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. 2008 Jun 19;453(7198):1127-31.
doi: 10.1038/nature06934. Epub 2008 Apr 16.

"VSports最新版本" Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins

Abbhirami Rajagopal  1 , Anita U Rao, Julio Amigo, Meng Tian, Sanjeev K Upadhyay, Caitlin Hall, Suji Uhm, M K Mathew, Mark D Fleming, Barry H Paw, Michael Krause, Iqbal Hamza
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Haem homeostasis is regulated by the conserved and concerted functions of HRG-1 proteins

Abbhirami Rajagopal et al. Nature. .

Abstract

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 VSports手机版. 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. .

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Figures

Figure 1
Figure 1. Identification of hrg-1 and hrg-4 in C. elegans
a, Fluorescent ZnMP (40 µM for 3 h) accumulation in worms grown in mCeHR-2 medium supplemented with 1.5 µM (left) and 500 µM (right) haem. Differential interference contrast (DIC, top) and rhodamine fluorescence (bottom). b, Total mean fluorescence intensity (filled circles) of ZnMP accumulated in worms (40 µM for 3 h) after 9 days of growth in mCeHR-2 medium supplemented with the indicated haem concentrations. Open diamonds, growth of worms in haemin. Results are means ± s.d. (n = 100). c, Northern blot analysis of hrg-1 and hrg-4 expression in response to 4 and 20 µM haem in mCeHR-2 medium. The blot was stripped and reprobed with gpd-2 as loading control. kb, kilobases. d, Expression of hrg-4 (circles) and hrg-1 (squares) mRNA estimated by quantitative RT–PCR from total RNA obtained from worms grown at the indicated haem concentrations. Each data point shows mean ± s.d. and the results are representative of three separate experiments. Inset: mRNA levels at higher haem concentrations. e, Multiple sequence alignment of C. elegans HRG-1 with its vertebrate orthologues. Asterisk, histidine (H90); circles, aromatic amino acids; box, putative transmembrane domains; YXXxφ, C-terminal tyrosine motif; D/EXXxLL, di-leucine motif. f, Phylogenetic analysis of HRG-1 proteins by using the neighbour-joining method. g, Predicted topology of C. elegans HRG-1 showing H90 in TMD2, and FARKY, the putative haem-interacting motif, in the cytoplasmic tail.
Figure 2
Figure 2. hrg-1 and hrg-4 are essential for haem homeostasis in C. elegans
a, IQ6011 hrg-1::gfp ‘heme sensor’ strain responds to exogenous haem after sequential exposure to E. coli grown on agar plates in the absence (left and right) and presence (middle) of 200 µM haem. UTR, untranslated region. b, Spectrofluorometric measurements of GFP in worm lysates from IQ6011 strain grown in the presence of indicated concentrations of haem plus 20 µM PPIX or 1 mM FeCl3. Each data point shows mean ± s.d. and the results are representative of three separate experiments. c–e, Depletion of hrg-1 or hrg-4 in worms by RNAi with feeding bacteria. c, Dysregulation of GFP (means ± s.e.m.; n = 35–45 worms per treatment) in IQ6011 when fed with bacteria grown in the presence of 0, 5 and 25 µM haem. Values with different letter labels are significantly different (P < 0.001) within each treatment. d, Aberrant ZnMP fluorescence accumulation in worms fed with 10 µM ZnMP for 16 h. Scale bar, 50 µm. e, Differences in viable progeny (mean ± s.d.; n = 30 P0 worms per treatment) after 5 days of exposure to 1 µM GaPP plus RNAi bacteria. Filled bars, viable eggs; open bars, larvae. The results for c–e are representative of at least four separate experiments.
Figure 3
Figure 3. HRG-1 is essential for erythropoiesis and development in zebrafish
a, Zebrafish hrg-1 expression by whole-mount in situ hybridization: left, 15 somites; right, 24 h after fertilization. b, Knockdown of zebrafish hrg-1 by using morpholinos (MO2) against zebrafish hrg-1 reveals severe anaemia with very few o-dianisidine-positive red cells (arrows, right panel), hydrocephalus, and a curved body with shortened yolk tube (arrows, left panel). WT, wild type. c, Decrease in haemoglobinized cells in MO2 morphants (arrows). d, e, Cehrg-1 cRNA injected along with MO2, shows restoration of haemoglobinized cells (d) and complete rescue of the developmental defects of hydrocephalus, body axis curvature, and yolk sac formation (e, arrows).
Figure 4
Figure 4. Expression, localization and functional studies of worm and mammalian hrg-1
a, b, mRNA expression of human HRG-1 in multiple adult human tissues (a) and human tissue-derived cell lines (b). The blots were stripped and reprobed with β-actin as loading control. c, Expression of C-terminally tagged proteins in transfected HEK-293 cells by SDS–PAGE and immunoblotting with antibodies against HA (lanes 1–3, 50 µg) and GFP (lanes 4– 6, 25 µg), or by in vitro expression with 35S fluorography (lanes 7–9, one-fifth of total extract). d, Cellular localization of C-terminally tagged fluorescent proteins in transfected HEK-293 cells by confocal microscopy. The plasma membrane (PM) was identified by using wheat germ agglutinin. Scale bar, 20 µm. e, HRG-1 proteins interact with haem as a function of pH. Cell lysates (lanes 1 and 4, one-tenth of total protein) from HEK-293 cells expressing the indicated HA-tagged proteins were incubated with haemin-agarose. Wash (lanes 2 and 5) depicts the final wash before elution of the bound protein (lanes 3 and 6) from the haemin-agarose column. Samples were subjected to SDS–PAGE followed by immunoblotting with anti-HA antisera. f, Flow-cytometry histograms show enhanced ZnMP uptake and accumulation after 30 min of incubation with 5 µM ZnMP in MEL cells stably expressing either hHRG-1–HA (right) or empty vector (left). g, Electrophysiological currents (means ± s.d., n = 4) elicited from Xenopus oocytes injected with cRNA encoding the indicated protein, when clamped at −110 mV in the presence of 20 µM haemin chloride. The y axis represents the difference in current in the presence and absence of haemin, normalized to the current observed in the absence of haem. Values with different letter labels are significantly different (P < 0.05) within each treatment compared with hKv1.1 control. h, Proposed model for the function of HRG-1 proteins in haem homeostasis in C. elegans intestinal cells. CeHRG-4 mediates haem uptake through the plasma membrane, whereas CeHRG-1 facilitates intracellular haem availability through an endosomal and/or lysosomalrelated compartment. The model does not exclude the possibilities that CeHRG-1 traffics through the plasma membrane and may be functional on the cell surface.
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