Structural and biochemical studies of protein poly(ADP-ribosyl)ation

Abstract

Protein poly(ADP-ribosyl)ation (PARylation) has been found to be involved in various cellular processes, such as DNA damage recognition and repair, chromatin structure dynamics, gene transcription and poly(ADP-ribose)-dependent cell death. Most recently, PARylation has been shown to control the polyubiquitination and degradation of Axin, a key regulator of the Wnt signaling pathway. RNF146, which contains a WWE domain and a RING domain, is the only proven E3 ubiquitin ligase to date that requires PARylation of the substrate for subsequent polyubiquitination. The RNF146 WWE domain has been shown to bind poly(ADP-ribose) (PAR). In this thesis, first I review the current knowledge on protein PARylation. Then I describe my doctoral research on the structural and biochemical studies on how WWE domain recognizes PAR polymer. My studies reveal the structural basis of the RNF146 WWE domain/iso-ADP-ribose (iso-ADPR, the internal unit of PAR) interaction and, for the first time, define the PAR/iso-ADPR binding as a bona fide function of the WWE domain family. This suggests that PAR may be a signal for protein ubiquitination and this signal is decoded by WWE domain-containing E3 ubiquitin ligases. In Chapter 3, I describe my structural studies on poly(ADP-ribose) glycohydrolase (PARG), which is the principal enzyme responsible for the degradation of PAR polymers in vivo. By solving the structures of the mouse PARG catalytic domain and its complex with its inhibitor ADP-HPD, I reveal how the PAR polymer as a substrate is recognized by PARG. The structures also suggest how the N terminal flexible peptide preceding the PARG catalytic domain regulates the enzymatic activity of PARG. This study helps the understanding of PARG catalytic and regulatory mechanisms as well as the rational design of PARG inhibitors.