Adenylylation
Adenylylation,[1][2] more commonly known as AMPylation, is a process in which an adenosine monophosphate (AMP) molecule is covalently attached to the amino acid side chain of a protein.
AMPylators have been shown to be comparable to kinases due to their ATP hydrolysis activity and reversible transfer of the metabolite to a hydroxyl side chain of the protein substrate.
However, AMPylation catalyse a nucleophilic attack on the α-phosphate group, while kinase in the phosphorylation reaction targets γ-phosphate.
[5]De-AMPylation is the reverse reaction in which the AMP molecule is detached from the amino acid side of a chain protein.
The bacterial GS-ATase (GlnE) encodes a bipartite protein with separate N-terminal AMPylation and C-terminal de-AMPylation domains whose activity is regulated by PII and associated posttranslational modifications.
This shows that Fic domains are highly conserved that indicates the important role of AMPylation in regulating cellular stress in bacteria.
Effectors such as VopS, IbpA, and DrrA, have been shown to AMPylate host GTPases and cause actin cytoskeleton changes.
Rho, Rab, and Arf GTPase families are involved in actin cytoskeleton dynamics and vesicular trafficking.
Vibrio parahaemolyticus is a Gram-negative bacterium that causes food poisoning as a result of raw or undercooked seafood consumption in humans.
[9] VopS, a type III effector found in Vibrio parahaemolyticus, contains a Fic domain that has a conserved HPFx(D/E)GN(G/K)R motif that contains a histidine residue essential for AMPylation.
The transfer of an AMP moiety using ATP to the threonine residue results in steric hindrance, and thus prevents Rho GTPases from interacting with downstream effectors.
[3][5] The AMPylation on a tyrosine residue of the switch 1 region blocks the interaction of the GTPases with downstream substrates such as PAK.
Widely assumed to be ADP-ribosylation, it turns out to be FICD-mediated AMPylation, as inactivating the FICD gene in cells abolished all measurable post-translational modification of BiP.
[8] An understanding of the structural basis of BiP AMPylation and de-AMPylation is gradually emerging,[18][19] as are clues to the allostery that might regulate the switch in FICD's activity[20] but important details of this process as it occurs in cells remain to be discovered.
[21] 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.
Initially, this defect was attributed to a role for FICD on the cell surface of capitate projections - a putative site of neurotransmitter recycling[22] however a later study implicated FICD-mediated AMPylation of BiP Thr366 in the visual problem[23] The presynaptic protein α-synuclein was found to be a target for FICD AMPylation.
To detect unrecognized modified protein and label VopS substrates, ATP derivatives with a fluorophore at the adenine N6 NH2 is utilized to do that.
AMPylation is a post-translational modification, so it will modify protein properties by giving the polar character of AMP and hydrophobicity.
[5][6] Previously, many science works used Mass Spectrometry (MS) in different fragmentation modes to detect AMPylated peptides.
Due to AMP's stability, peptide fragmentation spectra is easy to read manually or with search engines.
[5][6] Inhibitors of protein AMPylation with inhibitory constant (Ki) ranging from 6 - 50 μM and at least 30-fold selectivity versus HypE have been discovered.
