Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate
The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing me...
| Main Authors: | , , , , |
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| Format: | Journal article |
| Language: | English |
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Cell Press
2018
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| _version_ | 1797081744853172224 |
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| author | Drown, B Shirai, T Rack, J Ahel, I Hergenrother, P |
| author_facet | Drown, B Shirai, T Rack, J Ahel, I Hergenrother, P |
| author_sort | Drown, B |
| collection | OXFORD |
| description | The post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes. |
| first_indexed | 2024-03-07T01:18:19Z |
| format | Journal article |
| id | oxford-uuid:8f78333d-f05b-4230-a8b6-a04a606c5eb9 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:18:19Z |
| publishDate | 2018 |
| publisher | Cell Press |
| record_format | dspace |
| spelling | oxford-uuid:8f78333d-f05b-4230-a8b6-a04a606c5eb92022-03-26T23:04:31ZMonitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrateJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:8f78333d-f05b-4230-a8b6-a04a606c5eb9EnglishSymplectic Elements at OxfordCell Press2018Drown, BShirai, TRack, JAhel, IHergenrother, PThe post-translational modification (PTM) and signaling molecule poly(ADP-ribose) (PAR) has an impact on diverse biological processes. This PTM is regulated by a series of ADP-ribosyl glycohydrolases (PARG enzymes) that cleave polymers and/or liberate monomers from their protein targets. Existing methods for monitoring these hydrolases rely on detection of the natural substrate, PAR, commonly achieved via radioisotopic labeling. Here we disclose a general substrate for monitoring PARG activity, TFMU-ADPr, which directly reports on total PAR hydrolase activity via release of a fluorophore; this substrate has excellent reactivity, generality (processed by the major PARG enzymes), stability, and usability. A second substrate, TFMU-IDPr, selectively reports on PARG activity only from the enzyme ARH3. Use of these probes in whole-cell lysate experiments has revealed a mechanism by which ARH3 is inhibited by cholera toxin. TFMU-ADPr and TFMU-IDPr are versatile tools for assessing small-molecule inhibitors in vitro and probing the regulation of ADP-ribosyl catabolic enzymes. |
| spellingShingle | Drown, B Shirai, T Rack, J Ahel, I Hergenrother, P Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title | Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title_full | Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title_fullStr | Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title_full_unstemmed | Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title_short | Monitoring poly(ADP-ribosyl)glycohydrolase activity with a continuous fluorescent substrate |
| title_sort | monitoring poly adp ribosyl glycohydrolase activity with a continuous fluorescent substrate |
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