Project Summary/Abstract The production of functional proteins is a multistep process whereby newly synthesized polypeptides are enzymatically processed, folded, and assembled into oligomeric complexes. Key maturation processes occur co-translationally by coupling mRNA translation and nascent protein maturation on the ribosomes, assisted by a network of regulatory proteins. Despite decades of basic research, our understanding of co-translational processes is shaped predominantly by genetic manipulations in prokaryotic and eukaryotic cell models, but limited data exists in cells from intact mammalian physiological systems. To overcome this hurdle, this project aims to develop a comprehensive understanding of co-translational regulation of nascent proteins as they reach their functional state within the angiogenic signaling pathways of the endothelium in the heart during aging. We have recently developed an approach and tested the potential for simultaneous isolation of proteins present within the actively translating polyribosome/mRNA complexes within the endothelium of the heart. State-of-the-art multiplex labelling and quantification of these proteins has permitted the identification of concordant and discordant regulated cell-specific pathways based on actively translating mRNA and protein profiles associated with the endothelial stress. Leveraging this innovative advancement in “functional co- translatomics”, we aim to study the changes in co-translational complexes within endothelial cell angiogenic ligand-receptor and cell-cell interaction signaling networks within the aging heart. In Aim 1 , we will define changes in the co-translational regulation of concordantly regulated angiogenic signaling pathways during neonatal and adult life with aging and gender. In Aim 2 , we will define the post-translational modifications of proteins within discordantly regulated angiogenic signaling pathways which may contribute to the impairment angiogenesis in the heart during aging. Ultimately this knowledge could aid in the development of more effective means for diagnosing and treating the impairment of angiogenesis that occurs with aging and predisposes the heart to cardiovascular disease and injury.