The long-term goals of this proposal are to delineate how humans regulate and integrate heme homeostasis at the organismal level. Heme, an iron containing organic ring, functions as a vital cofactor responsible for diverse biological functions in addition to serving as the major source of bioavailable iron in human nutrition. Heme is a hydrophobic and cytotoxic cofactor synthesized in the mitochondria although acceptor hemoproteins reside in different cellular compartments. How is heme transported through cellular membranes and organelles? In the previous grant cycle, we pioneered the use of Caenorhabditis elegans, a genetic animal model which does not synthesize heme but utilizes environmental heme to manufacture hemoproteins, to identify seven new molecules and establish a model for heme homeostasis and trafficking in animals. In the next funding cycle, we will employ innovative tools developed in the previous grant cycle to bridge significant knowledge gaps in delineating how tissues and cells communicate their heme status to regulate organismal heme homeostasis in live animals. Although a systemic heme communication and transport system may be expected in C. elegans, a heme auxotroph, several lines of evidence in humans, mice and zebrafish indicate that such pathways must also exist in vertebrates. Herein, we propose to elucidate the mechanisms of inter-tissue communication and identify the molecular identity of the heme signal using C. elegans. The studies in this proposal will test the hypothesis that HRG-7 and HRG-8 communicate and integrate intestinal heme status with extraintestinal tissues by initiating a feedback response to regulate systemic heme homeostasis. To elucidate the mechanisms of HRG-7 function at the molecular and organismal level, we will determine if catalytic activity, intestinal secretion, and subcellular location of HRG-7 is essentil for systemic heme homeostasis. To elucidate the mechanisms of HRG-8 function at the molecular and organismal level, we will define its genetic requirement by functional complementation of hrg-8 mutants using ectopically expressed and membrane anchored versions of HRG-8, and assess the inter-dependence of HRG-8 and HRG-7 for heme signaling function. To identify the molecular interactors or substrates of HRG-7 and HRG-8, we will use multidimensional protein identification technology, validate candidate interactors with RNAi knockdown, and specifically characterize interactors which have homologs in humans. Our studies in C. elegans will overcome several obstacles posed by mammalian model systems namely the control of intracellular heme levels by external means, experimental manipulation of animals at subcellular resolution, and optical transparency for in vivo monitoring of heme signals between tissues during development. Our proposed project is transformative because the molecules and mechanisms for trafficking and distribution of heme to regulate systemic heme homeostasis in eukaryotes are largely unknown. Results from our studies may provide a heuristic paradigm to tackle similar questions for other micronutrients.

Public Health Relevance

Iron deficiency is the world's number one nutritional disorder, and heme is the most bioavailable form of iron for human consumption. Identification of how dietary heme is transported and coordinated with systemic heme levels will permit the design of synthetic heme-based nutraceuticals specifically targeted to iron-deficient individuals. Iron deficiency is exacerbated by blood-loss due to heme auxotrophic parasites such as intestinal hookworms which depend on host heme to survive. A drug that targets the heme transport and regulatory pathway in helminths would be a powerful anthelminthic. Thus, identification of heme trafficking pathways across species will have a profound and long-lasting impact on our understanding of fundamental, clinical and applied processes.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Integrative Nutrition and Metabolic Processes Study Section (INMP)
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Maruvada, Padma
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University of Maryland College Park
Veterinary Sciences
Schools of Earth Sciences/Natur
College Park
United States
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Samuel, Tamika K; Sinclair, Jason W; Pinter, Katherine L et al. (2014) Culturing Caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment. J Vis Exp :e51796
Korolnek, Tamara; Zhang, Jianbing; Beardsley, Simon et al. (2014) Control of metazoan heme homeostasis by a conserved multidrug resistance protein. Cell Metab 19:1008-19
Yuan, Xiaojing; Fleming, Mark D; Hamza, Iqbal (2013) Heme transport and erythropoiesis. Curr Opin Chem Biol 17:204-11
Chen, Caiyong; Samuel, Tamika K; Krause, Michael et al. (2012) Heme utilization in the Caenorhabditis elegans hypodermal cells is facilitated by heme-responsive gene-2. J Biol Chem 287:9601-12
Yuan, Xiaojing; Protchenko, Olga; Philpott, Caroline C et al. (2012) Topologically conserved residues direct heme transport in HRG-1-related proteins. J Biol Chem 287:4914-24
Chen, Caiyong; Samuel, Tamika K; Sinclair, Jason et al. (2011) An intercellular heme-trafficking protein delivers maternal heme to the embryo during development in C. elegans. Cell 145:720-31
Ihrig, Jessica; Hausmann, Anja; Hain, Anika et al. (2010) Iron regulation through the back door: iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in Saccharomyces cerevisiae. Eukaryot Cell 9:460-71
Severance, Scott; Rajagopal, Abbhirami; Rao, Anita U et al. (2010) Genome-wide analysis reveals novel genes essential for heme homeostasis in Caenorhabditis elegans. PLoS Genet 6:e1001044
Sinclair, Jason; Hamza, Iqbal (2010) A novel heme-responsive element mediates transcriptional regulation in Caenorhabditis elegans. J Biol Chem 285:39536-43
Schultz, Iman J; Chen, Caiyong; Paw, Barry H et al. (2010) Iron and porphyrin trafficking in heme biogenesis. J Biol Chem 285:26753-9

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