College of Agriculture & Natural Resources
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Item MRP5 AND MRP9 PLAY A CONCERTED ROLE IN MALE REPRODUCTION AND MITOCHONDRIAL FUNCTION(2021) Chambers, Ian George; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential iron-containing cofactor in proteins that perform diverse functions in biology. Free heme is not only hydrophobic but also generates cytotoxic peroxide radicals. In eukaryotes, heme synthesis occurs in the mitochondria but must be transported to different intracellular organelles to be utilized by hemoproteins, a process that remains poorly understood. In Caenorhabditis elegans, MRP5/ABCC5 is an essential heme exporter as mrp-5 knockout worms are unviable due to their inability to export nutritional heme from the intestine to extra-intestinal tissues. Heme supplementation restores viability of these mutants but fails to restore male reproductive deficits. By contrast, MRP5 in mammals regulates heme levels in the secretory pathway but shows no reproductive phenotypes. Phylogenetically, the closest homolog of MRP5 in vertebrates is MRP9/ABCC12, which is absent in C. elegans raising the possibility that MRP9 may genetically compensate for MRP5 lossin vertebrates. Here, we show that MRP5 and MRP9 double knockout (DKO) mice are viable but reveal significant male reproductive deficits, reminiscent of mrp-5 worms. Although MRP9 is highly expressed in sperm, MRP9 knockout mice show reproductive phenotypes only when MRP5 is absent. Unlike other ABCC transporters, these proteins localize to mitochondrial-associated membranes (MAMs), dynamic scaffolds that associate the mitochondria and endoplasmic reticulum. Consequently, combined loss of both transporters results in abnormal sperm mitochondria and reduced fertilization rates in DKO mice. Untargeted metabolomics show striking differences in metabolite profiles in the DKO testes, consistent with the localization of these transporters to MAMs where inter-organellar metabolite exchange occurs. RNA-seq results show significant alterations in genes related to mitochondria function and energy production, EIF2 signaling, and retinoic acid metabolism. Targeted functional metabolomics reveal retinoic acid levels are significantly lower in the DKO testes. These findings establish a model in which MRP5 and MRP9 play a concerted role in regulating normal male reproductive functions and mitochondrial sufficiency.Item FUNCTIONAL CHARACTERIZATION OF HEME TRANSPORTERS IN ZEBRAFISH(2017) Zhang, Jianbing; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hrg1 and Mrp5 are identified as eukaryotic heme importer and exporter, respectively. Two Hrg1 paralogs have been annotated in zebrafish genome, Hrg1a (Slc48a1b) and Hrg1b (Slc48a1a) with 84% homology in protein sequences. Hrg1a and hrg1b are widely expressed in embryonic and adult zebrafish. Yeast growth assays reveal that zebrafish Hrg1a and Hrg1b are both capable of heme import. However, hrg1a and hrg1b double knockout (hrg1 DKO) zebrafish generated by CRISPR/Cas9 has no overt defects in differentiation and maturation of erythroid cells. Knockdown of hrg1a in hrg1b mutants or vice versa does not impair erythroid lineage in zebrafish embryos. These genetic results suggest that Hrg1 is not required for maturation and hemoglobinization of primitive erythroid cells. Hrg1a and hrg1b mRNA are upregulated in adult kidneys and spleens upon PHZ-induced hemolysis, together with hmox1, a downstream heme degrading enzyme, suggesting that Hrg1 is involved in adult heme-iron recycling during erythrophagcytosis in kidney and spleen of adult zebrafish. DAB-enhanced Perl’s iron staining reveals that iron is accumulated in macrophages in the kidney and spleen in adult wild-type zebrafish. However, macrophages with positive Perl’s staining are rarely found in the kidney of hrg1 DKO and instead large amount of iron is deposited in renal tubules, suggesting defects in heme-iron recycling by kidney macrophages in hrg1 DKO under PHZ-induced hemolysis. Whole transcriptome sequencing of mRNA extracted from spleens and kidneys reveals massive differentially expressed genes in hrg1 DKO involved in immune response, lipid transport, oxidation-reduction process and proteolysis. These indicate that hrg1 DKO are deficient in recycling heme-iron derived from damaged RBCs in the absence of functional Hrg1. Phylogenetic analysis reveals that Mrp5 and Mrp9 are closed homologs in the zebrafish genome. Yeast growth assays reveal that both zebrafish Mrp5 and Mrp9 are capable of heme export. Morpholino knockdown of mrp5 and mrp9 in zebrafish showed severe anemia in developing embryos indicating their involvements in erythropoietic development. Subsequent generation and characterization of mrp5 and mrp9 mutants by CRISPR/Cas9 will further define the function of Mrp5 and Mrp9 during zebrafish development.Item Genetic Characterization of Toxic Resistant Mutants in C. elegans(2010) Walston, Jonathan Dean; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor that plays a key role in diverse biological processes. Free heme, however, is hydrophobic and toxic to cellular macromolecules. C. elegans lack the heme biosynthetic pathway, and therefore contains a highly regulated trafficking network to redistribute heme throughout the worm. A forward genetic screen in C. elegans identified thirteen mutants which grow at toxic concentrations of heme in axenic liquid media. These mutants, termed them for Toxic HEMe resistant, belong to five complementation groups of which IQ7280, IQ7310, IQ8280 and IQ9110 strains were characterized. them mutants exhibit abnormal responses to heme analogs, mating defects, and growth on heme-deficient bacteria. Pyrosequencing analysis mapped IQ7310, IQ8280, and IQ7280 to a common genetic interval on chromosome I and IQ9110 to chromosome V. Solexa deep sequencing identified mutations in novel genes which may play an essential role in organismal heme homeostasis.