Site-Specific Antibody for Protein Poly-ADP-Ribosylation

Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that is catalyzed by a family of enzymes known as Poly-ADP-ribose polymerases (PARPs). Among the various PARP family members, PARP1 is a nuclear enzyme that is critically involved in cell stress responses. Upon sensing DNA strand breaks, PARP1 becomes activated, and then utilizes the cofactor, NAD+ to synthesize a large array of PARylated proteins. These protein-linked PAR chains serve as a platform to recruit the DNA repair machinery, and to initiate the DNA damage response (DDR). These findings provide the rationale to develop PARP1 inhibitors to treat human malignancies. Indeed, four PARP1 inhibitors have been recently approved by the FDA to treat BRCA-deficient ovarian and/or breast cancer patients. Despite the exciting progresses of PARP1 inhibitors in the clinic, the basic signaling mechanism of PARP1 and the related PARP enzymes remains poorly understood. The analysis of protein PARylation represents a daunting challenge, owing to the low- abundance, labile and heterogeneous nature of this PTM. As a result, very few genuine PARP1 substrates (and their modification sites) have been identified, and in most of the cases, how PARylation regulates the function of the DNA damage responsive proteins is poorly defined. Recently, we developed the first large-scale mass spectrometric (MS) approach towards site-specific characterization of protein Asp-/Glu-PARylation. Although this strategy enables the quantitative assessment of the PARylated proteome, it is a quite lengthy procedure that requires the access to both mass spectrometry equipment and computational proteomics software. Furthermore, current anti-PAR antibodies only allow the analysis of the level of total PARylation, but not PARylation at a specific residue. In this proposal, we will develop a chemical biology approach towards the development of general site-specific PARylation antibodies. We will select a number of PARylation targets that are critically involved in DDR, and the corresponding antibodies will be evaluated using cell systems and animal models. We envision that the development of antibodies that allow for the simple, sensitive, specific and rapid detection of PARylation sites would greatly facilitate the understanding the role of PARylation in DDR and cancer. We also expect that this approach will be highly useful for the future development of biomarkers targeting these aberrant “protein modification signatures”.