Synthesis and Characterisation of NAD+ Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation

Loading...
Thumbnail Image
Date
2017
Editors
Contact
Journal ISSN
Electronic ISSN
ISBN
Bibliographical data
Publisher
Series
URI (citable link)
DOI (citable link)
ArXiv-ID
International patent number
Link to the license
EU project number
Project
Open Access publication
Collections
Restricted until
Title in another language
Research Projects
Organizational Units
Journal Issue
Publication type
Dissertation
Publication status
Published
Published in
Abstract
Within the last decade, research on poly(ADP-ribose) experienced a renaissance and various studies unveiled the importance of this biopolymer as a complex, functionally diverse protein modification and signalling molecule. Fundamental processes such as DNA repair and transcription are coordinated by poly(ADP-ribose) and its binding to proteins. Thus, different pathophysiological conditions and disease states, e.g. in cancer and inflammation, have been associated with this protein modification and motivated its targeting in clinical trials.
ADP-ribose chains are tightly regulated in a posttranslational manner by ADP-ribosyl transferases and hydrolases in response to external stimuli and thus cellular levels fluctuate rapidly. Built from NAD+, single or multiple ADP-ribose units are covalently attached onto arginines, glutamates, aspartates or lysines of acceptor proteins. Thereby, nicotinamide is released and mono(ADP-ribos)ylated or poly(ADP-ribos)ylated proteins are formed. Due to these dynamics and complexity, the visualisation of poly(ADP-ribos)ylation remains not only a challenging task, but would also be of high importance to understand these processes on a cellular level.
The aim of this PhD project was to explore the applicability of chemically modified NAD+ analogues for the detection of DNA damage induced poly(ADP-ribos)ylation. Thus, a perfect analogue should be an efficient substrate of ADP-ribosyl transferases in vitro and in cellula. It should be able to specifically label cellular formation of poly(ADP-ribose) in dependency of extrinsic stimuli. Ideally, the new analogue enables to monitor poly(ADP-ribos)ylation in realtime and in a dynamic fashion.
The challenge was met by synthesising NAD+ analogues that can be built into poly(ADPribose) by ARTD1, the major poly(ADP-ribos)ylating enzyme in DNA repair. Thus, the positions as well as the types of NAD+ modifications were investigated, and analogues substituted in adenine position 2 were found to be best-suited for this purpose. Using bioorthogonal reporter groups, the intracellular visualisation of poly(ADP-ribose) was demonstrated simultaneously in two colours, e.g. as required in time dependent or pulsechase experiments. Moreover, a fluorophore-modified NAD+ enabled the direct monitoring of poly(ADP-ribos)ylation inside of a living cell. Thereby, a full turnover of poly(ADP-ribose) was observed after laser-induced DNA damage in real-time. Additionally, protein-specific interaction with poly(ADP-ribose) was detected in intact cells using a powerful FLIM-FRET technique and the GFP-tagged protein of interest. Finally, the substrate scopes of other poly(ADP-ribose) synthesising enzymes like ARTD2, ARTD5 and ARTD6 were explored to broaden the applicability of the developed NAD+ analogues.
In summary, the chemical biology approaches developed in here proved powerful for biological applications and the novel tools will help to elucidate PAR biology by studying the polymer in its natural environment.
Summary in another language
Subject (DDC)
540 Chemistry
Keywords
ARTD; click chemistry; NAD+; poly(ADP-ribose); posttranslational modification
Conference
Review
undefined / . - undefined, undefined. - (undefined; undefined)
Cite This
ISO 690WALLRODT, Sarah, 2017. Synthesis and Characterisation of NAD+ Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Wallrodt2017Synth-38571,
  year={2017},
  title={Synthesis and Characterisation of NAD<sup>+</sup> Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation},
  author={Wallrodt, Sarah},
  address={Konstanz},
  school={Universität Konstanz}
}
RDF
<rdf:RDF
    xmlns:dcterms="http://purl.org/dc/terms/"
    xmlns:dc="http://purl.org/dc/elements/1.1/"
    xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#"
    xmlns:bibo="http://purl.org/ontology/bibo/"
    xmlns:dspace="http://digital-repositories.org/ontologies/dspace/0.1.0#"
    xmlns:foaf="http://xmlns.com/foaf/0.1/"
    xmlns:void="http://rdfs.org/ns/void#"
    xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > 
  <rdf:Description rdf:about="https://kops.uni-konstanz.de/server/rdf/resource/123456789/38571">
    <dspace:isPartOfCollection rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dcterms:abstract xml:lang="eng">Within the last decade, research on poly(ADP-ribose) experienced a renaissance and various studies unveiled the importance of this biopolymer as a complex, functionally diverse protein modification and signalling molecule. Fundamental processes such as DNA repair and transcription are coordinated by poly(ADP-ribose) and its binding to proteins. Thus, different pathophysiological conditions and disease states, e.g. in cancer and inflammation, have been associated with this protein modification and motivated its targeting in clinical trials.&lt;br /&gt;ADP-ribose chains are tightly regulated in a posttranslational manner by ADP-ribosyl transferases and hydrolases in response to external stimuli and thus cellular levels fluctuate rapidly. Built from NAD+, single or multiple ADP-ribose units are covalently attached onto arginines, glutamates, aspartates or lysines of acceptor proteins. Thereby, nicotinamide is released and mono(ADP-ribos)ylated or poly(ADP-ribos)ylated proteins are formed. Due to these dynamics and complexity, the visualisation of poly(ADP-ribos)ylation remains not only a challenging task, but would also be of high importance to understand these processes on a cellular level.&lt;br /&gt;The aim of this PhD project was to explore the applicability of chemically modified NAD+ analogues for the detection of DNA damage induced poly(ADP-ribos)ylation. Thus, a perfect analogue should be an efficient substrate of ADP-ribosyl transferases in vitro and in cellula. It should be able to specifically label cellular formation of poly(ADP-ribose) in dependency of extrinsic stimuli. Ideally, the new analogue enables to monitor poly(ADP-ribos)ylation in realtime and in a dynamic fashion.&lt;br /&gt;The challenge was met by synthesising NAD+ analogues that can be built into poly(ADPribose) by ARTD1, the major poly(ADP-ribos)ylating enzyme in DNA repair. Thus, the positions as well as the types of NAD+ modifications were investigated, and analogues substituted in adenine position 2 were found to be best-suited for this purpose. Using bioorthogonal reporter groups, the intracellular visualisation of poly(ADP-ribose) was demonstrated simultaneously in two colours, e.g. as required in time dependent or pulsechase experiments. Moreover, a fluorophore-modified NAD+ enabled the direct monitoring of poly(ADP-ribos)ylation inside of a living cell. Thereby, a full turnover of poly(ADP-ribose) was observed after laser-induced DNA damage in real-time. Additionally, protein-specific interaction with poly(ADP-ribose) was detected in intact cells using a powerful FLIM-FRET technique and the GFP-tagged protein of interest. Finally, the substrate scopes of other poly(ADP-ribose) synthesising enzymes like ARTD2, ARTD5 and ARTD6 were explored to broaden the applicability of the developed NAD+ analogues.&lt;br /&gt;In summary, the chemical biology approaches developed in here proved powerful for biological applications and the novel tools will help to elucidate PAR biology by studying the polymer in its natural environment.</dcterms:abstract>
    <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-04-24T08:07:20Z</dc:date>
    <dc:language>eng</dc:language>
    <dcterms:rights rdf:resource="https://rightsstatements.org/page/InC/1.0/"/>
    <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/38571/3/Wallrodt_0-405682.pdf"/>
    <dc:rights>terms-of-use</dc:rights>
    <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/>
    <dcterms:issued>2017</dcterms:issued>
    <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2017-04-24T08:07:20Z</dcterms:available>
    <dcterms:isPartOf rdf:resource="https://kops.uni-konstanz.de/server/rdf/resource/123456789/29"/>
    <dcterms:hasPart rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/38571/3/Wallrodt_0-405682.pdf"/>
    <bibo:uri rdf:resource="https://kops.uni-konstanz.de/handle/123456789/38571"/>
    <foaf:homepage rdf:resource="http://localhost:8080/"/>
    <dc:creator>Wallrodt, Sarah</dc:creator>
    <dc:contributor>Wallrodt, Sarah</dc:contributor>
    <dcterms:title>Synthesis and Characterisation of NAD&lt;sup&gt;+&lt;/sup&gt; Analogues for the Cellular Imaging of Poly(ADP-Ribos)ylation</dcterms:title>
  </rdf:Description>
</rdf:RDF>
Internal note
xmlui.Submission.submit.DescribeStep.inputForms.label.kops_note_fromSubmitter
Contact
URL of original publication
Test date of URL
Examination date of dissertation
March 31, 2017
University note
Konstanz, Univ., Doctoral dissertation, 2017
Method of financing
Comment on publication
Alliance license
Corresponding Authors der Uni Konstanz vorhanden
International Co-Authors
Bibliography of Konstanz
Refereed