A Chemical Footprinting Approach towards Poly-ADP-Ribosylation-regulated Biomolecular Condensation

Project Summary Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that is catalyzed by a family of enzymes called Poly-ADP-ribose polymerases (PARPs). Among the various PARP enzymes, PARP1 is a nuclear protein that is critically involved in cell stress responses. In response to genotoxic stress, PARP1 binds to nicked DNA and is rapidly activated, resulting in the synthesis of a large number of PARylated proteins and initiation of the DNA damage repair (DDR) mechanisms. Indeed, four PARP1 inhibitors have recently been approved by the FDA to treat BRCA-mutated ovarian and/or breast cancers. Besides the role in regulating DDR in the context of human malignancies, recent evidence suggests that PARylation serves as a death signal in neurons. Importantly, genetic deletion or pharmacological inhibition of PARP1 offers profound protection against brain dysfunction in the animal models of many neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis/ALS and frontotemporal dementia/FTD. PARP1 is directly activated by a variety of neurotoxic stimulants (e.g., pathologic protein aggregates), and aberrant PARylation promotes the formation of biomolecular condensates. Despite the established role of PARylation in the regulation of phase-transition, the structural aspects of this process are elusive. To address this, we will leverage our published work and the extensive experience of my lab. These preliminary data are largely focused on two different programs. First, PARylation is a notorious PTM for mass spectrometrists, because of its labile and heterogenous nature. We recently were able to overcome these challenges, and develop a large- scale mass spectrometric approach towards comprehensive characterization of the Asp- and Glu-PARylated proteome. Using this approach, we have defined the global PARylated proteome under various genotoxic conditions. Second, biomolecular condensates are a class of membrane-less organelles, whose structural dynamics are less amenable to traditional biophysical tools. To address this, we previously developed a mass spectrometry-based chemical “footprinting” method for the structural analysis of these protein fibrils. Based on these results, we will develop a novel, tunable footprinting approach for the characterization of the structural dynamics of biomolecular condensates that are relevant to ALS and FTD (Aim 1). Then we will use tunable footprinting to study how PARylation regulates phase-transition in vitro (Aim 2). Finally, we will use tunable footprinting to characterize PARylation-mediated phase-transition in induced pluripotent stem-cell-derived neurons (iPSN) and fly models of C9orf72-mediated ALS and FTD (Aim 3). The information garnered from these studies will provide a fundamental understanding of this critical biological process, paving the way for targeting PARP1 for the treatment of ALS and FTD, and more broadly, Alzheimer's disease related dementias.