Our long-term research objectives are to define the cellular and molecular determinants of heme homeostasis in humans. Understanding how heme-iron is utilized will permit the design of novel nutritional strategies to ameliorate iron deficiency anemia in humans and uncover novel therapeutic drug targets against parasites that exacerbate global iron deficiency.
At the University of Maryland, Dr. Hamza deliberately set out to uncover heme trafficking pathways in eukaryotes - which were unknown at the time. His pioneering work with the invertebrate animal model C. elegans has demonstrated that this roundworm is exceptional because it does not synthesize heme but rather utilizes environmental heme to manufacture heme-containing proteins, which have human homologs. This broke the existing paradigm that heme synthesis occurred in all free-living eukaryotes [PNAS 2005]. Using the worm model, we identified the first eukaryotic heme importer/transporter (HRG-1) which is conserved in zebrafish and humans [Nature 2008]. We uncovered how heme is exported from the intestine to other tissues including the embryos by HRG-3 and ABCC5/MRP5 [Cell 2011; Cell Metabolism 2014], and more recently how organs communicate to each other to maintain organismal heme homeostasis by HRG-7 [Nature Cell Biology 2017]. These findings represent major discoveries in heme trafficking and establish a heuristic paradigm for heme transport in animals. Beyond C. elegans, we have shown that related free-living and parasitic nematodes (helminths) do not synthesize heme, much like Trypanosomes and Leishmania. Thus, selective targeting of parasite heme transport pathways could be their Achilles heel. Our groundbreaking studies resulted not only in the identification of homologs for heme transporters in humans [Cell Metabolism 2013; PNAS 2016a; PNAS 2016b] but also in parasites such as hookworms, filarial worms and Leishmania, which rely on host heme for survival [Infect Immun 2006, PLoS NTD. 2009; PLoS Path. 2012]. We anticipate that our studies will lead to identification of inhibitors that can target heme transport pathway in parasites which infect humans, livestock, and plants, as well as in humans with genetic disorders of heme and iron metabolism.
WHY IS HEME IMPORTANT?
From a public-health perspective, iron deficiency is the most common nutritional disorder in the world. According to the World Health Organization, four out of five people in the world may be iron deficient, making nutritional iron deficiency one of the top ten risk factors in both developed and developing countries (WHO/UNICEF). A major contributing factor is the high prevalence of malaria and hookworm infections in developing countries where iron deficiency is endemic. Although iron is one of the most abundant metals on the planet, it is not readily bioavailable because iron is not easily soluble at the pH of the intestinal lumen and dietary phytates and phosphates retard iron absorption. By contrast, heme (iron-protoporphyrin IX), is a bioavailable source of iron because it is soluble in the intestinal lumen and heme absorption is not affected by dietary components. However, the intestinal heme transporter has yet to be identified.
From a clinical perspective, humans recycle more than 360 billion senescent red blood cells (RBCs) every day (5 million/second). Adults have ≈2.5 x 10^13 RBCs and >60% of iron in the body is used for hemoglobin production in these cells. Consequently, iron deficiency anemia is the primary clinical manifestation of iron deficiency. Each day, macrophages of the reticuloendothelial system recycle ≈25 mg of iron derived from heme by ingesting senescent RBCs; the bone marrow utilizes this recycled iron to daily produce new RBCs. Thus, the majority of iron reutilization by the body is via heme-iron, and genetic defects in the recycling of iron by macrophages result in anemia. Despite the importance of heme in macrophage-dependent iron recycling, the molecules and the pathways responsible for heme transport across intracellular membranes remain poorly understood.
From a cell biological perspective, heme is a crucial prosthetic group for a wide variety of proteins that function in key physiological processes ranging from gas sensing to xenobiotic detoxification to microRNA processing. Within most organisms, heme is synthesized by a highly regulated multistep pathway which is conserved throughout evolution from bacteria to mammals. The synthesis and degradation of heme have been extensively studied, yet neither the mechanisms nor the molecules responsible for assembling heme into hemoglobin and other hemoproteins are known. Free heme is hydrophobic and cytotoxic due to its peroxidase reactivity. Moreover, hemoproteins reside in different cellular compartments. Thus, specific intracellular pathways exist for the safe and directed trafficking of heme from the site of synthesis or entry into the cell to distinct target organelles and proteins. Identification of the intra- and intercellular heme trafficking pathways remains poorly understood due to a lack of genetically tractable model systems.
WHY USE WORMS?
We found that C. elegans is the only known genetic animal model that is unable to synthesize heme de novo, albeit requiring heme to sustain metabolic processes. Since this organism lacks the ability to make heme, it provides us with a clean genetic background devoid of endogenous heme, and the ability to externally control the metabolic flux of heme. More than 70% of all human genes are conserved in C. elegans, and methods are in place for identifying genes by forward and reverse genetic screens. Importantly, genetic analyses in this roundworm will provide us with a molecular blueprint to pinpoint orthologous genes and pathways in humans and in parasites that exacerbate human iron deficiency.
Our studies are inherently multi-dimensional in design and multi-organismal in scope, and not restricted by the genetic tractability of a particular model system. This strategy is based upon our broad research expertise and an in-depth knowledge of multiple genetic model systems. We simultaneously use C. elegans, mammalian cell lines, mice, zebrafish, and yeast to tackle questions related to heme homeostasis. Our collective expertise in biochemical, cell biological, and genomic studies are translational permitting us to work across species and exploit each system maximally in order to define the molecular basis of heme trafficking in eukaryotes.
One of the most important discoveries of the past century was that prenatal iron supplementation can prevent many serious birth defects and reduce the risk of premature and low-birth weight birth outcomes by 6 to 7 fold when maternal diet is supplemented with high iron during the first two trimesters. Thus, determining the underlying causes for the genetic and ecogenetic basis of nutritional iron deficiency in women of childbearing potential will likely have far greater consequences than the immediate effects of reducing birth defects or preterm births. Our pioneering work with the invertebrate animal model C. elegans has demonstrated that this roundworm is exceptional because it does not synthesize heme but rather utilizes membrane-bound permeases, HRG-1 and HRG-4, to import environmental heme. Correspondingly, tissues such as muscle, neurons, hypodermal cells, and embryos are dependent on intestinal heme to fulfill their metabolic requirements. Consistent with this concept, we uncovered HRG-3, a novel peptide secreted by the mother’s intestine and functions to transport maternal heme to developing oocytes ensuring that sufficient heme is available to sustain embryonic development. These results imply that HRG-3 functions as an intercellular chaperone to mobilize maternal heme and deliver it to the next generation. Although an intercellular heme transport system is to be expected in worms which are heme auxotrophs, we assert that an intercellular pathway for heme distribution must also exist in vertebrates. To tackle this question in vertebrates we will use zebrafish embryology and genetics coupled with developing highly specific genetically-encoded heme sensors and non-invasive, label-free imaging platforms to monitor and measure heme levels in living cells.
Membrane trafficking and compartmentalization
Heme is cytotoxic because of its intrinsic peroxidase activity. How then is heme transported through membranes and cellular organelles? What are the mechanisms for incorporating heme into specific hemoproteins that reside in the cytoplasm, peroxisomes, mitochondrial inter-membrane space, secretory pathway, and nucleus? Are these mechanisms specific to the target apo-proteins or to their sub-organellar milieu? Humans produce intracellular hemoproteins such as hemo-, myo-, neuro-, and cytoglobins, nuclear hormone receptors, and transcription factors, as well as heme enzymes including cytochrome P450s, soluble guanylyl cyclases, thyro- and myelo-peroxidases, catalases, and respiratory cytochromes. These proteins and enzymes are located in different cellular compartments and perform diverse functions, all of which depend upon heme as a prosthetic group. To uncover these trafficking pathways, we conducted a genomic screen in C. elegans which identified several hundred Heme Responsive Genes (HRGs). We recently demonstrated that HRG-1 and its paralogs HRG-4, HRG-5 and HRG-6 are four transmembrane permeases. They contain invariant histidines in TMD2 and the exoplasmic E2 loop, and a FARKY motif in the cytoplasmic C-terminal tail. These residues could potentially either directly bind heme or interact with the heme side chains. In addition, a conserved tyrosine and acidic-dileucine based sorting signal situated within ten residues after TMD4 in the carboxyl terminus. These motifs have been shown to sort transmembrane proteins to endosomes and lysosome-related organelles. Notably, HRG-4 lacks both sorting signals, and the histidine residue in TMD2 of CeHRG-1 is substituted with a tyrosine. Unlike histidines, tyrosine heme ligands have a lower redox potential, and the coordination stabilizes heme and prevents it from carrying out oxidative chemistry. These analyses suggest that heme transporters have intrinsic differences in their heme-binding properties which may be linked to intracellular or tissue localization. We are currently addressing these questions by identifying the mechanisms for protein localization and the cellular basis for heme compartmentalization.
Organismal heme transport
To identify regulators of organismal heme homeostasis, we conducted a functional RNAi screen by depleting >18,000 genes in C. elegans. Our primary, secondary, and tertiary screens identified a novel catalog of activators and repressors that modulate organismal heme homeostasis. These genes encode putative membrane transporters/channels, tethering complexes, redox regulators, soluble chaperones, and mitochondrial proteins. Over 200 candidate genes have homologs in humans and are highly expressed in hematopoietic tissues. Some of these genes are involved in regulating systemic heme homeostasis by using an inter-tissue communication pathway to coordinatively regulate heme levels within an animal. Using worms and zebrafish we are currently dissecting the functions and mechanisms of specific lead candidates which reveal significant perturbations in heme homeostasis.
Inhibitors of parasite heme transporters
Currently, there are no pharmacological tools to aid in the study of the cellular and physiological roles of eukaryotic heme transporters. We developed and validated a cell-based, high throughput screening (HTS)-compatible screen for small molecule antagonists of HRG-1-related proteins. Because parasitic nematodes and the kinetoplastids also acquire heme from the environment, it is anticipated that reagents identified in our HTS can be used to establish the feasibility of targeting the heme transport pathway for the treatment of helminth infections, Trypanosomiasis, intestinal nematodes, kinetoplastid diseases, lymphatic filariasis, onchocerciasis, and Leishmaniasis, as well as human genetic disorders of heme and iron metabolism. Our long-term goals are to advance strategies that target heme transport pathways unique to the parasite.
Our research is supported by grants from the National Institutes of Health.