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1.
PLoS Genet ; 16(12): e1008857, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33370780

RESUMEN

Studies of the severely pancytopenic scat mouse model first demonstrated the crucial role of RASA3, a dual RAS and RAP GTPase activating protein (GAP), in hematopoiesis. RASA3 is required for survival in utero; germline deletion is lethal at E12.5-13.5 due to severe hemorrhage. Here, conditional deletion in hematopoietic stem and progenitor cells (HSPCs) using Vav-iCre recapitulates the null phenotype demonstrating that RASA3 is required at the stem and progenitor level to maintain blood vessel development and integrity and effective blood production. In adults, bone marrow blood cell production and spleen stress erythropoiesis are suppressed significantly upon induction of RASA3 deficiency, leading to pancytopenia and death within two weeks. Notably, RASA3 missense mutations in two mouse models, scat (G125V) and hlb381 (H794L), show dramatically different hematopoietic consequences specific to both genetic background and molecular variant. The mutation effect is mediated at least in part by differential effects on RAS and RAP activation. In addition, we show that the role of RASA3 is conserved during human terminal erythropoiesis, highlighting a potential function for the RASA3-RAS axis in disordered erythropoiesis in humans. Finally, global transcriptomic studies in scat suggest potential targets to ameliorate disease progression.


Asunto(s)
Proteínas Activadoras de GTPasa/genética , Antecedentes Genéticos , Hematopoyesis , Mutación , Pancitopenia/genética , Animales , Células Cultivadas , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C
2.
Blood Cells Mol Dis ; 87: 102524, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33341069

RESUMEN

Inherited bone marrow failure syndromes (IBMFS) are heterogeneous disorders characterized by dysregulated hematopoiesis in various lineages, developmental anomalies, and predisposition to malignancy. The scat (severe combined anemia and thrombocytopenia) mouse model is a model of IBMFS with a phenotype of pancytopenia cycling through crises and remission. Scat carries an autosomal recessive missense mutation in Rasa3 that results in RASA3 mislocalization and loss of function. RASA3 functions as a Ras-GTPase activating protein (GAP), and its loss of function in scat results in increased erythroid RAS activity and reactive oxygen species (ROS) and altered erythroid cell cycle progression, culminating in delayed terminal erythroid differentiation. Here we sought to further resolve the erythroid cell cycle defect in scat through ex vivo flow cytometric analyses. These studies revealed a specific G0/G1 accumulation in scat bone marrow (BM) polychromatophilic erythroblasts and scat BM Ter119-/c-KIT+/CD71lo/med progenitors, with no changes evident in equivalent scat spleen populations. Systematic analyses of RNAseq data from megakaryocyte-erythroid progenitors (MEPs) in scat crisis vs. scat partial remission reveal altered expression of genes involved in the G1-S checkpoint. Together, these data indicate a precise, biphasic role for RASA3 in regulating the cell cycle during erythropoiesis with relevance to hematopoietic disease progression.


Asunto(s)
Células Eritroides/citología , Eritropoyesis , Proteínas Activadoras de GTPasa/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Células Eritroides/metabolismo , Proteínas Activadoras de GTPasa/genética , Ratones Endogámicos BALB C , Mutación Missense , Proteínas ras/metabolismo
3.
Nature ; 522(7557): 474-7, 2015 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-25970251

RESUMEN

Many acute and chronic anaemias, including haemolysis, sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia, are not treatable with erythropoietin (Epo), because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently, we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor, burst-forming unit erythroid (BFU-E), and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors, with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis, PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally, both in control and corticosteroid-treated BFU-E cells, PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists, additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias.


Asunto(s)
Células Precursoras Eritroides/citología , Eritropoyesis , PPAR alfa/metabolismo , Receptores de Glucocorticoides/metabolismo , Enfermedad Aguda , Anemia/tratamiento farmacológico , Anemia/metabolismo , Anemia/patología , Anemia Hemolítica/metabolismo , Animales , Butiratos/farmacología , Butiratos/uso terapéutico , Técnicas de Cultivo de Célula , Células Cultivadas , Cromatina/genética , Cromatina/metabolismo , Enfermedad Crónica , Modelos Animales de Enfermedad , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Eritropoyesis/efectos de los fármacos , Eritropoyetina/farmacología , Femenino , Fenofibrato/farmacología , Glucocorticoides/farmacología , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/embriología , Ratones , PPAR alfa/agonistas , PPAR alfa/deficiencia , Fenilhidrazinas/farmacología , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Transducción de Señal/efectos de los fármacos
4.
Development ; 144(3): 430-440, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28143845

RESUMEN

Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states.


Asunto(s)
Anemia Neonatal/genética , Factores de Transcripción de Tipo Kruppel/genética , Mutación , Sustitución de Aminoácidos , Anemia Neonatal/sangre , Anemia Neonatal/embriología , Animales , Animales Recién Nacidos , Citocinas/sangre , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Eritropoyesis/genética , Regulación del Desarrollo de la Expresión Génica , Heterocigoto , Humanos , Factores de Transcripción de Tipo Kruppel/sangre , Factores de Transcripción de Tipo Kruppel/deficiencia , Ratones , Ratones Noqueados , Ratones Mutantes , Modelos Biológicos , Proteínas Musculares/sangre , Proteínas Mutantes/sangre , Proteínas Mutantes/genética
5.
Nucleic Acids Res ; 45(3): 1130-1143, 2017 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-28180284

RESUMEN

The rules of engagement between zinc finger transcription factors and DNA have been partly defined by in vitro DNA-binding and structural studies, but less is known about how these rules apply in vivo. Here, we demonstrate how a missense mutation in the second zinc finger of Krüppel-like factor-1 (KLF1) leads to degenerate DNA-binding specificity in vivo, resulting in ectopic transcription and anemia in the Nan mouse model. We employed ChIP-seq and 4sU-RNA-seq to identify aberrant DNA-binding events genome wide and ectopic transcriptional consequences of this binding. We confirmed novel sequence specificity of the mutant recombinant zinc finger domain by performing biophysical measurements of in vitro DNA-binding affinity. Together, these results shed new light on the mechanisms by which missense mutations in DNA-binding domains of transcription factors can lead to autosomal dominant diseases.


Asunto(s)
ADN/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transcriptoma/genética , Dedos de Zinc/genética , Animales , Línea Celular , Supervivencia Celular/genética , Células Eritroides/metabolismo , Eritropoyesis/genética , Humanos , Factores de Transcripción de Tipo Kruppel/química , Ratones , Modelos Genéticos , Modelos Moleculares , Proteínas Mutantes/química , Mutación Missense , Unión Proteica
6.
J Biol Chem ; 289(11): 7835-43, 2014 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-24509859

RESUMEN

Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.


Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Regulación de la Expresión Génica , Proteína 1 Reguladora de Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Porfirias/metabolismo , Animales , Blastocisto/citología , Diferenciación Celular , Línea Celular Tumoral , Femenino , Genotipo , Células HEK293 , Hemo/química , Humanos , Hierro/química , Proteínas Hierro-Azufre/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Biosíntesis de Proteínas , Protoporfirinas/metabolismo , Pez Cebra
7.
Proc Natl Acad Sci U S A ; 109(30): 12099-104, 2012 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-22773809

RESUMEN

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.


Asunto(s)
Eritropoyesis/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Trombopoyesis/fisiología , Animales , Animales Modificados Genéticamente , Activación Enzimática/fisiología , Eritropoyesis/genética , GTP Fosfohidrolasas/metabolismo , Ratones , Mutación Missense/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Trombopoyesis/genética , Pez Cebra
8.
Physiol Genomics ; 45(9): 343-50, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23482811

RESUMEN

Studies of ion transport pathophysiology in hematological disorders and tests of possible new therapeutic agents for these disorders have been carried out in various mouse models because of close functional similarities between mouse and human red cells. We have explored strain-specific differences in erythrocyte membrane physiology in 10 inbred mouse strains by determining erythrocyte contents of Na(+), K(+), and Mg(2+), and erythrocyte transport of ions via the ouabain-sensitive Na-K pump, the amiloride-sensitive Na-H exchanger (NHE1), the volume and chloride-dependent K-Cl cotransporter (KCC), and the charybdotoxin-sensitive Gardos channel (KCNN4). Our data reveal substantial strain-specific and sex-specific differences in both ion content and trans-membrane ion transport in mouse erythrocytes. These differences demonstrate the feasibility of identifying specific quantitative trait loci for erythroid ion transport and content in genetically standardized inbred mouse strains.


Asunto(s)
Cationes/metabolismo , Eritrocitos/metabolismo , Animales , Femenino , Humanos , Transporte Iónico , Masculino , Ratones , Potasio/sangre , Caracteres Sexuales , Sodio/sangre , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Especificidad de la Especie , Simportadores/metabolismo , Cotransportadores de K Cl
9.
Proc Natl Acad Sci U S A ; 107(34): 15151-6, 2010 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-20696915

RESUMEN

Studies of mouse models of anemia have long provided fundamental insights into red blood cell formation and function. Here we show that the semidominant mouse mutation Nan ("neonatal anemia") carries a single amino acid change (E339D) within the second zinc finger of the erythroid Krüppel-like factor (EKLF), a critical erythroid regulatory transcription factor. The mutation alters the DNA-binding specificity of EKLF so that it no longer binds promoters of a subset of its DNA targets. Remarkably, even when mutant Nan and wild-type EKLF alleles are expressed at equivalent levels, the mutant form selectively interferes with expression of EKLF target genes whose promoter elements it no longer binds. This interference yields a distorted genetic output and selective protein deficiencies that differ from those seen in EKLF-heterozygous and EKLF-null red blood cells and presents a unique and unexpected mechanism of inherited disease.


Asunto(s)
Anemia/genética , Factores de Transcripción de Tipo Kruppel/genética , Sustitución de Aminoácidos , Anemia/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Mapeo Cromosómico , ADN/genética , ADN/metabolismo , Modelos Animales de Enfermedad , Eritrocitos/metabolismo , Femenino , Expresión Génica , Genes Reporteros , Hemoglobinas/genética , Heterocigoto , Humanos , Células K562 , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Fenotipo , Embarazo , Regiones Promotoras Genéticas , Activación Transcripcional , Dedos de Zinc/genética
10.
Proc Natl Acad Sci U S A ; 107(13): 6022-7, 2010 Mar 30.
Artículo en Inglés | MEDLINE | ID: mdl-20231455

RESUMEN

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain betaIII spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in betaIII spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact betaIII spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most Spnb3(-/-) animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluRdelta, IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of alphaII spectrin. It appears that betaIII spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y.


Asunto(s)
Ataxia/etiología , Convulsiones/etiología , Espectrina/deficiencia , Animales , Ataxia/genética , Ataxia/fisiopatología , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/fisiopatología , Encéfalo/ultraestructura , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Transportador 4 de Aminoácidos Excitadores/metabolismo , Femenino , Marcación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Fenotipo , Convulsiones/genética , Convulsiones/fisiopatología , Espectrina/genética , Espectrina/fisiología , Ataxias Espinocerebelosas/etiología , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/fisiopatología , Sinapsis/fisiología , Sinapsis/ultraestructura
11.
Nat Genet ; 34(1): 59-64, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12669066

RESUMEN

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.


Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/deficiencia , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Eritropoyesis/genética , Mitosis/genética , Mutación , Pez Cebra/embriología , Pez Cebra/genética , Secuencia de Aminoácidos , Anemia Diseritropoyética Congénita/genética , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fenotipo , Pez Cebra/sangre
12.
Genesis ; 50(12): 882-91, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22926980

RESUMEN

The three adducin proteins (α, ß, and γ) share extensive sequence, structural, and functional homology. Heterodimers of α- and ß-adducin are vital components of the red cell membrane skeleton, which is required to maintain red cell elasticity and structural integrity. In addition to anemia, targeted deletion of the α-adducin gene (Add1) reveals unexpected, strain-dependent non-erythroid phenotypes. On an inbred 129 genetic background, Add1 null mice show abnormal inward curvature of the cervicothoracic spine with complete penetrance. More surprisingly, a subset of 129-Add1 null mice develop severe megaesophagus, while examination of peripheral nerves reveals a reduced number of axons in 129-Add1 null mice at four months of age. These unforeseen phenotypes, described here, reveal new functions for adducin and provide new models of mammalian disease.


Asunto(s)
Proteínas de Unión a Calmodulina/genética , Proteínas del Citoesqueleto/genética , Acalasia del Esófago/genética , Cifosis/genética , Animales , Proteínas del Citoesqueleto/metabolismo , Acalasia del Esófago/patología , Eliminación de Gen , Cifosis/patología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Penetrancia , Especificidad de la Especie
13.
Blood ; 116(14): 2590-9, 2010 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-20585041

RESUMEN

The short actin filaments in the red blood cell (RBC) membrane skeleton are capped at their pointed ends by tropomodulin 1 (Tmod1) and coated with tropomyosin (TM) along their length. Tmod1-TM control of actin filament length is hypothesized to regulate spectrin-actin lattice organization and membrane stability. We used a Tmod1 knockout mouse to investigate the in vivo role of Tmod1 in the RBC membrane skeleton. Western blots of Tmod1-null RBCs confirm the absence of Tmod1 and show the presence of Tmod3, which is normally not present in RBCs. Tmod3 is present at only one-fifth levels of Tmod1 present on wild-type membranes, but levels of actin, TMs, adducins, and other membrane skeleton proteins remain unchanged. Electron microscopy shows that actin filament lengths are more variable with spectrin-actin lattices displaying abnormally large and more variable pore sizes. Tmod1-null mice display a mild anemia with features resembling hereditary spherocytic elliptocytosis, including decreased RBC mean corpuscular volume, cellular dehydration, increased osmotic fragility, reduced deformability, and heterogeneity in osmotic ektacytometry. Insufficient capping of actin filaments by Tmod3 may allow greater actin dynamics at pointed ends, resulting in filament length redistribution, leading to irregular and attenuated spectrin-actin lattice connectivity, and concomitant RBC membrane instability.


Asunto(s)
Citoesqueleto/ultraestructura , Eritrocitos Anormales/ultraestructura , Eritrocitos/metabolismo , Tropomodulina/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Actinas/ultraestructura , Anemia/metabolismo , Anemia/patología , Animales , Citoesqueleto/metabolismo , Citosol/metabolismo , Eritrocitos/patología , Eritrocitos/ultraestructura , Técnicas de Inactivación de Genes , Ratones , Fragilidad Osmótica , Tropomodulina/análisis , Tropomodulina/genética
14.
Blood ; 116(25): e139-49, 2010 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-20833975

RESUMEN

A substantial genetic contribution underlies variation in baseline peripheral blood counts. We performed quantitative trait locus/loci analyses to identify chromosome regions harboring genes influencing red cell hemoglobin concentration using the cell hemoglobin concentration mean (CHCM), a directly measured parameter analogous to the mean cell hemoglobin concentration. Fourteen significant loci (gene symbols Chcmq1-Chcmq14) were detected. Seven of these influenced CHCM in a sex-specific fashion, and 2 showed significant interactive effects (epistasis). For quantitative trait locus/loci detected in multiple crosses, confidence intervals were narrowed using statistical and bioinformatic approaches. Two strong candidate genes emerged and were further analyzed: adult ß-globin (Hbb) for Chcmq3 on Chr 7, and transferrin (Trf) for Chcmq2 on Chr 9. High and low allele parental strains in crosses detecting Chcmq3 segregate 100% with the known ancestral haplotype blocks, hemoglobin (Hb) diffuse (Hbb(d)) and Hb single (Hbb(s)), respectively. Hbb(d) consists of nonidentical major and minor polypeptides and exhibits an increased positive charge relative to Hbb(s) due to the net loss of 2 negative residues in the Hbb(dminor) polypeptide, resulting in a pI of 7.85 versus 7.13. Thus, as shown in human erythrocytes, positively charged Hbs are associated with cell dehydration and increased CHCM in mouse erythrocytes.


Asunto(s)
Cromosomas de los Mamíferos/genética , Eritrocitos/metabolismo , Variación Genética/genética , Haplotipos/genética , Hemoglobinas/genética , Sitios de Carácter Cuantitativo , Globinas beta/genética , Animales , Cruzamientos Genéticos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA
15.
Blood ; 115(9): 1804-14, 2010 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-20056793

RESUMEN

Five spontaneous, allelic mutations in the alpha-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph(1J), sph(2J), sph(2BC), sph(Dem)). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph(3J), a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sph(Ihj), a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent beta-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph(4J), a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, beta-adducin. The severity of anemia in sph(4J) indicates that the highly conserved cysteine residue at the C-terminus of alpha-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.


Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/deficiencia , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Mutación , Espectrina/deficiencia , Espectrina/genética , Esferocitosis Hereditaria/sangre , Esferocitosis Hereditaria/genética , Alelos , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Unión a Calmodulina/sangre , Codón sin Sentido , Cartilla de ADN/genética , Membrana Eritrocítica/metabolismo , Eritrocitos Anormales/metabolismo , Eritrocitos Anormales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Datos de Secuencia Molecular , Mutación Missense , Estabilidad del ARN/genética , Homología de Secuencia de Aminoácido , Esferocitosis Hereditaria/patología
16.
Nature ; 440(7080): 96-100, 2006 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-16511496

RESUMEN

Iron has a fundamental role in many metabolic processes, including electron transport, deoxyribonucleotide synthesis, oxygen transport and many essential redox reactions involving haemoproteins and Fe-S cluster proteins. Defective iron homeostasis results in either iron deficiency or iron overload. Precise regulation of iron transport in mitochondria is essential for haem biosynthesis, haemoglobin production and Fe-S cluster protein assembly during red cell development. Here we describe a zebrafish mutant, frascati (frs), that shows profound hypochromic anaemia and erythroid maturation arrest owing to defects in mitochondrial iron uptake. Through positional cloning, we show that the gene mutated in the frs mutant is a member of the vertebrate mitochondrial solute carrier family (SLC25) that we call mitoferrin (mfrn). mfrn is highly expressed in fetal and adult haematopoietic tissues of zebrafish and mouse. Erythroblasts generated from murine embryonic stem cells null for Mfrn (also known as Slc25a37) show maturation arrest with severely impaired incorporation of 55Fe into haem. Disruption of the yeast mfrn orthologues, MRS3 and MRS4, causes defects in iron metabolism and mitochondrial Fe-S cluster biogenesis. Murine Mfrn rescues the defects in frs zebrafish, and zebrafish mfrn complements the yeast mutant, indicating that the function of the gene may be highly conserved. Our data show that mfrn functions as the principal mitochondrial iron importer essential for haem biosynthesis in vertebrate erythroblasts.


Asunto(s)
Eritroblastos/metabolismo , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas de Pez Cebra/metabolismo , Anemia/sangre , Anemia/metabolismo , Animales , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Secuencia Conservada , Eritroblastos/citología , Eritroblastos/patología , Regulación de la Expresión Génica , Prueba de Complementación Genética , Hemo/metabolismo , Homeostasis , Humanos , Sobrecarga de Hierro , Proteínas Hierro-Azufre/biosíntesis , Proteínas Hierro-Azufre/genética , Proteínas de Transporte de Membrana/genética , Ratones , Proteínas Mitocondriales , Datos de Secuencia Molecular , Mutación/genética , Filogenia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Células Madre/citología , Células Madre/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética
17.
Physiol Genomics ; 43(1): 1-11, 2011 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-20823217

RESUMEN

Lung function detection in mice is currently most accurately measured by invasive techniques, which are costly, labor intensive, and terminal. This limits their use for large-scale or longitudinal studies. Noninvasive assays are often used instead, but their accuracy for measuring lung function parameters such as resistance and elastance has been questioned in studies involving small numbers of mouse strains. Here we compared parameters detected by two different methods using 29 inbred mouse strains: enhanced pause (Penh), detected by unrestrained plethysmography, and central airway resistance and lung elastance, detected by a forced oscillation technique. We further tested whether the phenotypic variations were determined by the same genomic location in genome-wide association studies using a linear mixed model algorithm. Penh, resistance, and elastance were measured in nonexposed mice or mice exposed to saline and increasing doses of aerosolized methacholine. Because Penh differed from airway resistance in several strains and because the peak genetic associations found for Penh, resistance, or elastance were located at different genomic regions, we conclude that using Penh as an indicator for lung function changes in high-throughput genetic studies (i.e., genome-wide association studies or quantitative trait locus studies) measures something fundamentally different than airway resistance and lung elastance.


Asunto(s)
Resistencia de las Vías Respiratorias/fisiología , Pletismografía/métodos , Resistencia de las Vías Respiratorias/efectos de los fármacos , Algoritmos , Animales , Femenino , Estudio de Asociación del Genoma Completo , Masculino , Cloruro de Metacolina/farmacología , Ratones , Sitios de Carácter Cuantitativo
18.
J Biol Chem ; 285(7): 4757-70, 2010 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-20007969

RESUMEN

Spectrin and protein 4.1 cross-link F-actin protofilaments into a network called the membrane skeleton. Actin and 4.1 bind to one end of beta-spectrin. The adjacent end of alpha-spectrin, called the EF-domain, is calmodulin-like, with calcium-dependent and calcium-independent EF-hands. It has no known function. However, the sph(1J)/sph(1J) mouse has very fragile red cells and lacks the last 13 amino acids in the EF-domain, suggesting the domain is critical for skeletal integrity. Using pulldown binding assays, we find the alpha-spectrin EF-domain either alone or incorporated into a mini-spectrin binds native and recombinant protein 4.2 at a previously identified region of 4.2 (G(3) peptide). Native 4.2 binds with an affinity comparable with other membrane skeletal interactions (K(d) = 0.30 microM). EF-domains bearing the sph(1J) mutation are inactive. Binding of protein 4.2 to band 3 (K(d) = 0.45 microM) does not interfere with the spectrin-4.2 interaction. Spectrin-4.2 binding is amplified by micromolar concentrations of Ca(2+) (but not Mg(2+)) by three to five times. Calmodulin also binds to the EF-domain (K(d) = 17 microM), and Ca(2+)-calmodulin blocks Ca(2+)-dependent binding of protein 4.2 but not Ca(2+)-independent binding. The data suggest that protein 4.2 is located near protein 4.1 at the spectrin-actin junctions. Because proteins 4.1 and 4.2 also bind to band 3, the erythrocyte anion channel, we suggest that one or both of these proteins cause a portion of band 3 to localize near the spectrin-actin junctions and provide another point of attachment between the membrane skeleton and the lipid bilayer.


Asunto(s)
Calcio/farmacología , Calmodulina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/metabolismo , Espectrina/metabolismo , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/genética , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Calcio/metabolismo , Bovinos , Proteínas del Citoesqueleto/genética , Motivos EF Hand/genética , Motivos EF Hand/fisiología , Electroforesis en Gel de Poliacrilamida , Humanos , Espectrometría de Masas , Proteínas de la Membrana/genética , Ratones , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrina/genética , Porcinos
19.
Br J Haematol ; 154(4): 492-501, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21689084

RESUMEN

Spherocytosis is one of the most common inherited disorders, yet presents with a wide range of clinical severity. While several genes have been found mutated in patients with spherocytosis, the molecular basis for the variability in severity of haemolytic anaemia is not entirely understood. To identify candidate proteins involved in haemolytic anaemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in red blood cells (RBCs) from normal and ß-adducin (Add2) knock-out mice. We detected seven proteins that were decreased and 48 proteins that were increased in ß-adducin null RBC ghosts. Since haemolytic anaemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Among the 48 proteins increased in Add2 knockout RBCs, only 11 were also increased in reticulocytes. Of the proteins decreased in Add2 knockout RBCs, α-adducin showed the greatest intensity difference, followed by SLC9A1, the sodium-hydrogen exchanger previously termed NHE1. We verified these mass spectrometry results by immunoblot. This is the first example of SLC9A1deficiency in haemolytic anaemia and suggests new insights into the mechanisms leading to fragile RBCs.


Asunto(s)
Proteínas de Transporte de Catión/deficiencia , Eritrocitos/metabolismo , Proteínas de Microfilamentos/deficiencia , Animales , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte de Catión/sangre , Proteínas del Citoesqueleto , Membrana Eritrocítica/metabolismo , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/sangre , Proteómica/métodos , Reticulocitos/metabolismo , Intercambiador 1 de Sodio-Hidrógeno , Intercambiadores de Sodio-Hidrógeno/sangre
20.
Blood Cells Mol Dis ; 47(2): 85-94, 2011 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-21592827

RESUMEN

Hemolytic anemia is one of the most common inherited disorders. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative proteomic approach to detect differences in RBCs from normal and beta-adducin (Add2) knock-out mice. We detected 7 proteins that were decreased and 48 proteins that were increased in the beta-adducin knock-out RBC ghost. Since hemolytic anemias are characterized by reticulocytosis, we compared reticulocyte-enriched samples from phenylhydrazine-treated mice with mature RBCs from untreated mice. Label-free analysis identified 47 proteins that were increased in the reticulocyte-enriched samples and 21 proteins that were decreased. Among the proteins increased in Add2 knockout RBCs, only 11 were also found increased in reticulocytes. Among the proteins decreased in Add2 knockout RBCs, beta- and alpha-adducin showed the greatest intensity difference, followed by NHE-1 (Slc9a1), the sodium-hydrogen exchanger. We verified these mass spectrometry results by immunoblot. This is the first example of a deficiency of NHE-1 in hemolytic anemia and suggests new insights into the mechanisms leading to fragile RBCs. Our use of label-free comparative proteomics to make this discovery demonstrates the usefulness of this approach as opposed to metabolic or chemical isotopic labeling of mice.


Asunto(s)
Anemia Hemolítica/genética , Proteínas de Unión a Calmodulina , Proteínas de Transporte de Catión/genética , Eritrocitos/metabolismo , Fragilidad Osmótica/genética , Fenilhidrazinas/efectos adversos , Isoformas de Proteínas/genética , Proteómica/métodos , Reticulocitos/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Secuencia de Aminoácidos , Anemia Hemolítica/inducido químicamente , Anemia Hemolítica/metabolismo , Anemia Hemolítica/patología , Animales , Western Blotting , Proteínas de Unión a Calmodulina/deficiencia , Proteínas de Unión a Calmodulina/genética , Proteínas de Transporte de Catión/deficiencia , Modelos Animales de Enfermedad , Recuento de Eritrocitos , Membrana Eritrocítica/genética , Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Perfilación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Fenilhidrazinas/farmacología , Isoformas de Proteínas/metabolismo , Recuento de Reticulocitos , Reticulocitos/citología , Intercambiador 1 de Sodio-Hidrógeno , Espectrometría de Masas en Tándem
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