Structures of the Human Poly (ADP-Ribose) Glycohydrolase Catalytic Domain Confirm Catalytic Mechanism and Explain Inhibition by ADP-HPD Derivatives

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA dam...

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Vydané v:PloS one Ročník 7; číslo 12; s. e50889
Hlavní autori: Tucker, Julie A., Bennett, Neil, Brassington, Claire, Durant, Stephen T., Hassall, Giles, Holdgate, Geoff, McAlister, Mark, Nissink, J. Willem M., Truman, Caroline, Watson, Martin
Médium: Journal Article
Jazyk:English
Vydavateľské údaje: United States Public Library of Science 10.12.2012
Public Library of Science (PLoS)
Predmet:
Adenosine
Adenosine diphosphate
Automation
Binding sites
Biology
Calorimetry
Catalysis
Catalytic Domain
Cell death
Computational Biology
Conservation
Crystallography
Deoxyribonucleic acid
DNA
DNA damage
Enzymes
Glycoside Hydrolases - chemistry
Health aspects
Humans
Hydrolysis
Inhibitors
Linkages
Monosaccharides
Poly(ADP-ribose)
Poly(ADP-ribose) glycohydrolase
Poly(ADP-ribose) polymerase
Polymers
Protein binding
Protein Conformation
Proteins
Reversing
Ribose
Structure-Activity Relationship
Surface plasmon resonance
Titration
Titration calorimetry
ISSN:1932-6203, 1932-6203
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Shrnutí:Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5'-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.
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Conceived and designed the experiments: JAT NB CB G. Hassall G. Holdgate MM CT MW. Performed the experiments: JAT NB CB G. Hassall CT MW. Analyzed the data: JAT NB CB G. Holdgate G. Hassall CT JWMN MW STD. Contributed reagents/materials/analysis tools: JAT CB G. Hassall CT JWMN. Wrote the paper: JAT STD NB G. Hassall G. Holdgate JWMN CT.
Current address: Conformetrix, Manchester, Greater Manchester, United Kingdom
Competing Interests: At the time of this work, all authors were employed by AstraZeneca UK Ltd. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0050889