Analysis of poly(ADP-ribose) function in the cytoplasmic stress response

DESCRIPTION (provided by applicant): Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It functions as both a covalent modification of acceptor proteins and as a scaffold that binds specific sets of proteins. The polymer plays a role in essential cellular functions including cell division and cell cycle progression and regulation of transcription and translation. pADPr also functions in cell stress responses such as apoptosis, DNA damage repair, and innate immune responses. In preliminary results we identified a new function for pADPr and the enzymes that polymerize it, pADPr polymerases (PARPs) in regulation of stress granule (SG) assembly and the post-transcriptional regulation of mRNAs. These results connect the fields of cellular stress, PARPs and mRNA regulation in an unexpected manner. They suggest that pADPr functions as a structural scaffold for SG assembly, similar to its function at the spindle pole and at sites of DNA damage. We anticipate that our results will have a high impact on all three fields and wish to extend our work to include mechanistic studies. One of our long-term goals is to understand how pADPr functions as a scaffold. SG assembly and mRNA regulation is an ideal process to study the scaffold function of pADPr. In this proposal, we seek to determine the mechanism of pADPr function in the assembly of a SG and begin to understand how pADPr binding to proteins regulates function. We do so using a combination of biochemical assays and cell biology. In specific Aim 1 we identify the sites of pADPr modification that are required for SG assembly and generate and determine the mechanism in which pADPr is synthesized for stress. In Aim 2 we determine the manner in which pADPr regulates mRNA binding and recruitment to the SG, and in Aim 3 we examine the structure- function relationships that govern binding of pADPr to proteins. We believe the proposed experiments will help elucidate the scaffold function of pADPr elsewhere in the cell. SGs have important disease relevance. PARP inhibition, already shown to be effective for breast and ovarian cancer therapies, might be equally effective for treatment of other stress-related diseases such as solid tumors, ischemia, and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It is polymerized by a family of proteins called pADPr polymerases (PARPs). pADPr and PARPs function in essential cellular functions and cell stress responses. These stress response pathways are common targets for human disease therapy. We recently identified a new stress function for pADPr - stress granule assembly. Stress granules are large multi-protein complexes that assemble in the cytoplasm in response to external stresses such as heat shock, oxidative stress, ischemia and viral infection. They are thought to regulate the stability and translation potential of mRNAs. Recently, pADPr has been implicated in cancer, prompting pharmaceutical companies to develop inhibitors that target members of the PARP family. Several PARP-1 inhibitors have progressed to Phase 3 trials for treatment of breast and ovarian cancers in near record time. The long-term goal of our studies is to understand how pADPr functions in the stress granule.

DESCRIPTION (provided by applicant): Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It functions as both a covalent modification of acceptor proteins and as a scaffold that binds specific sets of proteins. The polymer plays a role in essential cellular functions including cell division and cell cycle progression and regulation of transcription and translation. pADPr also functions in cell stress responses such as apoptosis, DNA damage repair, and innate immune responses. In preliminary results we identified a new function for pADPr and the enzymes that polymerize it, pADPr polymerases (PARPs) in regulation of stress granule (SG) assembly and the post-transcriptional regulation of mRNAs. These results connect the fields of cellular stress, PARPs and mRNA regulation in an unexpected manner. They suggest that pADPr functions as a structural scaffold for SG assembly, similar to its function at the spindle pole and at sites of DNA damage. We anticipate that our results will have a high impact on all three fields and wish to extend our work to include mechanistic studies. One of our long-term goals is to understand how pADPr functions as a scaffold. SG assembly and mRNA regulation is an ideal process to study the scaffold function of pADPr. In this proposal, we seek to determine the mechanism of pADPr function in the assembly of a SG and begin to understand how pADPr binding to proteins regulates function. We do so using a combination of biochemical assays and cell biology. In specific Aim 1 we identify the sites of pADPr modification that are required for SG assembly and generate and determine the mechanism in which pADPr is synthesized for stress. In Aim 2 we determine the manner in which pADPr regulates mRNA binding and recruitment to the SG, and in Aim 3 we examine the structure- function relationships that govern binding of pADPr to proteins. We believe the proposed experiments will help elucidate the scaffold function of pADPr elsewhere in the cell. SGs have important disease relevance. PARP inhibition, already shown to be effective for breast and ovarian cancer therapies, might be equally effective for treatment of other stress-related diseases such as solid tumors, ischemia, and neurodegenerative disease. PUBLIC HEALTH RELEVANCE: Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It is polymerized by a family of proteins called pADPr polymerases (PARPs). pADPr and PARPs function in essential cellular functions and cell stress responses. These stress response pathways are common targets for human disease therapy. We recently identified a new stress function for pADPr - stress granule assembly. Stress granules are large multi-protein complexes that assemble in the cytoplasm in response to external stresses such as heat shock, oxidative stress, ischemia and viral infection. They are thought to regulate the stability and translation potential of mRNAs. Recently, pADPr has been implicated in cancer, prompting pharmaceutical companies to develop inhibitors that target members of the PARP family. Several PARP-1 inhibitors have progressed to Phase 3 trials for treatment of breast and ovarian cancers in near record time. The long-term goal of our studies is to understand how pADPr functions in the stress granule.