Real‐Time Cellular Imaging of Protein Poly(ADP‐ribos)ylation

Buntz, Annette; Wallrodt, Sarah; Gwosch, Eva; Schmalz, Michael; Beneke, Sascha; Ferrando‐May, Elisa; Marx, Andreas; Zumbusch, Andreas

doi:10.1002/anie.201605282

Cited by 35 publications

(50 citation statements)

References 34 publications

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“…Functionalised full-length NAD + analogues have recently emerged as promising tools for fluorescence-based visualisation of ADP-ribosylation in cell-lysates 11,16,17,18 and in cells 19,20 .…”

Section: Discussionmentioning

confidence: 99%

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

Kalesh

Lukauskas

Borg

et al. 2019

Preprint

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ADP-ribosylation is integral to a diverse range of cellular processes such as DNA repair, chromatin regulation and RNA processing. However, proteome-wide investigation of its cellular functions has been limited due to numerous technical challenges including the complexity of the poly(ADP-ribose) (PAR) chains, low abundance of the modification and lack of sensitive enrichment methods. To facilitate livecell profiling of ADP-ribosylated proteins, we show that 2-alkyne-adenosine (2YnAd) is metabolically incorporated in mammalian cells and enables fluorescent detection and robust affinity enrichment of the modified proteins. We then present an integrated chemical biology approach that involves simultaneous metabolic incorporation of 2YnAd and the previously reported 6-alkyne-adenosine (6YnAd) in live cells followed by click chemistry with a capture reagent to facilitate highly sensitive and comprehensive enrichment of the modified proteins. By combining this dual metabolic labelling strategy with the tandem mass tag (TMT) isobaric mass spectrometry, we have quantified the responses of thousands of endogenous proteins to clinical PARP inhibitors Olaparib and Rucaparib. Our study provides insight into the wider scope of PARP targets and will help to advance further characterisation of their functional roles.Recently, it has been demonstrated that 6-alkyne adenosine (6YnAd), a compound that was previously demonstrated to be suitable for labelling poly(A) tails of mRNAs in mammalian cells 9 , enables sensitive fluorescence profiling of ADP-ribosylated proteins in live cells 10 . In this work, we analysed the labelling efficiency of 6YnAd for the first time using Tandem Mass Tag (TMT)-based quantitative proteomics and found that 6YnAd alone limits substrate coverage, but that inclusion of a similar adenosine analogue, 2-alkyne adenosine (2YnAd), allows a more comprehensive assessment of ADP-ribosylated proteins. We report an integrated chemical proteomics approach that enables the quantitative profiling of ADPribosylation of several thousands of endogenous proteins in a robust, reproducible and unbiased manner. Cell culture. MDA-MB-231 cells (ATCC) were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS) (Gibco) without antibiotics in a humidified incubator with 5% CO2 at 37 o C and passaged at every 2-3 days at 80-90% confluency. Metabolic labelling and preparation of cell extracts. In a 6-well plate, 4 x 10 5 cells were treated with 1 ml of fresh media (with 10% FBS) containing 2 µl of appropriate concentration of 6YnAd or 2YnAd (stock solutions in dimethyl sulfoxide [DMSO]) or DMSO control. For labelling experiments in 10 cm plates, 5 ml of fresh media with 10% FBS and 10 µl of the clickable adenosine analogue stock solutions were used. The cells were incubated at 37 o C and 5% CO2 for 1 h. The cells were then washed with PBS three times and lysed using 4% SDS lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 4% SDS and 500 U Benzonase) and whole-cell lysates were colle...

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Section: Discussionmentioning

confidence: 99%

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

Kalesh

Lukauskas

Borg

et al. 2019

Preprint

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“…For this purpose, we selected bulky, dye-modified NAD + analogues 5 and 6 , which were previously prepared by our group [21], in order to have a direct, fluorescent read-out. The outcome is summarised in Table 2 and the SDS PAGE gels obtained are depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD⁺ analogues

Wallrodt¹,

Simpson²,

Marx³

2017

Beilstein J. Org. Chem.

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ADP-ribosyl transferases with diphtheria toxin homology (ARTDs) catalyse the covalent addition of ADP-ribose onto different acceptors forming mono- or poly(ADP-ribos)ylated proteins. Out of the 18 members identified, only four are known to synthesise the complex poly(ADP-ribose) biopolymer. The investigation of this posttranslational modification is important due to its involvement in cancer and other diseases. Lately, metabolic labelling approaches comprising different reporter-modified NAD+ building blocks have stimulated and enriched proteomic studies and imaging applications of ADP-ribosylation processes. Herein, we compare the substrate scope and applicability of different NAD+ analogues for the investigation of the polymer-synthesising enzymes ARTD1, ARTD2, ARTD5 and ARTD6. By varying the site and size of the NAD+ modification, suitable probes were identified for each enzyme. This report provides guidelines for choosing analogues for studying poly(ADP-ribose)-synthesising enzymes.

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“…Previous studies report a series of chemically synthesized NAD + analogues for investigating NAD + ‐dependent ADP‐ribosylation (Buntz et al., ; Carter‐O'Connell et al., , ; Gibson et al., ; Jiang, Kim, Frizzell, Kraus, & Lin, ; Wallrodt, Buntz, Wang, Zumbusch, & Marx, ; Wang, Rösner, Grzywa, & Marx, ). These synthetic methods for preparing NAD + analogues have proven challenging because of the synthetic complexity, low efficiency for the last difficult pyrophosphate coupling step, and tedious and time‐consuming product purification.…”

Section: Commentarymentioning

confidence: 99%

Chemoenzymatic Preparation of 4′‐Thioribose NAD⁺

Zhang

Dai

Cheng

et al. 2019

CP Nucleic Acid Chemistry

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This chemoenzymatic procedure describes a strategy for the preparation of 4′‐thioribose nicotinamide adenine dinucleotide (S‐NAD+), including chemical synthesis of nicotinamide 4′‐riboside (S‐NR), recombinant expression and purification of two NAD+ biosynthesis enzymes nicotinamide riboside kinase (NRK) and nicotinamide mononucleotide adenylyltransferase (NMNAT), and enzymatic synthesis of S‐NAD+. The first basic protocol describes the procedures for introduction of nicotinamide onto 4′‐thioribose and subsequent deprotection to generate S‐NR as the key intermediate for enzymatically synthesizing S‐NAD+. In the second basic protocol, experimental methods are detailed for the production of recombinant human NRK1 and NMNAT1 to catalyze conversion of S‐NR to S‐NAD+. The third basic protocol presents the enzymatic approach for the generation of S‐NAD+ from S‐NR precursor. © 2019 by John Wiley & Sons, Inc.

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Real‐Time Cellular Imaging of Protein Poly(ADP‐ribos)ylation

Cited by 35 publications

References 34 publications

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD⁺ analogues

Chemoenzymatic Preparation of 4′‐Thioribose NAD⁺

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Real‐Time Cellular Imaging of Protein Poly(ADP‐ribos)ylation

Cited by 35 publications

References 34 publications

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

An Integrated Chemical Proteomics Approach for Quantitative Profiling of Intracellular ADP-Ribosylation

Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD+ analogues

Chemoenzymatic Preparation of 4′‐Thioribose NAD+

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Product

Resources

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Investigation of the action of poly(ADP-ribose)-synthesising enzymes on NAD⁺ analogues

Chemoenzymatic Preparation of 4′‐Thioribose NAD⁺