Abstract
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.
Highlights
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA resulting in covalent attachment of poly-ADP-ribose (PAR) polymers to a variety of amino acid residues on target proteins [1,2], or to 5’- and 3’-terminal phosphate residues at doubleand single-strand breaks of a DNA molecule [3,4,5]
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death
The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein
Summary
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA resulting in covalent attachment of poly-ADP-ribose (PAR) polymers to a variety of amino acid residues on target proteins [1,2], or to 5’- and 3’-terminal phosphate residues at doubleand single-strand breaks of a DNA molecule [3,4,5]. A turn-on split luciferase sensor utilizing APLF PBZ domains for PAR-binding [8] allows detection of PAR in cell lysates, without single cell resolution available for microscopic techniques. A work by Buntz et al [9] describes a method using fluorescent cell-permeable NAD+ analogue as a Förster resonance energy transfer (FRET) acceptor, and EGFP-labeled ARTD1 as a FRET donor to monitor PARylation of EGFP-ARTD1 with fluorescence lifetime imaging microscopy (FLIM). The drawback of this approach is the need for the exogenous ligand, which limits the applicability outside cell culture models. Translocation of PBZ tagged with a fluorescent protein was shown to highlight spots of PAR accumulation, whereas PBZ in combination with split GFP enabled a large-scale search for PARylated proteins
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