Abstract
Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 µM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival.
Highlights
From a nutritional perspective, heme is a readily bioavailable source of iron for human consumption [1,2]
Efforts to understand heme trafficking in multicellular organisms have failed primarily due to the inability to separate the processes of endogenous heme synthesis from heme uptake and transport
Caenorhabditis elegans is unique among model organisms because it cannot synthesize heme but instead eats environmental heme to grow and develop normally
Summary
Heme is a readily bioavailable source of iron for human consumption [1,2]. Heme transport molecules in animals are likely to be divergent from bacterial and yeast proteins at the genetic level; bacterial and yeast heme-binding proteins have no obvious orthologs in mammals [5,6,7,8]. This is demonstrated by the identification of a heme exporter, the feline leukemia virus subgroup C cellular receptor (FLVCR), which does not show any obvious similarities to known bacterial heme transport proteins [9,10]. That a viral receptor could be a potential heme exporter in developing erythroid cells underscores the divergence among heme transport proteins and emphasizes the importance of implementing unbiased genetic approaches to elucidate the heme homeostasis pathways in tractable model systems
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