Browsing by Subject "Iron"

Transferrin receptor 1 (Tfr1) serves as a receptor for transferrin, an iron-binding protein in the blood, in its canonical role of iron assimilation. Tfr1 is expressed ubiquitously in many tissues and is believed to be required for iron uptake by most cells.

The Tfr1 global knockout mouse highlights the requirement for Tfr1 in erythrocyte precursors. The erythron is the tissue with the highest iron requirement, to enable hemoglobin production. Tfr1-null embryos die by embryonic day 12.5 with anemia, which has been assumed to cause lethality of the knockout mice. Due to the embryonic lethality of the mice, the role of Tfr1 has not been well characterized in other tissues in vivo. This thesis examines the role of Tfr1 in other tissues through the generation and characterization of conditional knockout mouse models of Tfr1 deletion in the intestinal epithelium, pancreas, and skin.

Tfr1 is expressed on the basolateral surface of proliferating cells in the intestinal epithelium. Deletion of Tfr1 specifically in the intestinal epithelium resulted in the loss of intestinal epithelial homeostasis, loss of proliferation, lipid accumulation, gene expression indicating epithelial to mesenchymal transition of intestinal epithelial cells, and early neonatal lethality. These phenotypes were mostly alleviated by forced expression of a mutant Tfr1 allele which is unable to bind to iron-loaded transferrin, suggesting that Tfr1 has a novel role independent of its canonical iron-assimilatory ability.

Deletion of Tfr1 in the pancreas resulted in juvenile death due to perturbed homeostasis of both endocrine and exocrine tissues, resulting in symptoms associated with pancreatitis and diabetes. No diabetic phenotype was detected in the conditional knockout mouse model of Tfr1 deletion specifically in β-cells, suggesting that the primary effect of the loss of Tfr1 was limited to the exocrine tissue.

Deletion of Tfr1 in the epidermis of the skin caused neonatal lethality with abnormal hair follicle morphology and a significant reduction in dermal adipocytes.

These results indicate that the loss of Tfr1 has pleiotropic effects, depending on the cell type affected. Furthermore, Tfr1 appears to have non-canonical functions in the intestinal epithelium, a novel discovery.

Fluorescent sensors provide a powerful analytical tool for the intracellular detection of metal cations. In some cases, these fluorescent metal-chelating sensors have helped elucidate the function of metal cations within complicated cellular systems. However, most measure or sense changes in the bulk concentration of a metal species and do not respond to those involved in a specific cellular event. For instance, misregulated copper and iron are implicated in neurodegenerative disease and cancer because of their ability to catalytically propagate the formation of the hydroxyl radical through reaction with hydrogen peroxide. A fluorescent sensor that is unresponsive to metal binding until activation by intracellular hydrogen peroxide could potentially pinpoint the location of this oxidative reaction and provide an understanding of the relationship between copper/iron and hydrogen peroxide.

Described here is the development of two fluorescent prochelators that show a selective fluorescence response to iron or copper only in the presence of hydrogen peroxide. A boronic ester masked spirolactam-based prochelator displays a copper-selective turn-on response after oxidation with hydrogen peroxide in organic solvents as determined by absorbance and fluorescence spectroscopy. However, a competing mechanism occurs in aqueous solution due to hydrolytic instability of the masked prochelator and results in a separate copper-dependent turn-on response as verified by liquid chromatography-mass spectroscopy. A second fluorescent prochelator design relies on metal-dependent fluorescence quenching after oxidation of a self-immolative boronic ester in both organic and aqueous solvents. Cellular microscopy studies show that the sensor's fluorescence intensity is unchanged until incubation with exogenous hydrogen peroxide, which resulted in a decreased fluorescent signal that is restored upon competitive chelation. Both of these prochelators provide a template for future applications and designs with improved properties.

Two additional chapters describe the development of a UV-activated iron prochelator and a new fluorescently tagged metal chelator. The UV-activated prochelator is protected with two nitrophenyl groups that are photolyzed with 350 nm light within 10 minutes to reveal a high affinity iron triazole-base chelator. A chelator of this nature may provide protection from UV-induced iron liberation and oxidative stress. A second triazole-based chelator with an embedded coumarin fluorophore was prepared as a potential metal sensor. However, this design showed off-target fluorescence responses, thus it cannot be utilized in its current form for metal detection.

MicroRNAs are key post-transcriptional regulators that have been found to play critical roles in the regulation of cellular functions. There is an emerging concept that microRNAs may be just as essential for fine-tuning physiological functions and responding to changing environments and stress conditions as for viability or development. In this dissertation, two studies are presented: The first study demonstrates a role for microRNA in the regulation of oxidative stress response in erythroid cells and the functional consequences of dysregulated microRNA expression in Sickle Cell Disease (SCD) pathobiology. The second study examines a functional role for microRNA in the cellular response to changes in cellular iron concentration. Together these studies illustrate the scope of importance of microRNAs in the coordination of cellular responses to diverse stresses.

Homozygous Sickle Cell (HbSS) erythrocytes are known to have reduced tolerance for oxidative stress, yet the basis for this phenotype has remained unknown. Here we use erythrocyte microRNA expression profiles to identify a subset of HbSS patients with higher miR-144 expression and more severe anemia. We reveal that in K562 erythroid cells and primary erythroid progenitor cells, miR-144 directly regulates NRF2, a central regulator of cellular response to oxidative stress, and modulates the oxidative stress response. We further demonstrate that increased miR-144 is associated with the reduced NRF2 levels, decreased glutathione regeneration, and attenuated antioxidant capacity found in HbSS erythroid progenitors, thereby providing a mechanism for the reduced oxidative stress tolerance and increased anemia severity seen in HbSS patients.

The post-transcriptional regulation of the IRP2 regulon in the cellular response to iron deficiency is well characterized. Here we examine the potential role for microRNA-mediated regulation in the coordinated response to cellular iron deficiency.

The ability of iron (Fe) to easily transition between two valence states makes it a preferred co-factor for innumerable biochemical reactions, ranging from cellular energy production, to oxygen transport, to DNA synthesis and chromatin modification. While Fe is highly abundant on the crust of the earth, its insolubility at neutral pH limits its bioavailability. As a consequence, organisms have evolved sophisticated mechanisms of adaptation to conditions of scarce Fe availability.

Studies in the baker's yeast Saccharomyces cerevisiae have shed light into the cellular mechanisms by which cells respond to limited Fe-availability. In response to Fe-deficiency, the transcription factors Aft1 and Aft2 activate a group of genes collectively known as the Fe-regulon. Genes in this group encode proteins involved in the high-affinity plasma membrane Fe-transport and siderophore uptake systems, as well as Fe-mobilization from intracellular stores and heme re-utilization. Concomitant with the up-regulation of the Fe-regulon, a large number of mRNAs encoding Fe-dependent proteins as well as proteins involved in many Fe-dependent processes are markedly down regulated. Thus, in response to low Fe-levels the cell activates the Fe-uptake and mobilization systems, while down-regulating mRNAs involved in highly Fe-demanding processes leading to a genome-wide remodeling of cellular metabolism that permits the funneling of the limiting Fe to essential Fe-dependent reactions.

The Fe-regulon member Cth2 belongs to a family of mRNA-binding proteins characterized by an RNA-binding motif consisting of two tandem zinc-fingers of the CX8CX5CX3H type. Members of this family recognize and bind specific AU-rich elements (AREs) located in the 3'untranslated region (3'UTRs) of select groups of mRNAs, thereby promoting their rapid degradation. In response to Fe-limitation, Cth2 binds ARE sequences within the 3'UTRs of many mRNAs encoding proteins involved in Fe-homeostasis and Fe-dependent processes, thereby accelerating their rate of decay.

Work described in this dissertation demonstrates that the Cth2 homolog, Cth1, is a bona fide member of the Fe-regulon, binds ARE-sequences within the 3'UTRs of select mRNAs and promotes their decay. Cth1 and Cth2 appear to be only partially redundant; Cth1 preferentially targets mRNAs encoding mitochondrial proteins, while Cth2 promotes the degradation of most of Cth1 targets in addition to other mitochondrial and non-mitochondrial Fe-requiring processes. The coordinated activity of Cth1 and Cth2 results in dramatic changes in glucose metabolism. In addition, experiments described in this dissertation indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE-mediated regulation by the Cth1 and Cth2 proteins, creating an auto- and trans-regulatory circuit responsible for differences in their expression. Finally, work described here demonstrates that Cth2 is a nucleocytoplasmic shuttling protein and that shuttling is important for the early determination of cytosolic mRNA-fate.

Excessive amounts of labile metals such as iron, zinc, and copper have been found in the brains of patients diagnosed with various neurodegenerative diseases, which include Alzheimer's and Parkinson's disease. Iron and copper can participate in the Haber-Weiss cycle forming reactive oxygen species, ROS, which can induce cell death. In vivo, these labile metals act catalytically regenerating additional ROS.

It has recently become evident that these neurodegenerative diseases are complex and have various targets for therapy. Described herein are the newest approaches to tackling those multifaceted issues using multifunctional chelators as well as an in depth look at a specific class of iron binding pro-chelators. Researchers have taken advantage of properties that increase cell and blood brain barrier, BBB, permeability, increase antioxidant capabilities, increased selectivity for metals in damage prone environments, lower A-beta peptide aggregation, and inhibit key enzymes associated with disease pathways.

Our research group has described sets of masked chelators, which have the potential for selectivity binding iron and copper in damage prone environments. QBP, a hydroxyquinoline-based pro-chelator was recently shown to inhibit further Aβ aggregation and diminish copper's reactivity while BSIH, a salicylaldhyde isonicotinic hydrazone pro-chelator, has been shown to react selectively with hydrogen peroxide revealing an active chelator able to bind iron. Described in this thesis is a set of iron pro-chelator derivatives of the parent compound, BSIH. These masked chelators can potentially reveal high affinity metal ligands when oxidized by H2O2. Unlike traditional iron chelators, which can disrupt iron homeostasis, these pro-chelators have a protective mask consisting of various boronic esters that react and reveal a phenolic oxygen selectively by H2O2. Studies on kinetics of oxidation by H2O2, solubility, iron affinity, and stability in various conditions have been established.

In this dissertation several metalloproteins, both metal transport proteins and the classic metalloprotein hemoglobin, are investigated using a variety of biophysical and electrochemical techniques. In each case, thermodynamic measurements provide insight into the role and mode of action of the metalloprotein under investigation. In Chapters 2 and 3, we focus on the thermodynamic properties of the metal while bound by the protein. In Chapter 4, we focus on the thermodynamic properties of the protein with and without the metal. In Chapter 5, we utilize both the metal and the protein as our probe.

In Chapter 2, we probe the thermodynamic properties of the heme-bound iron to elucidate the structure-function relationships underlying two important physiological responses of hemoglobin (Hb): the Root Effect of hemoglobin from certain fish and the different nitrite reactivities of hemoglobins from clams. Hemoglobins of some fish exhibit significantly lowered oxygen affinity at low pH, allowing for proton-mediated release of O₂. This phenomenon, known as the Root Effect, serves as a proton-driven pump delivering O₂ to the swim bladders and eyes of the fish. The clam, ,L. pectinata, expresses functionally distinct Hb I that transports H₂S and Hb II that transports O₂. These two hemoglobins differ widely in their reactivity with nitrite, a reactant of great importance to the study of vasodilation in humans. The structural basis of the extreme pH-sensitivity of the Root Effect Hbs and the extreme reactivities of the Lucina Hbs with nitrite are debated. Focusing on the metal as the probe, we investigate the reduction potentials of these Hbs using spectroelectrochemistry and compare our findings with oxygen binding studies performed by our collaborators. In both cases, our data strongly suggest that steric hindrance is the determining factor governing the respective physiological response of each hemoglobin.

In Chapter 3, we again use the metal as the probe to determine the reduction potential of titanium bound by transferrin (Tf). Tf is the human iron transport protein that can also bind titanium. To address the possible mechanisms of titanium transport through the hypothesized redox-mediated Fe₂-Tf transport pathway, a modified spectroelectrochemistry (SEC) method was developed to measure the electrochemical properties of metalloproteins with very negative potentials. However, the reduction potential of Ti₂-Tf is far too negative to access with our system. As an alternative approach, the redox properties of several model titanium and iron compounds were characterized in order to develop a linear free energy relationship (LFER) allowing us to estimate the reduction potential of Ti₂-Tf to be ca. -900 mV vs. NHE. Our results indicate that the reduction potential of Ti₂-Tf is too low to be reduced by biological reducing agents and suggest that transferrin-mediated titanium transport follows a different mechanism than iron transport.

In Chapter 4, our focus shifts to the thermodynamic properties of the protein. Some pathogenic Gram-negative bacteria such as N. gonorrhoeae steal iron from their human host by expressing a receptor (TbpA/TbpB), which binds the human iron transport protein transferrin (Tf). Once iron crosses the outer membrane, ferric binding protein (FbpA) transports it across the periplasm to the cytosol. Focusing on the protein, we investigated the protein-protein interactions involved in this transport process and the roles that TbpA and TbpB play with the use of an H/D exchange and mass spectrometry based method termed SUPREX. We report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA and we demonstrate that both TbpA and TbpB individually can deferrate Tf without energy supplied from TonB, resulting in sequestration by apo-FbpA.

In Chapter 5, we extend our investigation of the N. gonorrhoeae iron uptake system by using the metal as the probe in one case and the protein as the probe in another case. TbpA, the β-barrel receptor protein that is required for utilization of Fe₂-Tf as an iron source, has a plug domain which we hypothesize binds iron and interacts with FbpA on the periplasmic side of the outer membrane. Utilizing SUPREX to monitor the thermodynamic properties of protein folding, we investigate 1) the possible interactions between the TbpA-plug and FbpA and 2) the ability of the TbpA-plug to bind iron.

Focusing on the metal as the probe, we designed an experimental apparatus to investigate the possible thermodynamic effects of the TbpA/TbpB receptor on the release of iron from Tf. We report the use of a competitive iron chelator and equilibrium dialysis allows for the spectroscopic monitoring of iron release from Tf in the absence of FbpA, but in the presence of opaque bacterial membrane preparations containing the receptor.