Translational control in heart and lung disease by RNase A family

Cardiovascular disease is the leading cause of death in the United States. Despite decades of research it isunclear how the RNase A family nuclease angiogenin stimulates blood vessel formation and why angiogenindysfunction is associated with heart failure and poor cardiovascular health. Recent work revealed thatangiogenins nuclease activity which is required for its angiogenic function is stimulated by binding to theribosome but it remains unclear whether angiogenins ribosome-dependent mechanism is involved inangiogenesis. A bacterial nuclease named ribocin with a strikingly angiogenin-like structure and ribosome-dependent activity holds similar potential for understanding lung health. Nearly all Cystic Fibrosis patientsexperience Pseudomonas aeruginosa bacterial pneumonia and subsequently suffer from lung tissueinflammation and lasting damage long after the infection has cleared. Ribocin encoded by P. aeruginosadamages human ribosomes specifically at central helix 69 of the 28S rRNA and inhibits translation. Wehypothesize that like other ribosome-inactivating proteins ribocin induces a ribotoxic stress response thatcauses inflammation and cell death in human lung tissues. To inform future therapeutic studies aimed attreating cardiovascular disease and post-infection pulmonary damage the mechanisms of translation controlby these RNase A-family nucleases must be elucidated in the context of their cellular functions. The goal of this project is to determine the structural basis of translation control by angiogenin during angiogenesis and determine the impact of translation control by ribocin on ribotoxic stress response and cell death. With guidance from the sponsor an expert in biochemical and structural basis of translation and collaborators whoare experts in the RNA developmental biology and cryo-EM method development the trainee will apply cuttingedge methods for in-cell cryogenic electron microscopy (cryo-EM) complemented by cell assay andbiochemical approaches to visualize structural changes to actively translating ribosomes during angiogenin-stimulated vascularization and ribocin-mediated tissue damage. Aim 1 will determine the contribution ofangiogenins ribosome-specific activity on tube formation (angiogenesis) in human umbilical vascularendothelial cells (HUVEC). Aim 2 will elucidate the structural mechanism of translation inhibition by ribocin and investigate the impact of selective ribosome damage by ribocin on ribotoxic stress response in human lungcells (IB3). The results of this study will reveal details of mechanisms underlying fundamental cardiovascularfunction and novel components of pulmonary disfunction necessary for future work in developing therapeuticsfor cardiovascular and lung disease.