Mechanisms of Regulation of Retinoic Acid Homeostasis - PROJECT ABSTRACT Vitamin A (retinol) is a diet derived nutrient that requires metabolic activation to all-trans-retinoic acid (atRA). Gene regulation by atRA is essential for reproduction, metabolic processes, immune responses, and epithelial maintenance. Dysregulated vitamin A homeostasis causes aberrant atRA concentrations that impact human health across lifespan. Tightly regulated pulses and gradients of atRA concentrations are necessary for appropriate atRA signaling. Despite decades of research, the mechanisms that generate these pulses and the processes that lead to dysregulated vitamin A homeostasis in humans are still poorly defined. We have employed detailed biochemical studies together with clinical research to fill this knowledge gap. Our preliminary studies suggest that lipid partitioning together with the Cellular Retinoic Acid Binding Proteins 1 and 2 (CRABP1 and CRABP2) have profound impact on tissue and cellular atRA concentrations. Further, our preliminary data indicates that the relative expression levels of the CRABPs and cytochrome P450 26 family (CYP26s) enzymes profoundly alter cellular atRA concentrations. The goal of this project is to establish a quantitative model on how lipid composition within a cell together with CRABPs and CYP26s regulates atRA concentrations in cellular compartments and atRA signaling via retinoic acid receptors (RARs). We hypothesize that the CRABPs 1) increase cytosolic atRA concentrations and counter lipid binding sinks in the cells, 2) directly regulate atRA metabolism via protein-protein interactions with CYP26 and 3) modulate atRA signaling in target cells to ensure establishment of spatiotemporal atRA concentration gradients. In our aim 1 we will define the distribution characteristics of atRA with consideration of different lipid compositions and variable CRABP expression. In aim 2 we will establish the biochemical details of the protein-protein interactions between ligand bound (holo) and ligand free (apo) CRABP1 and 2 and the key atRA clearance enzymes CYP26A1 and CYP26B1. We will also establish a model of how CRABPs alter tissue atRA metabolism and concentrations. In aim 3 we will identify the interacting residues between CRABPs and CYP26s and evaluate how covalent modifications of CRABPs alter these protein-protein interactions. Throughout our studies we will use innovative proteomics methods, in vitro- to-in vivo predictions and state-of-the-art kinetic modeling to advance knowledge of how atRA signaling, that is critical to life, is regulated. When completed, our results will provide unprecedented insight into how spatiotemporal atRA concentration gradients in specific cells and tissues are regulated and how measured tissue atRA concentrations can be linked to atRA signaling and pharmacological effects. This is significant as pulses and gradients of atRA concentrations are critical for development of many human diseases and for regulating cell cycle, cell differentiation and growth at all stages of a lifecycle, starting from reproduction to embryonic development, to maintenance of immunity, epithelial integrity and lipid metabolism across lifespan.