PROJECT SUMMARY Sulfur assimilation is an evolutionarily conserved pathway that plays an essential role in cellular and metabolic processes including sulfation, amino-acid biosynthesis and organismal development. Disruption of the sulfur assimilation pathway has been shown to result in a number of pathologies in drosophila, mice and humans. Here, we report that loss of a key enzymatic component of the pathway, bisphosphate 3'-nucleotidase BPNT1, in mice, both whole animal and intestine specific, leads to iron-deficiency anemia. The long-term goal of this research is to define the molecular mechanisms by which Bpnt1 loss-of-function, and subsequent accumulation of its substrate 3'-phosphoadenosine 5'-phosphate (PAP), leads to dysfunction of the iron regulatory machinery. Analysis of mutant enterocytes from intestine-specific Bpnt1 mutants demonstrate modulation of PAP influences levels of key iron homeostasis factors involved in dietary iron reduction, import and transport, that in part mimic those reported for the loss of hypoxic-induced transcription factor, HIF-2α. Our general hypothesis is that PAP accumulation leads to alterations in the iron-regulatory pathway, leading to iron deficiency anemia. Specifically, I hypothesize that PAP accumulation inhibits HIF- 2α−dependent and independent signaling. To test this hypothesis, I will employ biochemical, molecular, proteomic, and animal model approaches to obtain a mechanistic understanding of the molecular consequences of PAP accumulation. Then, I will apply these mechanistic insights to translational human genetics studies. I propose three aims: 1) Elucidate the molecular mechanisms by which PAP accumulation perturbs iron metabolism, 2) Characterize the role of Bpnt1-HIF-2α mediated iron homeostasis in mice and 3) Determine the role of Bpnt1 in human iron metabolism. The proposed studies are expected to define a new genetic basis for iron-deficiency anemia, delineate a molecular approach for rescuing the pathophysiology and provide an unanticipated link between nucleotide hydrolysis in the sulfur assimilation pathway and iron homeostasis. !