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HYPE mediated AMPylation profiling in human lysates

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Remigiusz Serwa

LAB HEAD: Edward W. Tate

PROVIDER: PXD002601 | Pride | 2015-12-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	AMPylation_HYPE.mgf	Mgf
	AMPylation_HYPEpeptides.pep.xml	Pepxml
	ATP1.mgf	Mgf
	ATP1peptides.pep.xml	Pepxml
	ATP2.mgf	Mgf

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1 - 5 of 62

Publications

Global Profiling of Huntingtin-associated protein E (HYPE)-Mediated AMPylation through a Chemical Proteomic Approach.

Broncel Malgorzata M Serwa Remigiusz A RA Bunney Tom D TD Katan Matilda M Tate Edward W EW

Molecular & cellular proteomics : MCP 20151124 2

AMPylation of mammalian small GTPases by bacterial virulence factors can be a key step in bacterial infection of host cells, and constitutes a potential drug target. This posttranslational modification also exists in eukaryotes, and AMP transferase activity was recently assigned to HYPE Filamentation induced by cyclic AMP domain containing protein (FICD) protein, which is conserved from Caenorhabditis elegans to humans. In contrast to bacterial AMP transferases, only a small number of HYPE subst ...[more]

PMID: 26604261

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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

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.

2017-03-29 | MSV000080782 | MassIVE

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

Diadenosine tetraphosphate as a cosubstrate for AMPylation mediated by FICD

Project description: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.

2023-01-27 | PXD036450 | 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

AMPylation profiling during neuronal differentiation reveals extensive variation on lysosomal proteins

Project description:Protein AMPylation is a prevalent posttranslational modification with an emerging role in neurodevelopment and neurodegeneration. Although in metazoans the two highly conserved protein AMP-transferases together with diverse group of AMPylated proteins have been identified using chemical proteomics and biochemical techniques the function of this modification remains largely unknown. Particularly problematic is a localisation of thus far identified AMPylated proteins and putative AMP-transferases. Here, we uncover protein AMPylation as a novel lysosomal protein posttranslational modification characteristic for differentiating neurons. The AMPylated soluble form of exonuclease PLD3 localised in lysosomes shows dramatic increase during the differentiation of neuronal cell lineages. Similar AMPylation pattern has been observed for a lysosomal acid phosphatase ACP2. Our discovery was enabled by combination of chemical proteomics and novel gel-based separation technique of modified and non-modified proteins. Together, our findings expose further the connection between the protein AMPylation and neurodevelopment and reveal a novel lysosomal posttranslational modification.

2021-12-01 | PXD023873 | Pride

AMpylation is a hallmark of human neuronal differentiation

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 (PAK-76) 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.

2024-05-21 | PXD008394 | 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

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

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

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