Bryan Mackenzie, PhD - US grants

[unreadable] DESCRIPTION (provided by applicant): DMT1 is a widely-expressed iron transporter that is essential for adequate intestinal absorption of iron and for transport of iron in red blood cell precursors for the production of hemoglobin. Rare mutations in DMT1 cause severe microcytic anemia. Conversely, dysregulation of DMT1 in hereditary hemochromatosis, the most common hereditary disease in Caucasians, results in toxic iron overload in vital organs. Since there exists no regulated mechanism for its excretion, iron homeostasis is achieved by regulating intestinal iron absorption. DMT1 is the gateway for iron absorption - it is the primary or only apical transporter of nonheme iron - making it the focus of this proposal. Iron deficiency affects as much as 10% of the U.S. population and is the most prevalent micronutrient deficiency worldwide. Important for this proposal, iron deficiency is a serious risk factor for cadmium intoxication, suggesting that cadmium and iron share a common absorptive mechanism. Under investigation in this proposal is the premise that DMT1 is a complex H+-coupled and voltage-dependent ferrous-iron (Fe2+) transporter and that an acidic microclimate at the intestinal brush-border membrane provides the proton-motive force energizing DMT1. We will probe the molecular basis of H+-coupling and explore a physiological role for the significant uncoupled H+ fluxes (slippage) through DMT1 using the voltage clamp, radiotracer (55Fe) assays, and fluorescence approaches in Xenopus oocytes expressing wildtype DMT1 or mutant proteins. Next, we will examine iron transport in the mouse intestine using radiotracer and fluorescence approaches; the use of specific inhibitors of intestinal Na+/H+ exchangers (NHE3 and NHE2) and mutant mouse models lacking NHE3, NHE2, or the gastric H+/K+-ATPase will permit us to evaluate the roles of gastric acid and the intestinal brush-border acidic microclimate in providing the H+ to drive DMT1-mediated Fe2+ transport. The second premise is that DMT1 serves absorption not only of iron but also of certain other essential metals such as Co and Mn (as well as trace metals such as Ni and V) but that this promiscuity also makes it a major route of entry for the toxic heavy metal Cd. Here, we will determine the comprehensive substrate profile and metal-ion selectivity of DMT1 using our oocyte assays. Next, we will use the Belgrade anemic rat (which bears a mutation in DMT1) to examine the physiological significance of DMT1 in the absorption of each of the metals it is capable of transporting. Finally, we will identify metal-coordination sites in DMT1. Studying DMT1 in this way will lead to a better understanding of the mechanisms of nonheme iron absorption, the conditions required for efficient absorption, and the role of DMT1 in the metabolism of other transition metals. The results of this work will help drive development of new strategies for improving metal nutrition, or for treating iron overload and heavy-metal intoxication. [unreadable] Public Health Relevance: Iron deficiency leads to anemia and the hereditary disease hemochromatosis leads to toxic iron overload, illustrating the importance of balancing intestinal iron absorption. The gateway for dietary iron to enter the cells lining the intestine is a protein called DMT1, which can also transport the toxic metal cadmium. In this project we will study iron and cadmium transport via DMT1 at the molecular level to drive development of new strategies to improve iron nutrition and prevent the toxic effects of cadmium or iron overload. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): The iron transporter ferroportin is critical for delivering iron into plasma from duodenal enterocytes absorbing dietary iron, macrophages recycling old red blood cells, and hepatocytes storing iron. The interaction of ferroportin with its ligand, the hepatic hormone hepcidin, is the key event in systemic iron homeostasis. After it binds hepcidin, ferroportin is degraded, thereby limiting iron entry into plasma. Dysregulation of the hepcidin- ferroportin axis underlies most common iron disorders, including anemia of inflammation, anemia of chronic kidney disease, anemia of cancer, hereditary hemochromatosis and iron-loading anemias. Ferroportin is the only known cellular iron exporter in vertebrates and is conserved down to invertebrates and plants. Despite its obvious biological importance, very little is known about the ferroportin structure and the mechanisms by which ferroportin transports iron. In a recent breakthrough, we identified the prokaryotic ortholog of Fpn and obtained its structure by X-ray crystallography. We are now poised to make rapid progress toward complete understanding of the structural basis of Fpn function by combining X-ray crystallography of mammalian Fpn with detailed structure-guided mutational and functional analyses of metal transport and hepcidin-ferroportin interaction in mammalian cells and Xenopus oocytes. Our Specific Aims are: Aim 1. Determine the structure of ferroportin by X-ray crystallography. While these efforts are ongoing, we will use the structure of prokaryotic Fpn ortholog to guide further studies of the transport mechanism, specific function of conserved residues, and as a framework for functional and mutagenesis work on the higher orthologs. Aim 2. Determine the mechanism of iron transport by ferroportin. These studies will determine the driving forces, ion coupling, and calcium gating of Fpn-mediated iron transport. Aim 3. Discover the structural determinants of ferroportin function and malfunction. We will use structure- and human disease guided mutagenesis to probe critical residues involved in iron binding and translocation, transporter gating, pH dependence, oligomerization, and hepcidin binding. Successful completion of the proposed studies is of fundamental importance for iron biology. Its significance also extends to general biology: identifying the structure of a new class of membrane transporters and defining the mechanism of iron transport will impact studies of other metal and ion transporters. Finally, understanding Fpn iron-transporting function and its regulation by hepcidin is biomedically significant. The proposed studies will generate much more definitive mechanistic and structural insights which will guide the development of improved small and large molecule therapeutics for iron-restrictive anemias and iron overload disorders.