Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Diadenosine tetraphosphate as a cosubstrate for AMPylation mediated by FICD

ABSTRACT: Diadenosine polyphosphates (ApnAs) are noncanonical nucleotides that are ubiquitous across all forms of life. Due to their strong accumulation upon cellular stress, they are considered alarmones to signal homeostatic imbalance. Nonetheless, their cellular role is poorly understood. In this work, we assessed ApnAs for their usage as cosubstrate for protein AMPylation, a post-translational modification. In humans, AMPylation mediated by the AMPylator FICD with ATP as cosubstrate is a response to ER stress. Here, we demonstrate that Ap4A is proficiently consumed for AMPylation by FICD. By chemical proteomics using a new chemical probe, we identified new potential AMPylation targets. Interestingly, we found that AMPylation targets of FICD may differ depending on the nucleotide cosubstrate. These results may suggest that signaling at elevated Ap4A levels during cellular stress differs from when Ap4A is present at low concentrations allowing response to extracellular cues.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Philip Saumer

LAB HEAD: Andreas Marx

PROVIDER: PXD036450 | Pride | 2023-01-27

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Structural basis of chaperone regulation by a post-translational modification

Project description:The Hsp70 chaperone BiP is covalently modified with adenosine monophosphate (referred to as AMPylation) in order to adapt its activity to the fluctuating folding load within the endoplasmic reticulum. This modification is catalyzed by the only human representative of the family of filamentation induced by cyclic adenosine monophosphate (Fic) enzymes HYPE/FICD. The structural basis for BiP binding and AMPylation has remained elusive due to the low affinity of enzyme substrate complexes.

2021-04-27 | PXD022869 | Pride

FICD activity and AMPylation remodelling modulate human neurogenesis

Project description:The AMPylation is novel protein post-translation modification. In consist of attachment of the adenosine monophosphate onto the serine, threonine or tyrosine amino acid residues of the protein. This process is catalysed by bacterial effectors or in human by protein FICD (also called HYPE) which contain conserved fic domain. In our study we have focused on identification of the unknown AMPylated proteins and their function in human cells (HeLa, A549, SH-SY5Y, iPSCs, NPCs and neurons) and cerebral organoids (COs). For this purpose we designed and synthesized the small molecule N6-propargyl phosphoramidate adenosine (pro-N6pA) which was used for in situ labelling of the AMPylation in cells and COs. Subsequnetly the the propargyl tag was employed for preparation of chemical-proteomic samples which were measured by LC-MS/MS. This approach confirmed the known AMPylation of heat shock protein HSPA5 and found diverse group of novel AMPylation targets. Several cathepsins found as AMPylation targets were further characterized by identification of AMP binding site. Based on our chemical proteomic data we found out that neurons and neuronal like cells contain distinct AMPylatino pattern from other studied cell types. This also suggested the importance of the AMPylation in these cell types which were then studied in cerebral organoids by overexpressing the FICD wt and E234G highly active mutant. This led to the accelerated differentiation of progenitors into the neurons. Overall our approach is suitable for identification of the AMPylated proteins in living cells and led to characterization of FICD function.

2019-12-04 | PXD015062 | Pride

HYPE mediated AMPylation site profiling with YnATP in human lysates

Project description:We describe here the first example of global chemoproteomic screening for HYPE mediated AMPylation sites of human proteins. Catalytically active mutant HYPE E234G was applied in combination with an alkynylated ATP and an alkynyl-biotinylated side ID reagent for capture of chemoenzymatically tagged proteins. AMPylation sites were detected by shotgun proteomics performed on a Q-Exactive instrument.

2015-12-02 | PXD003053 | Pride

The DNA-binding induced (de)AMPylation activity of a Coxiella burnetii Fic enzyme targets Histone H3

Project description:Intracellular bacterial pathogens such as Coxiella burnetii evade their host's immune response by secreting effector proteins that bind and modify host proteins, thereby reshaping cell signaling pathways that help establish a replication-friendly niche. Previous research has identified Filamentation induced by cyclic AMP (Fic) enzymes as effector proteins in bacterial infections that modify their target proteins via the post-translational modification AMPylation. Fic enzymes use adenosine triphosphate (ATP) as co-substrate to transfer the adenosine monophosphate (AMP) group to a hydroxyl-containing side chain of their target protein. Many representatives of this enzyme class contain an autoinhibitory helix that suppresses AMPylation activity in vitro by contacting the active site. The mechanisms reversing this inhibition are incompletely understood. Extensive studies of the only human Fic protein FICD have revealed that FICD can switch between AMPylation and the reversal of this modification, deAMPylation, by a monomer-dimer transition. However, so far FICD remained the only example where deAMPylation by dimerization could be shown. Here we identify the protein product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at S10 and S28. We show that CbFic2 is a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via its C-terminal helix-turn-helix domain. We propose that CbFic2 is capable of AMPylation in its monomeric form, and that binding to DNA induces a deAMPylating dimer.

2024-01-26 | PXD040330 | Pride

HYPE mediated AMPylation profiling in human lysates

Project description:We describe here the first example of global chemoproteomic screening and substrate validation for HYPE mediated AMPylation in mammalian cell lysate. Through quantitative mass spectrometry-based proteomics coupled with novel chemoproteomic tools providing MS/MS evidence of AMP modification we identified a total of 27 AMPylated proteins, including the previously validated substrate, ER chaperone BiP (HSPA5), along with novel substrates involved in pathways of gene expression, ATP biosynthesis and maintenance of cytoskeleton. This dataset represents the largest library of AMPylated human proteins reported to date and a foundation for substrate-specific investigations that can ultimately decipher the complex biological networks involved in eukaryotic AMPylation.

2015-12-02 | PXD002601 | Pride

Rab1b-AMPylation by Legionella DrrA is allosterically activated by Rab1

Project description:Legionella pneumophila infect eukaryotic cells by forming a replicative organelle – the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and posttranslational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP) – a process referred to as AMPylation. Here, we used a chemical approach for stabilizing low affinity Rab:DrrA complexes in a site-specific manner to gain insights into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The X-ray crystal structure of the Rab:DrrA complex revealed a previously unrecognized non-conventional Rab binding site (NC-RBS). Biochemical characterizations demonstrated allosteric stimulation of DrrA’s AMPylation activity via Rab-binding to the NC-RBS. We propose that allosteric control of DrrA not only prevents random and potentially cytotoxic AMPylation in the host, thereby ensuring an efficient infection process by Legionella, but also represents an unprecedented AMPylation activation mechanism.

2021-01-04 | PXD019043 | Pride

Pronucleotide probes reveal a diverging specificity for AMPylation vs. UMPylation of human and bacterial nucleotide transferases

Project description:AMPylation is a post-translational modification utilized by human and bacterial cells to modulate the activity and function of specific proteins. Major AMPylators such as human FICD and bacterial VopS have been studied extensively for their substrate and target scope in vitro. Recently, an AMP pronucleotide probe also facilitated the in situ analysis of AMPylation in living cells. Based on this technology we here introduce a novel UMP pronucleotide probe and utilize it in the profiling of uninfected and Vibrio parahaemolyticus infected human cells. Mass spectrometric analysis of labeled protein targets reveals an unexpected promiscuity of human nucleotide transferases with an almost identical target set of AMP- and UMPylated proteins. Vice versa, studies in cells infected by V. parahaemolyticus and its effector VopS revealed solely AMPylation of host enzymes highlighting a so far unknown specificity of this transferase for ATP. Taken together, pronucleotide probes provide an unprecedented insight into the in situ activity profile of crucial nucleotide transferases which can largely differ compared to their in vitro activity.

2024-02-27 | PXD045925 | Pride

MS-based in situ proteomics reveals AMPylation of host proteins during bacterial infection

Project description:Bacteria utilize versatile strategies to propagate infections within human cells, e.g. by the injection of effector proteins which alter crucial signaling pathways. One class of such virulence-associated proteins is involved in the AMPylation of eukaryotic Rho GTPases with devastating effects on viability. In order to get an inventory of AMPylated proteins, several technologies have been developed. However, as they were designed for the analysis of cell lysates, knowledge about AMPylation targets in living cells is largely lacking. We here implement a chemical-proteomic method for deciphering AMPylated host proteins in situ during bacterial infection. HeLa cells treated with a previously established cell permeable pronucleotide probe (pro-N6pA) were infected with Vibrio parahaemolyticus and modified host proteins were identified upon probe enrichment and LC-MS/MS analysis. Three already known targets of the AMPylator VopS - Rac1, RhoA and Cdc42 - could be confirmed and several other Rho GTPases were additionally identified. Applying an inactive VopS mutant validated these hits. The utility was further demonstrated by connecting the virulence phenotype of cell rounding with VopS mediated AMPylation of essential targets as well as deciphering the sites of modification. Overall, the methodology provides a reliable detection of host AMPylation in situ and thus a versatile tool in monitoring infection processes.

2020-12-08 | PXD022078 | Pride

Chemoproteomic discovery of a human RNA ligase

Project description:RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5'-PO4 and 3'-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as the first human enzyme promoting RNA ligation between 5'-PO4 and 3'-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. This data provides the groundwork for establishing a human RNA repair pathway.

2023-01-27 | PXD038132 | Pride

Pronucleotide probe for in vivo imaging of protein AMPylation.

Project description:Protein AMPylation is a prevalent posttranslational modification in human cells involved in the regulation of unfolded protein response and neural development. Here we present a novel pro-N6azA probe suitable for in vivo imaging and chemical proteomics profilling of AMPylated proteins. The pro-N6azA probe provides stable fluorescent labelling in living cells accesible by straightforward strain-promoted azide-alkyne click reaction. Application of this probe may contribute significantly to the analysis of protein AMPylation during various metabolic processes including neurodevelopment and unfolded protein response.

2019-12-10 | PXD016011 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data