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Probing Heme Signaling Dynamics Using Fluorescent Heme Sensors

(Georgia Institute of Technology, 2021-12-13) Moore, Courtney

Long considered to be a static protein cofactor, a growing body of evidence suggests that heme may function as a dynamic signaling molecule. To elucidate heme-based signal transduction, a library of genetically encoded fluorescent heme sensors with a wide range of heme binding affinities was developed. Moreover, new targets of heme signaling, namely the ubiquitin-proteasome system (UPS), were characterized and a novel role for heme oxygenase-2 (HO-2) in regulating heme availability independent of its role in catalyzing heme degradation was discovered. Together, the results reported herein expand the toolkit of reagents available to probe heme signaling, revealed novel heme homeostatic mechanisms, and demonstrated that heme plays important roles in signaling beyond its canonical function as an enzyme cofactor.
Parsing the Dual Roles of Cu/Zn Superoxide Dismutase (Sod1) in Oxidative Stress Protection and Redox Signaling

(Georgia Institute of Technology, 2021-01-04) Montllor Albalate, Claudia

Superoxide dismutases (SODs) are a highly conserved class of antioxidant enzymes that serve on the frontline of defense against reactive oxygen species (ROS). SODs, which detoxify superoxide radicals (O2•-) by catalyzing their disproportionation into molecular oxygen (O2) and hydrogen peroxide (H2O2), are rather unusual “antioxidant” enzymes in that they catalyze the production of one ROS – H2O2 – while scavenging another – O2•-. While much is known about the necessity for O2•- detoxification, it is less clear what the physiological consequences of SOD-derived H2O2 are. Given that hormetic levels of H2O2 are important for physiological redox signaling and also act as a pro-growth signal, SODs, especially Cu/Zn SOD (Sod1), which accounts for the majority of intracellular SOD activity in eukaryotes and is localized virtually everywhere in the cell except the mitochondrial matrix, may be key to promote various redox signaling pathways. My thesis work has been focused on parsing the dual roles of Sod1 in oxidative stress protection and redox signaling. I have found that the vast majority of Sod1 is dispensable for protection against O2•- toxicity using Saccharomyces cerevisiae and human cell lines as model organisms (Chapter 2 and 3, respectively). However, the bulk of Sod1 is required for proteome-wide H2O2-based redox signaling, including regulation of the of yeast casein kinase (Yck1, Chapter 2), the canonical Wnt signaling pathway (Chapter 3) and a molecular circuit that links O2 availability to the production of NADPH, a key cellular reductant that regenerates thiol-based antioxidant enzymes (Chapter 4). Altogether, my work finally explains the physiological necessity for an “antioxidant” enzyme like Sod1 to produce an oxidant – namely that the H2O2 that Sod1 produces is used to stimulate NADPH-dependent ROS scavenging and redox regulate a large network of metabolic enzymes. In the first part of my thesis work, described in Chapter 2, I discovered that < 1% of the total cellular Sod1 pool is required for protection against superoxide damage in yeast. Superoxide toxicity stems from the oxidative inactivation of 4Fe-4S clusters, resulting in defects in a number of pathways containing Fe/S dependent metabolic enzymes, and toxicity from the iron released from damaged clusters. The iron leads to deleterious redox reactions that oxidatively damage DNA, lipids, and proteins. By profiling cell wide markers of superoxide toxicity, including Fe/S cluster enzyme activity, DNA damage, and membrane fragmentation, in cell lines expressing a regulatable SOD1 promoter, I found that an undetectable (< 1%) amount of Sod1 is sufficient for superoxide resistance in air. Instead, the bulk of Sod1 is required to promote the stabilization of Yck1, a glucose sensing plasma membrane casein kinase previously found to be stabilized in a Sod1-dependent manner. This led me to conclude that the majority of Sod1 plays a more important role as a source for hormetic H2O2 than for scavenging O2•-. In Chapter 3, I extend my findings from yeast to mammalian cells, and found that ~50-80% depletion of Sod1 in human embryonic kidney cells using RNA interference does not result in oxidative stress but does impact casein kinase signaling in the Wnt pathway. The Yck1 homology in humans, CK1, is an integral component of the Wnt signaling pathway, which is necessary for embryonic development and is prooncogenic when hyperactivated. I found that silencing Sod1 does not affect cell proliferation or the expression of a panel of antioxidant enzymes, consistent with bulk Sod1 being dispensable for oxidative stress protection. However, Sod1 silencing resulted in reduced CK1 expression and attenuated Wnt signaling and Wnt-dependent cell proliferation. Thus, as in yeast, bulk Sod1 is dispersible for oxidative stress protection but seemingly required for redox signaling in human cell lines. Having established that bulk Sod1 is dispensible for protection from superoxide toxicity, in the third section of my thesis, described in Chapter 4, I probed the physiological roles of Sod1-derived spatio-temporal bursts of H2O2. As part of this effort, I discovered that an important but previously unknown antioxidant function of Sod1 is to integrate O2 availability to stimulate production of NADPH, a key cellular reductant that regenerates peroxide-scavenging thiol peroxidases and catalases. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the catalytic Cys residue in the glycolytic enzyme, glyceraldehyde phosphate dehydrogenase (GAPDH), which in turn re-routes carbohydrate flux through the oxidative phase of the pentose phosphate pathway (oxPPP) to increase NADPH. Sod1 senses O2 via O2.- generated from mitochondrial respiration and a NADPH oxidase, Yno1. The oxidation of GAPDH is exclusively dependent on and rate limited by Sod1, suggesting that Sod1 provides a highly localized pool of H2O2 in close proximity to GAPDH, likely via transient interactions between Sod1 and GAPDH. These findings broaden the antioxidant role of Sod1 to include stimulation of NADPH production, which offers more expansive protection against redox stress than just defending against O2.-, which most Sod1 is dispensable for as determined in Chapter 2. Moreover, in collaboration with the laboratory of Matthew Torres, we employed mass spectrometry based redox proteomics approaches to identify cell-wide targets of Sod1-dependent protein oxidation. This analysis revealed that Sod1 is a master regulator of metabolism and the thiol redoxome. Overall, my findings shed light on a broader role for Sod1 that extends beyond just superoxide scavenging as was previously thought. Given that changes in Sod1 expression and activity are a central aspect of the pathogenesis of a number of diseases, including many cancers and neurodegenerative disorders, my thesis work highlights how metabolic rewiring due to Sod1-based redox control may underlie the progression of human disease and may inspire new Sod1-based therapeutics and aid to understand the effectiveness of anti-Sod1 therapeutic interventions in cancer.
Probing Heme Trafficking Using Genetically Encoded Fluorescent Heme Sensors

(Georgia Institute of Technology, 2019-11-05) Martinez-Guzman, Osiris

In this thesis, we describe genetic screens in Baker’s yeast to identify factors that regulate a. steady-state heme availability, b. the dynamics of heme mobilization to different subcellular compartments as it is being synthesized, and c. heme uptake. Regarding steady-state heme availability, the Saccharomyces cerevisiae haploid gene deletion library was transformed with cytosolic heme sensors and mutants with altered heme availability were discovered in order to identify possible heme transporters, trafficking factors, and buffering proteins. The screen identified 114 strains with high cytosolic heme and 323 strains with low cytosolic heme. Amongst the deletion mutants with high heme, we found that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) is responsible for buffering intracellular heme and regulating the activity of the nuclear heme-dependent transcription factor heme activator protein (Hap1p). Amongst the deletion mutants with low heme, we found that Golgi-to-vacuolar vesicular trafficking is a key requirement to for ensuring heme is accessible to the cytosol. We propose a model in which heme is transported from endosomes into the cytosol. Altogether, for the first time, we identified genome-wide determinants of heme bioavailability. In order to probe heme distribution dynamics as heme is being synthesized, we developed a live-cell assay in yeast to monitor inter-compartmental heme trafficking kinetics to different subcellular compartments, including the mitochondrial matrix, cytosol, or nucleus. Surprisingly, we found that heme trafficking rates from the matrix side of the IM, where heme is made, to the mitochondrial matrix and cytosol are similar, while trafficking to the nucleus is ~25% faster. Moreover, we discovered that the heme biosynthetic enzyme, aminolevulinic acid synthase (ALAS) negatively regulates mitochondrial-nuclear heme trafficking, highlighting the close coordination of heme synthesis and trafficking. In addition, we identified GTPases that directly (Gem1) and indirectly (Dnm1 and Mgm1) regulate ERMES as being modulators of nuclear heme transport. Based on our results, we propose a model in which heme is trafficked via ER- mitochondrial membrane contact sites to other organelles such as the nucleus. Most eukaryotes have the capacity to both make and import heme. We used fluorescent heme sensors in Saccharomyces cerevisiae to determine if heme availability and utilization differs whether heme is derived from exogenous or endogenous sources. Our results demonstrate that S. cerevisiae is capable of acquiring and utilizing exogenous heme under high concentrations but not in an efficient way. Biosynthesized heme is more available than exogenous heme and is more efficient at activating heme dependent processes such as catalase activity and Hap1 activity, a heme dependent transcription factor. We observed conditions such as iron starvation promoted heme availability from exogenous sources in a heme oxygenase (HO) dependent manner. In addition, we screened the yeast deletion collection for factors that altered heme uptake and utilization and identified 33 deletion mutants that displayed an improvement in heme uptake, including mfm1∆ cells, which lacks a magnesium transporter and has a defect in mitochondrial membrane potential. Overall, our work probing heme homeostasis with genetically encoded heme sensors have identified a host of new factors that regulate heme trafficking dynamics and availability from both endogenous and exogenous heme sources. The tools and approaches described herein to identify new heme trafficking factors in Baker’s yeast are now being applied to probe heme homeostasis in human cell lines, where defects in heme metabolism cause a number of diseases, and in bacterial and fungal pathogens, which require heme for virulence.
Characterization and use of genetically encoded fluorescent heme sensors to interrogate heme trafficking, dynamics, and signaling

(Georgia Institute of Technology, 2019-05-17) Hanna, David Andrew

Heme is an essential yet cytotoxic iron containing metallonutrient. Well recognized for its role as a protein prosthetic group, more recent genetic and biochemical evidence indicate heme can act as a dynamic signaling molecule. Due to the cytoxicity associated with free or misregulated heme, the bioavailable heme pool utilized for signaling is tightly regulated and buffered to low levels, making heme acquisition and heme dependent signaling reliant on the ability to safely mobilize heme. However, the factors involved in mobilizing heme have remained poorly understood. Utilizing novel heme sensor technology put forth by this work, we revealed that heme is a highly dynamic molecule and that nitric oxide (NO), a well-established and ubiquitous signaling molecule, mobilizes cytosolic and nuclear heme pools. Additionally, we discovered that under Pb stress the regulatory heme pool increases while total heme is diminished. Having identified several physiological and pathophysiological conditions that mobilize labile heme, in collaboration with Prof. Matt Torres, we are now developing mass spectrometry-based techniques to identify proteins that bind and release heme in these contexts to define new heme signaling networks. Further, using our heme sensors in HEK293 cells we reveal a heme sequestering role for HO2 that is independent of its catalytic function.
Expansion of Mitochondrial and Nuclear Heme Sensor Library

(Georgia Institute of Technology, 2019-05) Atuluru, Pranusha

The long-term objective of the work in the lab is to determine the mechanisms by which cells sense and respond to the utilization of heme, an essential nutrient. Heme is an iron-containing compound of the porphyrin class that enables proteins to carry out an array of functions. Heme-dependent processes require that heme be dynamically mobilized to hemoproteins in almost every subcellular compartment. Although it is understood that the cytotoxicity and hydrophobicity of heme requires heme be tightly regulated by the cell, the method by which this is done is unknown [1]. The primary factor that limits the understanding of heme mobilization and trafficking is the lack of tools available to sense heme, more specifically labile heme. The Reddi lab is working to develop ratiometric fluorescent sensors to offer better insight into subcellular labile heme pools relevant for heme trafficking and signaling. HS1 (Heme Sensor 1) is mutated at either the His or Met in the heme-binding coordinating bundle of cytochrome to create sensors of different affinity. Ten new mutant sensors were created from the original HS1 and HS1-M7A, and it is seen that two sensors, H102C and H102C-M7H, are the most suitable sensors to be used in the mitochondria, nucleus and cytosol. With the use of these sensors, different pathways of heme trafficking and signaling can be studied in the cell.
Probing Heme Trafficking Factors via Organellar Contact Points Using Genetically Encoded Fluorescent Heme Sensors

(Georgia Institute of Technology, 2019-05) Saini, Arushi

Heme is an important protein cofactor and signaling molecule that plays diverse roles in biological systems. The hydrophobicity and cytotoxicity of heme necessitates that it is transported and trafficked in a regulated manner. However, the molecules and mechanisms responsible for mediating heme trafficking remain poorly understood. Until recently, the tools to study heme in vivo did not exist, but the emergence of genetically encoded fluorescent sensors has enabled comprehensive real time analysis of heme in model organisms such as Saccharomyces cerevisiae. This study showcases a new a protocol that allows investigation of heme trafficking from its site of synthesis in the matrix side of the mitochondrial inner membrane to the outer matrix, cytosol, and nucleus over time. The method allows for the simultaneous examination of heme re-population in three cellular compartments after chemically depleting it. The study revealed that mitochondrial contact points play central roles in regulating heme availability and illuminates novel approaches to heme trafficking. These methods have the potential to be adapted to more inclusive compartmental analyses and enable a better understanding of heme trafficking which can empower innovative approaches to study infectious diseases, neurodegenerative disorders, and anemias associated with perturbations in heme cellular dynamics.
Utilization of Cytochrome b562 as a Localized Labile Heme Chelator

(Georgia Institute of Technology, 2019-05) Jenkin, Bryan

Heme is an essential, but toxic cofactor required for virtually all aerobic life. As a consequence, cells are challenged to safely traffic heme to hemoproteins that reside in every subcellular compartment. However, the mechanisms underlying heme transport and trafficking are largely unknown. Moreover, it is unclear how various subcellular compartments communicate their requirement for heme to the mitochondria, where heme is synthesized. In order to determine how different subcellular compartments sense and respond to heme deficiency, I have been developing a heme chelator to induce local heme deficiencies. Once this is achieved, we can employ transcriptome and proteome profiling to determine pathways that enable various organelles to adapt to heme deficiency. Altogether, we seek to better understand how cells appropriate and distribute heme to diverse compartments that require this essential nutrient.
Hemoprotein discovery through proteomic techniques

(Georgia Institute of Technology, 2018-12-11) Stapleton, Cole S.

Heme is an essential cofactor and signaling molecule. Due to hydrophobicity and toxicity, heme must be tightly regulated to prevent cellular dysfunction. Despite this fact, the manner by which heme is regulated is not well known. Understanding the molecules and mechanisms required to maintain heme homeostasis would be greatly beneficial in understanding heme regulation in health and disease. Historically, hemin-agarose resin has been utilized to identify new hemoproteins. However, this approach is susceptible to the non-specific binding of a large number of proteins in the proteome, making the identification of new hemoproteins difficult. In order to improve upon this technique, we have utilized biorthogonally tagged heme analogs to enrich heme-binding proteins. These heme analogs, which contain an azide, can be fed to cells and distributed using the cell’s own trafficking machinery. Then, through phosphine-azide copper-free click chemistry, biotin can be conjugated to the azide-tagged heme analogs for subsequent enrichment on streptavidin-resin. We demonstrate that these azide-linked heme analogs can enrich model hemoproteins in vitro and can be imported by cells. We also were able to optimize the method of hemoprotein enrichment in Saccharomyces cerevisiae. This technique could be extended to many cell types, in numerous contexts to gain a more thorough understanding of the heme-binding proteome in health and disease.
Creating a library of genetically encoded heme sensors with varying binding affinities

(Georgia Institute of Technology, 2016-12-09) Ashworth, Jessie D

Heme is an important biological metallo-cofactor that also works as a signaling molecule in cells. Despite its importance, heme trafficking and mobilization in the cell are currently not well understood, in part due to the limited ability to visualize and quantify heme in vivo. This inconvenience has recently been overcome by the development of genetically encoded heme sensors by the Reddi lab. Using these sensors, the Reddi lab has identified new heme trafficking factors and probed the spatio-temporal dynamics of heme mobilization. While the heme sensors are powerful tools for imaging and quantifying heme, improvements could be implemented that would allow for greater utility than is currently available due to particular limitations in the prototype sensors. A current limitation in heme sensing is that the heme dissociation constants of the prototype sensors span a limited range. The high affinity heme sensor, HS1 (KdII ~ 10 pM and KdIII = 9 nM) is quantitatively saturated in all sub-cellular compartments, including the cytosol, nucleus, and mitochondria, making it unsuitable for heme monitoring. The low affinity sensor, HS1-M7A (KdII = 20 nM and KdIII = 2 uM) is ~20-50% saturated with heme in the cytosol, but ~ 0 % saturated in the nucleus and mitochondria, making it unsuitable for heme monitoring in these compartments. In an effort to broaden the utility of the heme sensors, we report herein an expanded library of sensors engineered to span a wide-array of heme affinities. Heme binding residues were mutated to Ala, Cys, His, Lys, Met, Ser and Tyr to generate a panel of 47 new mutants which were found to be between 0 and 100% saturated when expressed in the yeast cytosol. These new tools will enable unprecedented access to cellular heme pools for probing heme homeostasis in health and disease.
Development of genetically encoded heme sensors

(Georgia Institute of Technology, 2015-04-24) Harvey, Raven Mariah

Due to the biological importance of heme and its implication in various disease states, uncovering how it is transported throughout the cell is of vital importance. Some of the strongest in vivo tools present in the literature are FRET-based sensors using a number of chromophores that are optimized and expanded from GFP. In order to elucidate the movement of heme throughout the cell, GFP FRET -based heme sensors were designed, expressed, and purified to be further characterized in vitro. This series of heme sensors were expressed in Saccharomyces cerevisiae to monitor the in vivo movement of heme. Different growth conditions were explored to monitor the effect of these changes to cytosolic heme availability. These heme sensors are now poised to address the movement of heme from the mitochondria to other targets in the cell under a variety of conditions. This will provide insight into heme trafficking pathways, as well as the role heme plays in neurodegenerative diseases and aging