Four out of five people in the world may be iron-deficient, making nutritional iron deficiency the most common nutritional disorder. Although considerable experimental and nutritional data support an essential role for heme (iron-protoporphyrin IX) as a bioavailable source of iron in human nutrition, the pathways for heme absorption and utilization are currently unknown. From a cell biological perspective, heme is the prosthetic group for many important biological processes, and in eukaryotes it is synthesized in the mitochondrial matrix. Because heme is a hydrophobic molecule and is cytotoxic due to its intrinsic peroxidase activity, we hypothesize that heme does not merely diffuse through lipid bilayers but is actively transported via specific intracellular pathways. We propose to use the animal model, Caenorhabditis elegans, to identify the cellular pathways for heme transport and the molecules mediating heme trafficking, because this animal is the only known genetic animal model that is unable to synthesize heme eye no^o albeit requiring heme to sustain metabolic processes. Since C. elegans lacks the ability to make heme, it provides us with a clean genetic background devoid of endogenous heme, and the capacity to externally control the metabolic flux of heme. Thus, C. elegans is an obligate heme auxotroph and will be an excellent animal model to study dietary heme absorption as well as intracellular heme trafficking for transport, sequestration and incorporation into critical hemoproteins. The cellular pathways for heme transport will be mapped in C. elegans by characterizing phenoclusters of mutants isolated from forward genetic screens by biochemically assaying for heme levels and hemoprotein activity, evaluating viability and tracking heme transport in live animals with heme analogs, and determining heme distribution at the ultrastructural level using electron microscopy. The molecular identities of the mutated genes will be determined by categorizing mutants in genetic complementation groups, mapping the mutated genes using SNPs, localizing the mutant gene by RNA interference and gene rescue, and by sequencing candidate genes to determine the molecular lesion. The heme regulated genes in C. elegans, identified from our DMA microarrays, will be characterized by quantitatively validating subsets of genes by real-time PCR and RNA blot analysis, determining the function of the candidate genes by RNA interference, and defining the temporal and spatial expression of these gene products in response to heme by synthesizing transcriptional and translational reporter gene fusions. The results from these studies will provide new mechanistic insights into heme homeostasis in eukaryotes and may aid in the development of heme-based nutritional interventions for human iron deficiency, and potential drug targets for human helminthic infections that exacerbate iron deficiency.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK074797-05
Application #
7777349
Study Section
Integrative Nutrition and Metabolic Processes Study Section (INMP)
Program Officer
Maruvada, Padma
Project Start
2006-03-01
Project End
2012-02-28
Budget Start
2010-03-01
Budget End
2012-02-28
Support Year
5
Fiscal Year
2010
Total Cost
$258,109
Indirect Cost
Name
University of Maryland College Park
Department
Veterinary Sciences
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
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Soares, Miguel P; Hamza, Iqbal (2016) Macrophages and Iron Metabolism. Immunity 44:492-504
Luck, Ashley N; Yuan, Xiaojing; Voronin, Denis et al. (2016) Heme acquisition in the parasitic filarial nematode Brugia malayi. FASEB J 30:3501-3514
Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna et al. (2016) Regulation of intracellular heme trafficking revealed by subcellular reporters. Proc Natl Acad Sci U S A 113:E5144-52
Korolnek, Tamara; Hamza, Iqbal (2015) Macrophages and iron trafficking at the birth and death of red cells. Blood 125:2893-7
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
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
Yuan, Xiaojing; Fleming, Mark D; Hamza, Iqbal (2013) Heme transport and erythropoiesis. Curr Opin Chem Biol 17:204-11
Huynh, Chau; Yuan, Xiaojing; Miguel, Danilo C et al. (2012) Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1. PLoS Pathog 8:e1002795
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

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