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HYPE mediated AMPylation site profiling with YnATP in human lysates

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Remigiusz Serwa

LAB HEAD: Edward Tate

PROVIDER: PXD003053 | Pride | 2015-12-02

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	ATP1.mgf	Mgf
	ATP1peptides_1_1_0.mzid.gz	Mzid
	ATP1peptides_1_1_0.pride.mztab.gz	Mztab
	ATP2.mgf	Mgf
	ATP2peptides_1_1_0.mzid.gz	Mzid

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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

Similar Datasets

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

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

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

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

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

Evolution of the eye transcriptome under constant darkness in Sinocyclocheilus cavefish

Project description:we report a transcriptome-wide comparative investigation between surface and cave species in Sinocyclocheilus. De novo transcriptome assemblies were performed on surface and cave species; then the Sinocyclocheilus contigs were annotated with Gene Ontology. RNA-Seq assays revealed reduced transcription of a series of visual phototransduction and retinal disease related genes in cave-dwelling species compared with surface species. Degeneration of the retina in Sinocyclocheilus cavefish might occur in a lens-independent way by the down-regulation of several transcriptional factors, which have direct roles in retina development and maintenance, such as crx, rorb and Wnt pathway members. Examination of 2 different eye samples in 2 Sinocyclocheilus species.

2013-03-31 | E-GEOD-40446 | biostudies-arrayexpress

Identification of auto mono-ADP-ribosylation sites on SIRT6 and SIRT7

Project description:The aim of this project was to identify the auto mono-ADP-ribosylation sites on SIRT6 and SIRT7 to study the effect of the point mutations S56A and N189A respectively on their ADP-ribosylation activity. In addition, the mono-ADP-ribosylation pattern of SIRT7 was obtained in cells under different stress conditions (UV, H2O2, ionizing Irradiation and glucose starvation).

2021-04-19 | PXD018344 | Pride

Gene Expression Defines Natural Changes in Mammalian Lifespan

Project description:Mammals differ more than hundred fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life history traits, and identified the processes and pathways involved. These findings provide direct insights into how Nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages. RNA-seq gene expression profiling in normal liver, kidney and brain of 33 mammalian species.

2015-02-17 | E-GEOD-43013 | biostudies-arrayexpress

Developmental gene expression of Hymenochirus boettgeri

Project description:RNA-seq experiments to construct Hymenochirus bettgeri transcripts Collect mRNA from whole eggs/embryos/tadpoles, with poly-A enrichment method

2016-10-24 | E-GEOD-76089 | biostudies-arrayexpress

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