Dissertation

Probing Heme Signaling Dynamics using Fluorescent Heme Sensors

Moore, Courtney M

2021

Overview

Long considered to be a static protein cofactor, a growing body of evidence suggests that heme may function as a dynamic signaling molecule. Heme is an essential yet cytotoxic signaling molecule1 . Consequently, cells must regulate heme bioavailability in a manner that allows for essential function while mitigating heme’s toxic effects. Many human diseases including, cancers2 , neurodegenerative disorders3, and cardiovascular diseases4 , stem from defects in heme homeostatic mechanisms. Unfortunately, these mechanisms are poorly understood, in part due to the difficulty of imaging heme within live cells. Genetically encoded ratiometric heme sensors have been developed to probe bioavailable heme and utilized to identify heme trafficking factors and physiological processes that dynamically mobilize subcellular heme pools. These processes include cell catabolic mechanisms; labile heme has been implicated to control the heme catabolic enzymes and the Ubiquitin-Proteasome system.To elucidate heme-based signal transduction, my thesis work focused on: the development of a library of genetically encoded fluorescent heme sensors with a wide range of heme binding affinities, characterizing new targets of heme signaling, and the discovery of a new role for heme oxygenase-2 (HO-2) in regulating heme availability independent of its role in catalyzing heme degradation. The first chapter will cover the importance of heme in cell biology, including its roles as a signaling molecule and in human disease. In the second chapter, I will discuss the development and characterization of a library of heme sensors with differing heme affinities for broader applications. For example, the sensor library can be used to measure heme in a variety of compartments and cell types and distinguish between ferric and ferrous heme species. Chapter Three will cover a newly discovered role for heme in regulating the Ubiquitin-Proteasome system. Specifically, this chapter will focus on heme’s ability to regulate the activity of the yeast E1-ligase Uba1 and regulation of proteasome activity. The fourth chapter is focused on understanding the physiological role of the heme catabolic enzyme HO-2 under conditions in which heme is limiting. To this end, I found that HO-2 acts to bind and buffer heme, under basal conditions, rather than enzymatically degrade it. The final chapter will be a discussion and summary of this work.To elucidate the mechanisms underlying heme trafficking and signaling, the Reddi lab previously developed a pair of genetically encoded heme sensors, a high affinity sensor, HS1, which tightly binds both oxidation states of hem, KDII= 1 pM and KDIII= 3 nM, and a moderate affinity heme sensor, HS1-M7A, which exhibits a KDIIvalue of 25 nM and KDIII= 1 µM.5In yeast, HS1 quantitatively saturates with heme in the cytosol, nucleus, and mitochondria. In contrast, HS1-M7A is ~50% bound in the cytosol and < 30% bound in the nucleus and mitochondrial matrix. Since heme levels vary between different cell types and subcellular compartments, I sought to develop an expanded library of heme sensors with varying affinities for ferric and ferrous heme to measure and image labile heme in many different organisms, cell types, and physiological contexts. After screening 40 variants, results indicate that heme loading of the sensor is under both kinetic and thermodynamic control, and different subcellular locales exhibit microenvironments that favor rapid equilibration of heme with the sensor whereas others do not. In addition, my results thus far have identified at least one heme sensor variant that selectively binds ferrous heme.

• Publisher: ProQuest Dissertations & Theses
• Subject: Acids / Bioinformatics / Cells / Cellular biology / Chloride / Eukaryotes / Flow cytometry / Free radicals / Hemodialysis / Medicine / Mitochondria / Plasmids / Proteomics / Sodium / Software / Toxicity / Toxicology
• ISBN: 9798263323981