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  • A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation.

A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation.

Nature communications (2019-09-15)
Xiao-Nan Zhang, Qinqin Cheng, Jingwen Chen, Albert T Lam, Yanran Lu, Zhefu Dai, Hua Pei, Nikolai M Evdokimov, Stan G Louie, Yong Zhang
ANOTACE

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3'-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3'-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3'-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3'-azido NAD+ provides an important tool for studying cellular PARylation.

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Sigma-Aldrich
PARP2 Active human, recombinant, expressed in baculovirus infected insect cells, ≥60% (SDS-PAGE)
Sigma-Aldrich
PARG human, recombinant, expressed in Sf21 cells, His tagged, >95% (SDS-PAGE)
Sigma-Aldrich
Anti-poly-ADP-ribose binding reagent, Anti-poly-ADP-ribose binding reagent is a reagent that selectively binds to ADP ribose for use in Western Blotting, Immunocytochemistry and Dot Blot.
Sigma-Aldrich
Adenosine 5′-diphosphoribose sodium salt, ≥93%