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

ABSTRACT: 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).

INSTRUMENT(S): Orbitrap Eclipse, Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Joan Josep Bech-Serra

LAB HEAD: Alex Vaquero

PROVIDER: PXD018344 | Pride | 2021-04-19

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	ADPribosylpeptides.xlsx	Xlsx
	SIRT6_WT_S56A_2020MB000_JUMO_001_02_5pto.raw	Raw
	SIRT6_WT_S56A_2020MB000_JUMO_002_02_5pto.raw	Raw
	SIRT6_WT_S56A_peptide.csv	Csv
	SIRT6_WT_S56A_proteins.csv	Csv

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Publications

SirT7 auto-ADP-ribosylation regulates glucose starvation response through mH2A1.

Simonet Nicolás G NG Thackray Joshua K JK Vazquez Berta N BN Ianni Alessandro A Espinosa-Alcantud Maria M Morales-Sanfrutos Julia J Hurtado-Bagès Sarah S Sabidó Eduard E Buschbeck Marcus M Tischfield Jay J De La Torre Carolina C Esteller Manel M Braun Thomas T Olivella Mireia M Serrano Lourdes L Vaquero Alejandro A

Science advances 20200724 30

Sirtuins are key players of metabolic stress response. Originally described as deacetylases, some sirtuins also exhibit poorly understood mono-adenosine 5'-diphosphate (ADP)-ribosyltransferase (mADPRT) activity. We report that the deacetylase SirT7 is a dual sirtuin, as it also features auto-mADPRT activity. SirT7 mADPRT occurs at a previously undefined active site, and its abrogation alters SirT7 chromatin distribution. We identify an epigenetic pathway by which ADP-ribosyl-SirT7 is recognized ...[more]

PMID: 32832656

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A study into the ADP-ribosylome of IFN--stimulated THP-1 human macrophage-like cells identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-ribosylation

Project description:ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN--induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the anti-poly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN- increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN- and ADP-ribosylation signaling pathways.

2020-01-27 | PXD011690 | Pride

Genome-wide analysis of ADP-ribosylation by ADPr-ChAP reveals its association with chromatin after oxidative stress

Project description:Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scaled with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation-dependent. In combination with deep-sequencing and conventional ChIP, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes.

2016-02-04 | GSE69885 | GEO

Temporal and site-specific ADP-ribosylation dynamics upon different genotoxic stresses

Project description:The DNA damage response revolves around transmission of information via post-translational modifications, including reversible protein ADP-ribosylation. Here, we applied a mass spectrometry-based Af1521 enrichment technology for the identification and quantification of ADP-ribosylation sites as a function of various DNA damage stimuli and time. In total, we detected 1,681 ADP-ribosylation sites residing on 716 proteins in U2OS cells, and determined their temporal dynamics after exposure to the genotoxins H2O2 and MMS. Intriguingly, we observed a widespread but low-abundant serine ADP-ribosylation response at the earliest time point, with later time points centered on increased modification of the same sites. This suggests that early serine ADP-ribosylation events may serve as a platform for an integrated signal response. While treatment with H2O2 and MMS induced homogenous ADP-ribosylation responses, we observed temporal differences in the ADP-ribosylation site abundancies. Exposure to MMS-induced alkylating stress induced the strongest ADP-ribosylome response after 30 minutes prominently modifying proteins involved in RNA processing, whereas in response to H2O2-induced oxidative stress ADP-ribosylation peaked after 60 minutes, while mainly modifying proteins involved in DNA damage pathways. Collectively, the dynamic ADP-ribosylome presented here provides valuable insight into the temporal cellular regulation of ADP-ribosylation in response to DNA damage.

2021-11-04 | PXD028902 | Pride

An advanced strategy for comprehensive profiling of ADP-ribosylation sites using mass spectrometry-based proteomics

Project description:ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. While ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered >11,000 unique ADP-ribosylated peptides mapping to >7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. High-resolution MS/MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADP-ribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADP-ribosylation linked to the mitochondrion.

2019-02-26 | PXD012243 | Pride

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome (part 1)

Project description:Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADPribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.

2021-10-07 | PXD023835 | Pride

The regulatory landscape of the human HPF1- and ARH3-dependent ADP-ribosylome (part 2)

2021-10-11 | PXD027504 | Pride

Preservation of Ester-linked Modifications Reveals Glutamate/Aspartate mono-ADP-ribosylation by PARP1 and its Reversal by PARG

Project description:Ester-linked post-translational modifications, including serine and threonine ubiquitination, have gained recognition as important cellular signals. However, their detection remains a significant challenge due to the chemical lability of the ester bond. This is the case even for long-known modifications, such as ADP-ribosylation on aspartate and glutamate, whose role in PARP1 signaling has recently been questioned. We have developed easily implementable methods for preserving ester-linked modifications, which, when combined with a specific and sensitive modular antibody and mass spectrometry, has enabled us to uncover DNA damage-induced aspartate/glutamate mono-ADP-ribosylation. This previously elusive signal represents an initial wave of PARP1 signaling, contrasting with the more enduring nature of serine mono-ADP-ribosylation. Unexpectedly, we have discovered that the poly-ADP-ribose hydrolase PARG is capable of reversing ester-linked mono-ADP-ribosylation in cells. Our methodology enables broad investigations of various ADP-ribosylation writers and, as illustrated here for noncanonical ubiquitination, it paves the way for exploring other emerging ester-linked modifications.

2024-05-08 | PXD048274 | Pride

ADP-ribosylation of STAT1α by PARP-1 is Required for IFNγ-stimulated Transcriptional Responses in Macrophages [H3K27ac_ChIP-Seq_BMDM]

Project description:A key determinant of the pro-inflammatory responses in macrophages is the Signal Transducers and Activators of Transcription (STAT) family member, STAT1α. STAT1α activation by interferon-gamma (IFNγ) leads to induction of a transcriptional program that is coordinated in a large part by post-translational modifications such as phosphorylation. Poly(ADP-ribose) Polymerases (PARPs), which include PARP-1, catalyze the addition of ADP-ribose moieties (ADP-ribosylation) to target proteins and modulate their function. We found that PARP-1 mediates IFNγ-stimulated transcription by regulating genome-wide binding of STAT1α and its IFNγ-dependent phosphorylation. We identified STAT1α as a target of PARP-1 and found sites of ADP-ribosylation on its DNA-binding (DBD) and Transcription Activation (TA) domains. Surprisingly, ADP-ribosylation on the DBD and TA domains had distinct functional consequences on STAT1α transcriptional activity. Moreover, loss of ADP-ribosylation on either site led to diminished pro-inflammatory responses. These results suggest that PARP-1-driven ADP-ribosylation of STAT1α is a critical mediator of inflammation in macrophages.

2021-05-13 | GSE147952 | GEO

ADP-ribosylation of STAT1α by PARP-1 is Required for IFNγ-stimulated Transcriptional Responses in Macrophages [STAT1_ChIP-Seq_BMDM]

2021-05-13 | GSE147951 | GEO