Identifying and characterizing metabolic control mechanisms

PROJECT SUMMARY/ABSTRACT Mechanisms controlling metabolism are critical for maintaining homeostasis and are highly varied. Many metabolic control mechanisms involve metabolite sensors, which directly interact with the metabolite being sensed, eliciting a response that impacts the availability of the sensed molecule. Several metabolite sensors, such as mTORC1 and AMPK, are altered in human disease and targeted by pharmacologic agents, underscoring the importance of understanding the mechanisms by which cells and organisms sense key nutrients. Our laboratory has made fundamental contributions to our understanding of metabolite sensors that control de novo pyrimidine biosynthesis and iron metabolism, and identified mechanisms that enable biological systems to respond to changes in nutrient availability. Our proposal focuses on developing cell and mouse models in which these sensing mechanisms have been perturbed to better understand the physiologic importance of these metabolic sensing mechanisms. In the first project, we are studying the physiologic importance of an evolutionarily conserved mechanism by which cells sense pyrimidine levels via regulation of the rate limiting enzyme of this pathway, CAD. Through careful structure-function analysis, we have engineered mutants of CAD that lack this regulatory mechanism and confirmed that they exhibit increased activity with an inability to regulate biosynthetic flux. In cell-based models engineered to express CAD allosteric mutants, we will delineate the role of CAD allosteric regulation on the many connected biosynthetic pathways that support nucleotide synthesis, as well as the effects of nucleotide imbalance on the DNA mutagenesis rate. Then, we will develop animal models lacking this evolutionarily conserved sensing mechanism to delineate the impact of physiology, focusing on the function of proliferative cells, such as immune cells and barrier tissues. In the second project, we will define how dysregulation of the cellular iron-sensing rheostat impacts normal physiology. In particular, we will study how a specific component of the sensor, iron-sulfur cluster (ISC) cofactors, sense and respond to cellular iron levels by suppressing the synthesis of these cofactors in intact animals. We will study the impact of perturbed sensing on cells and tissues responsible for detecting and responding to changes in iron availability, and dissect whether these effects are mediated by two major iron response proteins, IRP1 and IRP2, using genetic knockout mice. In the third project, we will mechanistically dissect, in vitro, a novel iron sensing pathway that we identified by which ISCs control IRP2 activity. We anticipate that these iron sensing and response pathways are altered in multiple neurodegenerative diseases in which altered ISC or iron metabolism have been implicated, including Friedreich’s Ataxia.