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Pertussis toxin-catalyzed auto- and trans-ADP-ribosylation

ABSTRACT: This dataset was created to identify amino acids in pertussis toxin ADP-ribosyltransferase subunit (PtxS1) that get auto-modified during catalysis. In parallel, amino acids that get ADP-ribosylated by PtxS1 in the host cell target protein, G-protein inhibitory alpha subunit, were identified.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Bordetella Pertussis (strain Tohama I / Atcc Baa-589 / Nctc 13251)

SUBMITTER: Arto Pulliainen

LAB HEAD: Arto Tapio Pulliainen

PROVIDER: PXD050185 | Pride | 2025-05-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	PTX.msf	Msf
	PTX.raw	Raw
	PTX_NAD.msf	Msf
	PTX_NAD.raw	Raw
	PTX_NAD_Galphai.msf	Msf

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1 - 5 of 7

Publications

ADP-ribosyltransferase-based biocatalysis of nonhydrolyzable NAD+ analogs.

Sakari Moona M Bhadane Rajendra R Kumar Sujit S Azevedo Rita R Malakoutikhah Morteza M Masoumi Ahmadreza A Littler Dene R DR Härmä Harri H Kopra Kari K Pulliainen Arto T AT

The Journal of biological chemistry 20241218 1

Enzyme promiscuity is the ability of an enzyme to catalyze an unexpected side reaction in addition to its main reaction. Here, we describe a biocatalytic process to produce nonhydrolyzable NAD+ analogs based on the ADP-ribosyltransferase activity of pertussis toxin PtxS1 subunit. First, in identical manner to normal catalysis, PtxS1 activates NAD+ to form the reactive oxocarbenium cation. Subsequently, the electrophilic ribose 1' carbon of the oxocarbenium cation is subject of an attack by the n ...[more]

PMID: 39706271

Similar Datasets

Gas-phase Fragmentation of ADP-Ribosylated Peptides: Arginine-Specific Side-Chain Losses and their Implication in Database Searches

Project description:ADP-ribosylation is a reversible post-translational modification of proteins that has been linked to many biological processes. The identification of ADP-ribosylated proteins and of their acceptor amino acids remains a significant challenge. The attachment sites of the modification are difficult to study by mass spectrometry (MS) because of its labile nature and its complex fragmentation pattern in MS/MS experiments. In this study we performed a detailed analysis of higher-energy collisional dissociation (HCD) spectra acquired from ADP-ribosylated peptides which were modified on arginine, serine, glutamic acid, aspartic acid, tyrosine or lysine. In addition to the fragmentation of the peptide backbone, various cleavages of bonds within the ADP-ribose, and between the modification and the amino acid residue, have to be considered. We focused on gas-phase fragmentations that are specific either to ADP-ribosylated arginine or to ADP-ribosylated serine and other O-linked ADP-ribosylations. The O-glycosidic linkage between ADP-ribose and serine, glutamic acid or aspartic acid is the major cleavage site, making localization of these modification sites difficult. In contrast, the bond between ADP-ribose and arginine is relatively stable. The main cleavage site is the inner bond of the guanidine which results in the formation of ADP-ribosylated carbodiimide and of ornithine in place of modified arginine. Taking this specific cleavage into account, a considerably larger number of peptides containing ADP-ribosylated arginine were identified in database searches, and their modification sites were assigned with increased confidence.

2021-05-19 | PXD017013 | Pride

Development of a mass-spectrometry based method for the identification of the in vivo whole blood and plasma ADP-ribosylomes

Project description:Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modifications of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove the modification after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we established that plasma phosphodiesterases and ADP-ribosylhydrolases rapidly demodified in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a novel mass-spectrometry based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

2021-05-26 | PXD022156 | Pride

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site

Project description:Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-site identification and localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1 dependent in vivo ADPr-acceptor. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

2018-06-28 | PXD009948 | Pride

Tankyrase-mediated ADP-ribosylation is a novel regulator of TNF-induced death

Project description:TNF is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1, or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146 resulting in proteasomal degradation of complex 2 thereby limiting cell death. Intriguingly, expression of the ADP-ribose binding/hydrolyzing SARS-CoV-2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

2022-06-09 | PXD032131 | Pride

Proteomic characterization of the heart and skeletal muscle reveals widespread Arginine ADP-ribosylation by the ectopic ADP-ribosyltransferase ARTC1

Project description:ADP-ribosylation has thus far been studied on the proteomic level mainly in cell lines and in combination with genotoxic stress. The clostridium-like ADP-ribosyltransferases (i.e. ARTC) are GPI-anchored or secreted proteins that are expressed in a highly tissue specific manner. Transcriptomic data revealed that ARTC1 is expressed in skeletal muscle and heart tissue. Although ARTC1 is highly active in vitro, identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level has been challenging and only a few ADP-ribosylated targets are known. Using a new MS-based workflow, we provide the thus far most extensive identification of endogenous ADP-ribosylomes with hundreds of ARTC1-ADP-ribosylated proteins in C2C12 myotubes and in skeletal muscle and heart tissues from wild type and newly generated ARTC1 knockout mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport and muscle function. Together we provide the first comprehensive systems-level analysis of endogenous ADP-ribosylation in two different muscle tissues revealing a widespread function of ARTC1 and its targets.

2018-08-17 | PXD008041 | Pride

Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase

Project description:ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine are still missing. Here, we report that in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometry approach showed that ARH3-deficient MEFs are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen-peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress induced ADP-ribosylome.

2017-12-14 | PXD008083 | Pride

Salmonella antibacterial Rhs polymorphic toxin inhibits translation through ADP-ribosylation of EF-Tu P-loop

Project description:Rearrangement hot spots (Rhs) proteins are members of the broad family of polymorphic toxins. Polymorphic toxins are modular proteins composed of an N-terminal region that specifies their mode of secretion into the medium or into the target cell, a central delivery module and a C-terminal domain that has toxic activity. Here, we structurally and functionally characterize the C-terminal toxic domain of the antibacterial Rhsmain protein, which is delivered by the Type VI secretion system of Salmonella enterica Typhimurium. We show that this domain adopts an ADP-ribosyltransferase fold and inhibits protein synthesis by transferring an ADP-ribose group from NAD+ to the elongation factor EFTu. This modification is specifically placed on the sidechain of the conserved D21 residue located on the P-loop of the EF-Tu G-domain. Finally, we demonstrate that its cognate immunity protein neutralizes Rhsmain C-terminal toxin activity by acting like a lid that closes the catalytic site and traps the NAD+.

2023-03-11 | PXD033100 | Pride

Lack of the mitochondrial NAD transporter SLC25A51 redistributes NAD+ compartmentalization and cellular ADP-ribosylation

Project description:Nicotinamide adenine dinucleotide (NAD+) is a vital small molecule with important redox capacity in oxidative phosphorylation (OXPHOS) and a key co-factor in various enzymatic reactions. The recent identification of the mitochondrial NAD+ transporter SLC25A51 provides strong evidence for a direct regulation of the mitochondrial NAD+ pool. Though the effect of this transporter on glucose metabolism has been described, its contribution to other NAD+-dependent processes such as ADP-ribosylation remains elusive. Here, we report that knockdown of SLC25A51 decreased the NAD+ concentration in mitochondria but increased the NAD+ concentration in the cytoplasm and nucleus. The increase in nuclear and cytoplasmic NAD+ was not due to the upregulation of the salvage pathway, thus pointing towards an overall redistribution of NAD+ from the mitochondria towards the cyto/nuclear compartment. Furthermore, the NAD+ redistribution induced by knockdown or knockout of SLC25A51 resulted, as quantified by immunofluorescence or analyzed by mass-spectrometry, in a loss of mitochondrial ADP-ribosylation and an increase of PARP1-mediated nuclear ADP-ribosylation under basal conditions. Further, MMS/Olaparib induced PARP1 chromatin retention and the sensitivity of triple-negative MDA-MB-436 breast cancer cells to PARP inhibition were both reduced upon knockdown of SLC25A51. In addition, H2O2-induced PARP1-dependent nuclear ADP-ribosylation was prolonged while phosphorylation of H2AX was unexpectedly reduced. Together these results provide evidence that lack of SCL25A51 and subsequently altered NAD+ compartmentalization affects not only mitochondrial and nuclear ADP-ribosylation but also other chromatin associated events.

2023-10-24 | PXD031373 | Pride

Serine ADP-ribosylation depends on HPF1

Project description:We report that serine ADPr is strictly dependent on HPF1 (histone PARylation factor 1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. We identified three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on hundreds of other targets indicates that serine ADPr is a widespread modification.

2017-02-10 | PXD005627 | Pride

System-wide identification of enzyme substrates by thermal analysis (SIESTA)

Project description:Despite the immense importance of enzyme-substrate reactions, there is a lack of general and unbiased tools for identifying the molecular components participating in these reactions on a cellular level. Here we developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that post-translational modification of the substrate protein changes its thermal stability, and applies the concept of specificity to reveal the potential substrates. For selenoprotein thioredoxin reductase 1, SIESTA confirmed several known protein substrates and suggested novel candidates. For poly-(ADP-ribose) polymerase-10, SIESTA revealed a number of PARP10 putative substrates, which were confirmed by targeted mass spectrometry and functional assays. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, and facilitate drug discovery.

2021-01-11 | PXD010554 | Pride

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