutamine synthetase. De-AMPylation occurs at the N-terminus of the domain.

GS-ATase activity is controlled by the signal transducing primer PII, which exists in two forms. When nitrogen levels are high, PII interacts with GS-ATase to induce AMPylation. GS-ATase AMPylation of glutamine synthetase occurs at the tyrosine 397 site, causing the termination of glutamine synthesis. In low nitrogen levels, PII undergoes UMPylation and the UMPylated PII protein inhibits GS-ATase, causing the GS-ATase to be de-AMPylated. Once this occurs, glutamine synthesis takes place.

AMPylation in Eukaryotes
Most research on endogenous protein AMPylation in eukaryotes has focused on Huntingtin associated protein E (HypE) in humans, CG9523 in D. melanogaster, and Fic-1 in C. elegans. Most metazoans contain only one fic domain in their genome, and according to phylogenetic analysis, the domain was evolved independently or was acquired through horizontal gene transfer events. Despite this, their structures are similar: they contain an N-terminal followed by (usually two) tetratricopeptide repeats (TPRs), which are linked to a C-fic domain by four core α-helices.

CG9523 in Drosophila melanogaster
CG9523 is an endoplasmic reticulum (ER)-resident type II transmembrane. It is N-glycosylated on Asn288, proteolytically processed, and are then secreted through the ER secretory pathway to the cell surface of capitate projections - a putative site of neurotransmitter recycling. Blind flies unable to receive light stimulation from photosynaptic neurons were found to be responsive to light once again once the gene expression of a wild-type fic was activated, indicating that AMPylation could play a role in neurotransmitter recycling.

In vitro studies show that AMPylation can also lock Grp78 (an ER-resident heat shock chaperone protein) into an inactive state under stress by altering its ATPase domain, preventing the structural change required for protein folding. However, this is not a permanent structural change, as Grp78 AMPylation is reversible and can be de-AMPylated on demand to support protein folding within the endoplasmic reticulum.

HypE (FICD) in Homo sapiens
Huntingtin associated protein E (HypE) was identified as an AMPylase in 2009. HypE is an ER-resident AMPylase predominantly found in the ER-nuclear envelope continuum with the general structure of a type II membrane protein and is N-glycosylated at Asn275. It consists of a single α-helix linking the fic and TPR domains, which are made up of multiple α-helices. HypE's activity is at its optimum in the presence of Mn2+ and Mg2+, while high Ca2+ levels are correlated with an increase in ATP levels within the endoplasmic reticulum, creating favorable conditions for HypE to AMPylate its targets.

HypE and UPRER
UPRER consists of three components: IRE1, ATF6, and PERK. These three are normally bound by Grp78 and rendered inactive, but are activated in times of ER stress when Grp78 is led to misfolded proteins. The activation of UPRER results in reduced protein synthesis, reduction of the protein load entering the ER, and the induction of a transcriptional program that increases ER capacity to resolve stress.

Cell survival rates decrease under ER stress when UPRER is released and HypE is inactive, while overexpression of HypE is cytotoxic and leads to caspase-dependent apoptosis. HypE is evidenced to regulate UPRER through modification of Grp78, though the sites of modification and the effects of Grp78 modifications have not yet been confirmed. Two theories have been put forward:


 * 1) Grp78 AMPylation is an activating modification induced by ER stress. ATPase activity is enhanced by HypE-mediated AMPylation, but there is no effect on the binding of Grp78 on denatured proteins. ER stress increases HypE levels, causing the induction of ATF6 and PERK-dependent pathways.
 * 2) Grp78 AMPylation is an inactivating modification that maintains a readily accessible but inactive Grp78 pool when there is low ER stress. In times of high ER stress, AMPylation of Grp78 increases after proteostasis is established. As a result, low HypE levels result in elevated levels of Grp78 in the ER, increasing the buffer capacity of the ER to deal with an increased load of unfolded proteins and attenuating the induction of UPR.

HypE and Parkinson's Disease
The presynaptic protein α-synuclein was found to be a target for HypE. During HypE-mediated adenylylation of αSyn, aggregation of αSyn decreases and both neurotoxicity and ER stress were discovered to decrease in vitro. Thus, adenylylation of αSyn is possibly a protective response to ER stress and αSyn aggregation. However, further research needs to be done to discover whether this process is independent or whether adenylylation occurs in response to ER stress.

Fic-1 in Caenorhabditis elegans
Fic-1 is the only Fic protein present in the genetic code of C. elegans. It is primarily found in the ER nuclear envelope of adult germline cells and embryotic cells, but small amounts can be found within the cytoplasm as well. It is responsible for the AMPylation of core histones and eEF1-A type translation factors within the nematode.

Though varying AMPylation levels did not create any noticeable effects within the nematode's behaviour or physiology, Fic-1 knockout worms were more susceptible to infection by Pseudomonas aeruginosa compared to the counterparts with active Fic-1 domains, implying a link between AMPylation of cellular targets and immune responses within nematodes.