Iron is an essential co-factor for many enzymes that play a vital role in cellular processes. Unbound iron catalyzes the formation of harmful reactive oxygen species;therefore, most biological iron is sequestered to keep it in an unreactive form. However, a very small amount of iron exists in the free form within the cell and is known as the labile iron pool. Iron in this pool is hypothesized to associate with low-molecular-weight siderophores and this liganded iron is a target for carrier proteins such as lipocalin 24p3. 24p3 is a member of the lipocalin family a group of carrier proteins that bind and transport a variety of ligands and function in disparate biological processes ranging from vitamin delivery to apoptosis. We originally discovered 24p3 as the gene undergoing maximum transcriptional stimulation following induction of apoptosis by cytokine deprivation of interleukin-3 (IL-3) dependent cells. 24p3 induces apoptosis through a novel pathway culminating in a decrease in intracellular iron levels. 24p3 is a unique iron-binding protein, in that it lacks an intrinsic ability to bind iron. Instead a small molecule, a siderophore, such as enterobactin an E.coli gene product, mediates iron- binding. However, the basis by which 24p3 acquires intracellular is not known nor siderophores like enterobactin in E.coli have been identified in mammalian cells. Our long-term goal is to identify and characterize a mammalian ligand for 24p3 as a prerequisite to understanding the role of 24p3 and its ligand in cellular iron metabolism. Mass spectrometry (MS) of mammalian-derived 24p3 identified Dihydroxy Benzoic Acid (DHBA) as a co-purifying factor, however, subsequent MS analysis under non-denaturing conditions indicated that DHBA is a component of a larger molecule. We will perform MS and nuclear magnetic resonance (NMR) analyses to identify and determine the structure of this larger molecule (Aim 1). We have also identified several novel genes that are necessary for the biogenesis of the mammalian siderophore. These genes are highly homologous to a group of genes that constitute an operon controlling E. coli enterobactin synthesis. Moreover, RNAi-based knockdown of these homologues in several cultured cell lines resulted in alteration of cellular iron levels, suggesting a new and unanticipated role for the mammalian siderophore in cellular iron homeostasis. We will probe the role of IRE-IRP network in the regulation of the mammalian siderophore levels and in addition we will determine the relationship between ferritin and the mammalian siderophore in cellular iron homeostasis (Aim 2). Morpholino-based knockdown of the putative orthologue of entA, the gene controlling the rate-limiting step of enterobactin synthesis, in developing zebrafish embryos resulted in embryonic lethality, characterized by dramatically decreased hemoglobin levels. We will perform experiments to determine the role of a metazoan siderophore in heme biogenesis and in blood cell lineage specification in developing zebrafish embryos (Aim 3). Collectively, these results suggest that the metazoan/mammalian siderophore is essential for cellular iron homeostasis and hemoglobin formation.

Public Health Relevance

Iron deficiency anemia is the most common form of anemia affects about one in five women, half of pregnant women, 3 percent of men in the United States. The bone marrow - a red, spongy material found within the cavities of many of large bones - makes hemoglobin. Hemoglobin is an iron rich protein that gives red color to the blood and it also carries oxygen to the rest of the body. This grant aims to study the molecular mechanisms underlying hemoglobin formation in the bone marrow. If successful, this grant could help explain the causes of iron deficiency anemia and could help in designing better drugs to combat iron deficiency anemia.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK081395-05
Application #
8296067
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Wright, Daniel G
Project Start
2008-08-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
5
Fiscal Year
2012
Total Cost
$326,985
Indirect Cost
$118,714
Name
Case Western Reserve University
Department
Pathology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Liu, Zhuoming; Velpula, Kiran K; Devireddy, Lax (2014) 3-Hydroxybutyrate dehydrogenase-2 and ferritin-H synergistically regulate intracellular iron. FEBS J 281:2410-21
Liu, Zhuoming; Ciocea, Alieta; Devireddy, L (2014) Endogenous siderophore 2,5-dihydroxybenzoic acid deficiency promotes anemia and splenic iron overload in mice. Mol Cell Biol 34:2533-46
Liu, Zhuoming; Reba, Scott; Chen, Wei-Dong et al. (2014) Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection. J Exp Med 211:1197-213
Liu, Zhuoming; Petersen, Robert; Devireddy, Laxminarayana (2013) Impaired neutrophil function in 24p3 null mice contributes to enhanced susceptibility to bacterial infections. J Immunol 190:4692-706
Liu, Zhuoming; Yang, Amy; Wang, Zhengqi et al. (2011) Multiple apoptotic defects in hematopoietic cells from mice lacking lipocalin 24p3. J Biol Chem 286:20606-14
Devireddy, Laxminarayana R; Hart, Daniel O; Goetz, David H et al. (2010) A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production. Cell 141:1006-17