Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins.

Haems are metalloporphyrins that serve as prosthetic groups for various biological processes including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control, metabolic reprogramming and microRNA processing. With a few exceptions, haem is synthesized by a multistep biosynthetic pathway comprising defined intermediates that are highly conserved throughout evolution. Despite our extensive knowledge of haem biosynthesis and degradation, the cellular pathways and molecules that mediate intracellular haem trafficking are unknown. The experimental setback in identifying haem trafficking pathways has been the inability to dissociate the highly regulated cellular synthesis and degradation of haem from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural haem auxotrophs that acquire environmental haem for incorporation into haemoproteins, which have vertebrate orthologues. Here we show, by exploiting this auxotrophy to identify HRG-1 proteins in C. elegans, that these proteins are essential for haem homeostasis and normal development in worms and vertebrates. Depletion of hrg-1, or its paralogue hrg-4, in worms results in the disruption of organismal haem sensing and an abnormal response to haem analogues. HRG-1 and HRG-4 are previously unknown transmembrane proteins, which reside in distinct intracellular compartments. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations and, most strikingly, profound defects in erythropoiesis-phenotypes that are fully rescued by worm HRG-1. Human and worm proteins localize together, and bind and transport haem, thus establishing an evolutionarily conserved function for HRG-1. These findings reveal conserved pathways for cellular haem trafficking in animals that define the model for eukaryotic haem transport. Thus, uncovering the mechanisms of haem transport in C. elegans may provide insights into human disorders of haem metabolism and reveal new drug targets for developing anthelminthics to combat worm infestations.

Genome-wide analysis reveals novel genes essential for heme homeostasis in Caenorhabditis elegans.

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 microM 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.

Development and validation of an electrochemiluminescent ELISA for quantitation of oral insulin tregopil in diabetes mellitus serum.

AIM: Tregopil, a novel PEGylated human insulin is in clinical development for oral delivery in diabetes treatment. The aim of the study was to develop and validate a sensitive and specific ELISA method for quantitating Tregopil in diabetes subjects on basal Glargine, since most commercially available insulin kits either do not detect Tregopil or show significant reactivity to Glargine. METHODS: An electrochemiluminescent ELISA was developed and validated for Tregopil quantitation in diabetes serum. RESULTS: The method has a LLOQ of 0.25 ng/ml, shows minimum cross-reactivity to Glargine and was successfully tested using a subset of samples from Tregopil-dosed Type 1 diabetes mellitus patients. CONCLUSION: The ELISA method is sensitive and can be used to support accurate measurement of Tregopil with no cross-reactivity to Glargine and its metabolites in clinical studies.

The PRY/SPRY/B30.2 domain of butyrophilin 1A1 (BTN1A1) binds to xanthine oxidoreductase: implications for the function of BTN1A1 in the mammary gland and other tissues.

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (KD=101+/-31 nM in 10 mM HEPES, 150 mM NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.

The heme biosynthetic pathway of the obligate Wolbachia endosymbiont of Brugia malayi as a potential anti-filarial drug target.

BACKGROUND: Filarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs. METHODS AND FINDINGS: Genome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a approximately 600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5'-aminolevulinic acid dehydratase (ALAD, the second step). Similarly, Escherichia coli hemH (FC) deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening. CONCLUSIONS: Our studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm) might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues.

Lack of heme synthesis in a free-living eukaryote.

In most free-living eukaryotes studied thus far, heme is synthesized from a series of intermediates through a well defined evolutionarily conserved pathway. We found that free-living worms, including the model genetic organism Caenorhabditis elegans, and parasitic helminths are unable to synthesize heme de novo, even though these animals contain hemoproteins that function in key biological processes. Radioisotope, fluorescence labeling, and heme analog studies suggest that C. elegans acquires heme from exogenous sources. Iron-deprived worms were unable to grow in the presence of adequate heme unless rescued by increasing heme levels in the growth medium. These data indicate that although worms use dietary heme for incorporation into hemoproteins, ingested heme is also used as an iron source when iron is limiting. Our results provide a biochemical basis for the dependence of worm growth and development on heme, and they suggest that pharmacologic targeting of heme transport pathways in worms could be an important control measure for helminthic infections.