The objective of this proposal is to determine how stress granules (SGs), components of an integrated stress response program implicated in the pathogenesis of cancer, neurodegenerative disease, and virus infection, enhance the survival of cells exposed to adverse environmental conditions. Our central hypothesis is that SGs constitute RNA-centric signaling hubs analogous to classical multiprotein signaling complexes (e.g., lipid rafts, transmembrane receptor signaling complexes), and that SG assembly communicates a "state of emergency" by intercepting and sequestering components of multiple signaling pathways. This is based upon recent results showing that signaling proteins possessing low complexity (LC)/intrinsically disordered (ID) aggregation regions are recruited to SGs thereby altering major signaling pathways that modulate survival. The rationale for this research is that, once we know how SGs recruit signaling molecules to alter the stress response program, we will be able to modulate these events to treat cancer, neurodegenerative diseases and viral infections. We will test our central hypothesis by the completion of three specific aims:
AIM 1. To test the hypothesis that SGs promote the survival of stressed cells by modulating multiple signaling pathways. Our working hypothesis is that SG dependent alteration of multiple signaling pathways is sufficient to promote the survival of cells exposed to adverse conditions.
AIM 2. To test the hypothesis that nutrient stress inhibits SG formation via autophagic and non-autophagic pathways. Our working hypothesis is that nutrient stress- activated AMPK and ULK1 both directly (via phosphorylation of SG components) and indirectly (via induction of autophagic vacuoles that target SGs for degradation) inhibit SG formation.
AIM 3. To test the hypothesis that phosphorylation and 14-3-3 protein binding to LC/ID regions of signaling proteins modulates SG composition and function. Our working hypothesis is that these modifications stabilize the structure of LC/ID regions in a way that prevents their recruitment to SGs.
These aims will be completed using a USP10 point mutation that does not bind G3BP or inhibit SG formation to probe the relationship between SG assembly and cell survival. We will determine the role of nutrient stress in modulating SG formation in WT and autophagy- defective mouse embryo fibroblasts with or without dominant negative inhibitors of AMPK and ULK1. Finally, we will determine how phosphorylation and 14-3-3 binding alter the structure of LC/ID regions of SG proteins by monitoring in vitro hydrogel formation, binding to 5-aryl-isoxazole-3-carboxyamide, and NMR spectroscopy. These results are significant because they will provide a molecular basis for the development of pharmacologic strategies to modulate SG formation and cell survival in disease. This research is innovative because it shifts the paradigm of SGs from one of translational control to one of cell signaling.
This project will determine how stress granules, discrete RNA- and protein-containing regions found in the cytoplasm of stressed cells, communicate a state of emergency by sequestering components of multiple signaling pathways to enhance the survival of cells exposed to adverse conditions. As stress granules have been implicated in the pathogenesis of cancer, neurodegenerative disease, and virus infection, we expect to identify targets for the development of drugs that modulate stress granule assembly to increase (motoneurons in patients with neurodegenerative disease) or decrease (cancer) cell survival in different disease states.