Proteome-wide microarray-based screening of PAR-binding proteins

Abstract

Poly(ADP-ribose) (PAR) plays a crucial role in intracellular signaling and scaffolding through covalent modification or non-covalent binding to target proteins. The non-covalent binding PARylome has not been extensively characterized. Here we performed a PAR-binding screen using a human protein microarray that covers most of the human proteome to characterize the non-covalent binding PARylome. A total of 356 PAR-binding proteins were identified. The PAR-binding PARylome suggests that PAR-binding regulates a variety of biological processes beyond well-characterized DNA damage signaling and DNA repair. Proteins that may be reprogrammed by PAR-binding include signaling molecules, transcription factors, nucleic acid binding proteins, calcium binding proteins, ligases, oxidoreductases, enzymes, transferases, hydrolases, and receptors. The global database of PAR-binding proteins that we established will be a valuable tool for further in-depth analysis of the role of PARylation in a wide range of biological contexts.

ZNF432 stimulates PARylation and inhibits DNA resection to balance PARPi sensitivity and resistance

“…As evidence accumulates, it is very likely that some of these extra fingers possess a significant affinity for PAR polymers as seen for CTCF ( 56 ), ZBTB24 ( 19 ), or E4F1 ( 20 ). The identification of several ZFPs as specific readers of PAR suggests that the affinity of these proteins for PAR might be a widespread feature applicable to multiple members of the C2H2-type family of proteins ( 23 , 62 ), including ZNF432 for which we observed significant PAR-binding activity in vitro . A simulation study has shown that the affinity of ZFPs to a specific DNA target is larger than the sum of affinities of individual fingers and depends on multiple factors such as specific site and linker length between ZNFs ( 63 ).…”

“…PARylation has been recognized to coordinate the recruitment of several critical proteins that participate in the DDR and guide the DNA repair pathways. To find PAR readers affecting DSB repair, we searched for PAR readers in a data set of non-covalent PAR-binding proteins identified in a proteome-wide microarray-based screen ( 23 ). The C2H2-type ZNF protein domain was identified as the most statistically significant ontology term within PAR readers.…”

“…Five serine PARylation sites were identified in ZNF432, of which three are located in ZNF domains ( 18 , 37 , 38 , 39 ). ZNFs 4, 7, 12 and 15 contain the recently identified CNxC PAR-binding motif common to several C2H2-type ZFPs ( 23 ). As a first step towards the systematic functional characterization of ZNF432 in DSB repair and HR, we expressed GFP-ZNF432 as a fusion protein in 293T cells and determined its subcellular localization.…”

“…Because PARP-1-dependent PARylation has major implications in the DDR, including the regulation of DSBs that represents one of the most lethal types of DNA damage ( 22 ), we recently directed our attention to a group of ZFPs found to possess an affinity for PAR. Using a protein microarray that covers most of the human proteome, Kang and colleagues identified a group of C2H2-type ZFPs that could be characterized as PAR readers ( 23 ). Based on this observation, we explored the possibility that some of these ZFPs could act as effectors of HR-based DNA repair through which PARylation could play a potential regulatory role.…”

Histone H1.2 Dependent Translocation of Poly (ADP-ribose) Initiates Parthanatos

“…There are many nuclear proteins that are PARylated or bind PAR itself that could be candidates for carrying PAR out of the nucleus [21][22][23][24][25][26][27][28][29][30][31] . One of the major classes of proteins that are PARylated and bind PAR are histones 23,26,32 . Of the histones, the most abundant that are extensively ribosylated are the histone H1 family members 33 .…”