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1.
J Clin Invest ; 130(10): 5245-5256, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32634119

ABSTRACT

The congenital sideroblastic anemias (CSAs) can be caused by primary defects in mitochondrial iron-sulfur (Fe-S) cluster biogenesis. HSCB (heat shock cognate B), which encodes a mitochondrial cochaperone, also known as HSC20 (heat shock cognate protein 20), is the partner of mitochondrial heat shock protein A9 (HSPA9). Together with glutaredoxin 5 (GLRX5), HSCB and HSPA9 facilitate the transfer of nascent 2-iron, 2-sulfur clusters to recipient mitochondrial proteins. Mutations in both HSPA9 and GLRX5 have previously been associated with CSA. Therefore, we hypothesized that mutations in HSCB could also cause CSA. We screened patients with genetically undefined CSA and identified a frameshift mutation and a rare promoter variant in HSCB in a female patient with non-syndromic CSA. We found that HSCB expression was decreased in patient-derived fibroblasts and K562 erythroleukemia cells engineered to have the patient-specific promoter variant. Furthermore, gene knockdown and deletion experiments performed in K562 cells, zebrafish, and mice demonstrate that loss of HSCB results in impaired Fe-S cluster biogenesis, a defect in RBC hemoglobinization, and the development of siderocytes and more broadly perturbs hematopoiesis in vivo. These results further affirm the involvement of Fe-S cluster biogenesis in erythropoiesis and hematopoiesis and define HSCB as a CSA gene.


Subject(s)
Anemia, Sideroblastic/genetics , Molecular Chaperones/genetics , Mutation , Adolescent , Anemia, Sideroblastic/congenital , Anemia, Sideroblastic/metabolism , Animals , Child , DNA Mutational Analysis , Female , Frameshift Mutation , Gene Knockdown Techniques , Humans , Iron-Sulfur Proteins/deficiency , Iron-Sulfur Proteins/genetics , K562 Cells , Male , Mice , Mice, Knockout , Molecular Chaperones/metabolism , Pedigree , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Young Adult , Zebrafish
3.
J Adolesc Health ; 57(6): 574-9, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26422289

ABSTRACT

We present guidance on electronic nicotine delivery systems (ENDS) for health care professionals who care for adolescents. ENDS provide users with inhaled nicotine in an aerosolized mist. Popular forms of ENDS include e-cigarettes and vape-pens. ENDS range in disposability, customization, and price. Growth of ENDS usage has been particularly rapid in the adolescent population, surpassing that of conventional cigarettes in 2014. Despite surging use throughout the United States, little is known about the health risks posed by ENDS, especially in the vulnerable adolescent population. These products may potentiate nicotine addiction in adolescents and have been found to contain potentially harmful chemicals. The growth in these products may be driven by relaxed purchasing restrictions for minors, lack of advertising regulations, and youth friendly flavors. Taken together, ENDS represent a new and growing health risk to the adolescent population, one that health care professionals should address with their patients. We suggest a patient centered strategy to incorporate ENDS use into routine substance counseling.


Subject(s)
Adolescent Medicine , Electronic Nicotine Delivery Systems , Adolescent , Communication , Counseling , Humans
4.
Cell ; 161(4): 858-67, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25957689

ABSTRACT

The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.


Subject(s)
Endopeptidase Clp/metabolism , Erythropoiesis , Eukaryota/metabolism , Heme/biosynthesis , 5-Aminolevulinate Synthetase/metabolism , Amino Acid Sequence , Aminolevulinic Acid/metabolism , Animals , Biological Evolution , Endopeptidase Clp/chemistry , Endopeptidase Clp/genetics , Eukaryota/genetics , Humans , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Molecular Chaperones/metabolism , Molecular Sequence Data , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Zebrafish/metabolism
5.
J Clin Invest ; 124(10): 4294-304, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25157825

ABSTRACT

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.


Subject(s)
Erythropoiesis/genetics , Heme/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Anemia/metabolism , Animals , Cell Line , Erythroid Cells/metabolism , Gene Expression Regulation , Hemoglobins/metabolism , Liver/embryology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membranes/metabolism , Porphyrins/metabolism , Protoporphyrins/metabolism , RNA, Small Interfering/metabolism
6.
Am J Hum Genet ; 93(5): 906-14, 2013 Nov 07.
Article in English | MEDLINE | ID: mdl-24119684

ABSTRACT

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia.


Subject(s)
Anemia, Macrocytic/genetics , Membrane Proteins/genetics , Mitochondrial Diseases/genetics , Adolescent , Animals , Child , Erythropoiesis/genetics , Exome , Female , Gene Knockdown Techniques , Humans , Mitochondrial Proteins/genetics , Mutation , Zebrafish/genetics
7.
Cell Metab ; 17(3): 343-52, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23416069

ABSTRACT

Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.


Subject(s)
Anemia/genetics , Iron/metabolism , Receptors, Transferrin/metabolism , Sorting Nexins/metabolism , Analysis of Variance , Animals , Blotting, Western , Cells, Cultured , Fluorescein-5-isothiocyanate , Fluorescent Antibody Technique , Gene Silencing , Mice , Sorting Nexins/genetics , Zebrafish
8.
Dev Biol ; 373(2): 431-41, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-22960038

ABSTRACT

Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , Conserved Sequence/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/metabolism , Epistasis, Genetic , Erythropoiesis/genetics , Evolution, Molecular , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Hematopoietic System/embryology , Hematopoietic System/metabolism , Models, Biological , Molecular Sequence Data , Zebrafish/embryology , Zebrafish Proteins/chemistry , Zebrafish Proteins/metabolism
9.
Proc Natl Acad Sci U S A ; 109(30): 12099-104, 2012 Jul 24.
Article in English | MEDLINE | ID: mdl-22773809

ABSTRACT

Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ~94% succumb during the second crisis (~30 d of age) from catastrophic hematopoietic failure in the spleen and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis.


Subject(s)
Erythropoiesis/physiology , Receptors, Cytoplasmic and Nuclear/genetics , Thrombopoiesis/physiology , Animals , Animals, Genetically Modified , Enzyme Activation/physiology , Erythropoiesis/genetics , GTP Phosphohydrolases/metabolism , Mice , Mutation, Missense/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Thrombopoiesis/genetics , Zebrafish
10.
Biochemistry ; 50(51): 11047-57, 2011 Dec 27.
Article in English | MEDLINE | ID: mdl-22091532

ABSTRACT

Spontaneous glycation of bovine heart cytochrome c (cyt c) by the sugar ribose 5-phosphate (R5P) weakens the ability of the heme protein to transfer electrons in the respiratory pathway and to bind to membranes. Trypsin fragmentation studies suggest the preferential sites of glycation include Lys72 and Lys87/88 of a cationic patch involved in the association of the protein with its respiratory chain partners and with cardiolipin-containing membranes. Reaction of bovine cyt c with R5P (50 mM) for 8 h modified the protein in a manner that weakened its ability to transfer electrons to cytochrome oxidase by 60%. An 18 h treatment with R5P decreased bovine cyt c's binding affinity with cardiolipin-containing liposomes by an estimated 8-fold. A similar weaker binding of glycated cyt c was observed with mitoplasts. The reversal of the effects of R5P on membrane binding by ATP further supports an A-site modification. A significant decrease in the rate of spin state change for ferro-cyt c, thought to be due to cardiolipin insertion disrupting the coordination of Met to heme, was found for the R5P-treated cyt c. This change occurred to a greater extent than what can be explained by the permanent attachment of the protein to the liposome. Turbidity changes resulting from the multilamellar liposome fusion that is readily promoted by cyt c binding were not seen for the R5P-glycated cyt c samples. Collectively, these results demonstrate the negative impact that R5P glycation can have on critical electron transfer and membrane association functions of cyt c.


Subject(s)
Cytochromes c/metabolism , Mitochondrial Membranes/metabolism , Ribosemonophosphates/metabolism , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Cardiolipins/metabolism , Cattle , Cytochromes c/antagonists & inhibitors , Cytochromes c/chemistry , Electron Transport , Electron Transport Complex IV/chemistry , Electron Transport Complex IV/metabolism , Fungal Proteins/antagonists & inhibitors , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Glycosylation , Liposomes , Lysine/chemistry , Lysine/metabolism , Membrane Fusion , Mitochondrial Membranes/enzymology , Nephelometry and Turbidimetry , Organelles/enzymology , Organelles/metabolism , Oxidation-Reduction , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Ribosemonophosphates/chemistry , Solubility
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