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ENPP1 processes protein ADP-ribosylation in vitro
Luca Palazzo1, Casey M Daniels2, Joanne E Nettleship3,4
1Sir William Dunn School of Pathology, University of Oxford, UK.
The FEBS Journal
|July 14, 2016
Summary
ADP-ribosylation, a key protein modification in cellular processes, can be reversed by enzymes. This study identifies ENPP1 as a novel mammalian enzyme that converts ADP-ribosylation into ribose-5 phosphate (pR), aiding in site identification.
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Biology
Background:
- ADP-ribosylation is a crucial post-translational modification involved in fundamental cellular processes like DNA repair and cell death.
- Dysregulation of ADP-ribosylation is implicated in human diseases, including cancer and neurodegenerative disorders.
- Known reversal mechanisms involve macrodomain-containing proteins, while NUDIX domain proteins offer an alternative pathway to generate protein-conjugated ribose-5'-phosphate (pR).
Purpose of the Study:
- To investigate the mechanism and physiological relevance of protein phosphoribosylation in mammalian tissues.
- To identify mammalian enzymes capable of generating pR from ADP-ribosylated proteins.
- To explore the utility of pR tags for identifying ADP-ribosylation sites.
Main Methods:
- In vitro enzymatic assays using ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and ADP-ribosylated proteins.
- Mass spectrometry to analyze the products of enzymatic reactions.
- Comparison of ENPP1's activity with known NUDIX domain enzymes.
Main Results:
- ENPP1, a mammalian enzyme lacking a NUDIX domain, was identified as capable of generating pR from ADP-ribose on modified proteins in vitro.
- This finding indicates at least two enzyme families (Nudix and ENPP/NPP) can metabolize protein-conjugated ADP-ribose to pR.
- ENPP1 facilitates the conversion of mono- and poly(ADP-ribose) into mass spectrometry-friendly pR tags, simplifying ADP-ribosylation site identification.
Conclusions:
- Mammalian cells possess at least two distinct enzymatic pathways (NUDIX and ENPP/NPP) for generating protein phosphoribosylation (pR).
- The existence of these pathways suggests pR may be a conserved modification from bacteria to mammals.
- ENPP1 serves as a valuable tool for mass spectrometry-based detection and mapping of ADP-ribosylation sites.
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