Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets

Abstract

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor “tagged” cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

PARPs in genome stability and signal transduction: implications for cancer therapy

“…In particular, the consensus hexapeptide motif RxxPDG (where ‘x’ refers to any amino acid) within the ANK domain was shown to be necessary and sufficient for interaction with protein targets [178]. Several targets of Tankyrases were identified, among them the t elomeric- r epeat b inding f actor-1 (TRF1) [168], the i nsulin- r esponsive a mino- p eptidase (IRAP) [178], the 182-kDa t ankyrase- b inding protein (TAB182) [178], the n uclear m itotic a pparatus protein-1 (NuMA1) [178], AXIN1/2 [171], 3BP2 mutated in Cherubism disease [179,180], (Bhardwaj 2017) [172], and the CBP80/CBP20-dependent t ranslation i nitiation f actor (CTIF) [181]. Thus, the interaction with protein substrates can be considered the specificity determinant for Tankyrases.…”

Poly-ADP-ribosylation drives loss of protein homeostasis in ATM and Mre11 deficiency

“…To quantify differences in PARylation in ATM-depleted cells, we utilized a recently developed live-cell PARylation sensor (Krastev et al, 2018) (Fig. 2) that fuses PAR-binding domains (PBZ) to split Venus proteins.…”

“…The PBZ-PBZ sensor was derived from pBiFC-PBZ-VC and pBiFC-PBZ-VN (Addgene #110648 and 110646, respectively) but combined into one bacmam plasmid, pTP4623, using the backbone of pAceBac1 (Bieniossek et al, 2008). The original pBiFC PBZ constructs were gifts from Chris Lord (Krastev et al, 2018).…”

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

“…The drawback of this approach is the need for the exogenous ligand, which limits the applicability outside cell culture models. Very recently, PBZ (PAR-binding zinc finger) domains were used to construct genetically encoded fluorescent PAR sensor variants [ 10 ]. 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.…”