User:Giangpham17/Adenylylation

General intro
AMPylation uses ATP as a substrate. Another function of adenylylation is amino acids activation, which is catalyzed by tRNA aminoacyl synthetase.

Adenylylation  + adenylylators
This enzyme is created by two catalytic homologous halves. One half is responsible for catalyzing the adenylylation reaction, while the other half catalyzes the phosphorolytic deadenylylation reaction.

PII is the regulatory protein that controls the specificity of AMPylators. PII is the trimeric protein. There are two forms of PII, including unmodified PII and PII - UMP. PII binds to AMPylator catalyzes the attachment of AMP to the post-translational enzyme ( the most famous one is glutamine synthetase). This attachment initiates the adenylylation, which converts glutamine synthetase from active form to the inactive form. Conversely, the complex PII-AMP and AMPylators deadenylylated by removing AMP from the adenylated enzyme, leading to converting inactive glutamine synthetase to active form.

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 and bacterial pathogenicity
VopS also adenylates RhoA and cell division cycle 42 (CDC42), leading to a disaggregation of the actin filament network. As a result, the host cell’s actin cytoskeleton control is disabled, leading to cell rounding.

DrrA is the Dot/Icm type IV translocation system substrate DrrA from Legionella pneumophila. It is the effector secreted by L.pneumophila to modify GTPases of the host cells. This modification increases the survival of bacteria in host cells. DrrA is composed of Rab1b specific guanine nucleotide exchange factor (GEF) domain, a C-terminal lipid binding domain and an N-terminal domain with unclear cytotoxic properties. Research works show that N-terminal and full-length DrrA shows AMPylators activity toward host’s Rab1b protein (Ras related protein), which is also the substrate of Rab1b GEF domain. Rab1b protein is the GTPase Rabs to regulate vesicle transportation and membrane fusion. The adenylation by bacteria AMPylators prolongs GTP-bound state of Rab1b. Thus, the role of effector DrrA is connected toward the benefits of bacteria's vacuoles for their replication during the infection.

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.

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.

Chemical Handles
Chemical handles are used to detect post-translationally modified proteins. Recently, there is a N6pATP that contains an alkynyl tag (propargyl) at the N6 position of the adenine of ATP. This N6pATP combines with the click reaction to detect adenylylated proteins.To detect unrecognized modified protein and label VopS substrates, ATP derivatives with a fluorophore at the adenine N6 NH2 is utilized to do that.

Antibody-based Methods
Antibody is famous for its high affinity and selectivity, so it is the good way to detect adenylylated proteins. Recently, ɑ- AMP antibodies is used to detect and isolate adenylylated protein from cells and cell lysates. Adenylylation is a post-translational modification, so it will modify protein properties by giving the polar character of AMP and hydrophobicity. Thus, instead of using antibodies that detect a whole peptide sequence, raising AMP antibodies directly targeted to specific amino acids are prefered.

Mass Spectrometry Techniques
Previously, many science works used Mass Spectrometry (MS) in different fragmentation modes to detect adenylylated peptides. In responses to the distinctive fragmentation techniques, adenylylated protein sequences disintegrated at different parts of AMP. While electron transfer dissociation (ETD) creates minimum fragments and less complicated spectra, collision-induced dissociation (CID) and high-energy collision (HCD) fragmentation generate characteristic ions suitable for adenylyated proteins identification by generating multiple AMP fragments. Due to AMP’s stability, peptide fragmentation spectra is easy to read manually or with search engines.

Small Molecule Inhibition of Adenylyl Transferase
The first inhibitor of protein adenylylation is found recently by Thompson and coworkers. This is believed as an effective way to defeat infections. The inhibitor has an inhibitory constant (Ki) ranging from 6 - 50 µM and at least 30-fold selectivity versus HYPE ( the only known human adenylyl transferase).