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1.
Nature ; 553(7689): 506-510, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29342143

RESUMO

All haematopoietic cell lineages that circulate in the blood of adult mammals derive from multipotent haematopoietic stem cells (HSCs). By contrast, in the blood of mammalian embryos, lineage-restricted progenitors arise first, independently of HSCs, which only emerge later in gestation. As best defined in the mouse, 'primitive' progenitors first appear in the yolk sac at 7.5 days post-coitum. Subsequently, erythroid-myeloid progenitors that express fetal haemoglobin, as well as fetal lymphoid progenitors, develop in the yolk sac and the embryo proper, but these cells lack HSC potential. Ultimately, 'definitive' HSCs with long-term, multilineage potential and the ability to engraft irradiated adults emerge at 10.5 days post-coitum from arterial endothelium in the aorta-gonad-mesonephros and other haemogenic vasculature. The molecular mechanisms of this reverse progression of haematopoietic ontogeny remain unexplained. We hypothesized that the definitive haematopoietic program might be actively repressed in early embryogenesis through epigenetic silencing, and that alleviating this repression would elicit multipotency in otherwise lineage-restricted haematopoietic progenitors. Here we show that reduced expression of the Polycomb group protein EZH1 enhances multi-lymphoid output from human pluripotent stem cells. In addition, Ezh1 deficiency in mouse embryos results in precocious emergence of functional definitive HSCs in vivo. Thus, we identify EZH1 as a repressor of haematopoietic multipotency in the early mammalian embryo.


Assuntos
Células-Tronco Embrionárias/citologia , Inativação Gênica , Hematopoese , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Células-Tronco Multipotentes/citologia , Complexo Repressor Polycomb 2/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Cromatina/metabolismo , Desenvolvimento Embrionário , Feminino , Humanos , Linfócitos/metabolismo , Camundongos , Células-Tronco Pluripotentes/citologia , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/deficiência , Complexo Repressor Polycomb 2/genética
2.
Infect Immun ; 84(6): 1761-1774, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27045035

RESUMO

Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.


Assuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Malária Falciparum/prevenção & controle , Malária/prevenção & controle , Plasmodium berghei/imunologia , Plasmodium falciparum/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antígenos de Protozoários/administração & dosagem , Antígenos de Protozoários/genética , Sulfatos de Condroitina/química , Sulfatos de Condroitina/imunologia , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Feminino , Peso Fetal/efeitos dos fármacos , Imunização , Imunoglobulina G/biossíntese , Malária/imunologia , Malária/patologia , Malária Falciparum/imunologia , Malária Falciparum/patologia , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Parasitemia/imunologia , Parasitemia/patologia , Parasitemia/prevenção & controle , Placenta , Plasmodium berghei/genética , Plasmodium falciparum/genética , Gravidez , Complicações Parasitárias na Gravidez/imunologia , Complicações Parasitárias na Gravidez/patologia , Complicações Parasitárias na Gravidez/prevenção & controle , Domínios Proteicos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Natimorto
3.
Biochim Biophys Acta ; 1817(7): 1072-82, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22561116

RESUMO

The respiratory nitrate reductase complex (NarGHI) from Marinobacter hydrocarbonoclasticus 617 (Mh, formerly Pseudomonas nautica 617) catalyzes the reduction of nitrate to nitrite. This reaction is the first step of the denitrification pathway and is coupled to the quinone pool oxidation and proton translocation to the periplasm, which generates the proton motive force needed for ATP synthesis. The Mh NarGH water-soluble heterodimer has been purified and the kinetic and redox properties have been studied through in-solution enzyme kinetics, protein film voltammetry and spectropotentiometric redox titration. The kinetic parameters of Mh NarGH toward substrates and inhibitors are consistent with those reported for other respiratory nitrate reductases. Protein film voltammetry showed that at least two catalytically distinct forms of the enzyme, which depend on the applied potential, are responsible for substrate reduction. These two forms are affected differentially by the oxidizing substrate, as well as by pH and inhibitors. A new model for the potential dependence of the catalytic efficiency of Nars is proposed.


Assuntos
Biocatálise , Cloratos/metabolismo , Marinobacter/enzimologia , Nitrato Redutase/metabolismo , Nitratos/metabolismo , Percloratos/metabolismo , Adsorção , Azidas/farmacologia , Biocatálise/efeitos dos fármacos , Cristalografia por Raios X , Técnicas Eletroquímicas , Eletrodos , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/enzimologia , Grafite , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Cinética , Marinobacter/efeitos dos fármacos , Modelos Biológicos , Nitrato Redutase/química , Oxirredução/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Soluções , Espectrofotometria , Especificidade por Substrato/efeitos dos fármacos
5.
J Biol Inorg Chem ; 16(2): 209-15, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20963615

RESUMO

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.


Assuntos
Carbono/química , Eletroquímica/métodos , Eletrodos , Hemeproteínas/química , Citocromo-c Peroxidase/química , Paracoccus pantotrophus/enzimologia , Estrutura Secundária de Proteína
6.
Cell Rep ; 36(3): 109408, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34289374

RESUMO

The molecular mechanisms that govern the choreographed timing of organ development remain poorly understood. Our investigation of the role of the Lin28a and Lin28b paralogs during the developmental process of branching morphogenesis establishes that dysregulation of Lin28a/b leads to abnormal branching morphogenesis in the lung and other tissues. Additionally, we find that the Lin28 paralogs, which regulate post-transcriptional processing of both mRNAs and microRNAs (miRNAs), predominantly control mRNAs during the initial phases of lung organogenesis. Target mRNAs include Sox2, Sox9, and Etv5, which coordinate lung development and differentiation. Moreover, we find that functional interactions between Lin28a and Sox9 are capable of bypassing branching defects in Lin28a/b mutant lungs. Here, we identify Lin28a and Lin28b as regulators of early embryonic lung development, highlighting the importance of the timing of post-transcriptional regulation of both miRNAs and mRNAs at distinct stages of organogenesis.


Assuntos
Pulmão/embriologia , Pulmão/metabolismo , Morfogênese , Proteínas de Ligação a RNA/metabolismo , Homologia de Sequência de Aminoácidos , Embrião de Mamíferos/metabolismo , Retroalimentação Fisiológica , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Biológicos , Morfogênese/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/genética
7.
J Biol Inorg Chem ; 15(6): 967-76, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20422435

RESUMO

The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.


Assuntos
Biocatálise , Domínio Catalítico , Cobre/metabolismo , Óxido Nitroso/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Pseudomonas/enzimologia , Grupo dos Citocromos c/metabolismo , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Ativação Enzimática , Cinética
8.
Dev Cell ; 52(4): 446-460.e5, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032546

RESUMO

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Endotélio Vascular/citologia , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mecanotransdução Celular , Fatores de Transcrição/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Proteínas de Ciclo Celular/genética , Diferenciação Celular , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Hemodinâmica , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Fatores de Transcrição/genética , Peixe-Zebra , Proteínas rho de Ligação ao GTP/metabolismo
9.
Cell Stem Cell ; 22(4): 575-588.e7, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625070

RESUMO

While gene expression dynamics have been extensively cataloged during hematopoietic differentiation in the adult, less is known about transcriptome diversity of human hematopoietic stem cells (HSCs) during development. To characterize transcriptional and post-transcriptional changes in HSCs during development, we leveraged high-throughput genomic approaches to profile miRNAs, lincRNAs, and mRNAs. Our findings indicate that HSCs manifest distinct alternative splicing patterns in key hematopoietic regulators. Detailed analysis of the splicing dynamics and function of one such regulator, HMGA2, identified an alternative isoform that escapes miRNA-mediated targeting. We further identified the splicing kinase CLK3 that, by regulating HMGA2 splicing, preserves HMGA2 function in the setting of an increase in let-7 miRNA levels, delineating how CLK3 and HMGA2 form a functional axis that influences HSC properties during development. Collectively, our study highlights molecular mechanisms by which alternative splicing and miRNA-mediated post-transcriptional regulation impact the molecular identity and stage-specific developmental features of human HSCs.


Assuntos
Processamento Alternativo/genética , Proteína HMGA2/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteína HMGA2/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
10.
Cell Rep ; 17(12): 3178-3192, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-28009288

RESUMO

Hematopoietic stem cell (HSC) transplantation is curative for malignant and genetic blood disorders, but is limited by donor availability and immune-mismatch. Deriving HSCs from patient-matched embryonic/induced-pluripotent stem cells (ESCs/iPSCs) could address these limitations. Prior efforts in murine models exploited ectopic HoxB4 expression to drive self-renewal and enable multi-lineage reconstitution, yet fell short in delivering robust lymphoid engraftment. Here, by titrating exposure of HoxB4-ESC-HSC to Notch ligands, we report derivation of engineered HSCs that self-renew, repopulate multi-lineage hematopoiesis in primary and secondary engrafted mice, and endow adaptive immunity in immune-deficient recipients. Single-cell analysis shows that following engraftment in the bone marrow niche, these engineered HSCs further specify to a hybrid cell type, in which distinct gene regulatory networks of hematopoietic stem/progenitors and differentiated hematopoietic lineages are co-expressed. Our work demonstrates engineering of fully functional HSCs via modulation of genetic programs that govern self-renewal and lineage priming.


Assuntos
Imunidade Adaptativa/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Autorrenovação Celular/genética , Redes Reguladoras de Genes/genética , Hematopoese/genética , Hematopoese/imunologia , Células-Tronco Hematopoéticas/imunologia , Proteínas de Homeodomínio/imunologia , Humanos , Células-Tronco Pluripotentes Induzidas/imunologia , Camundongos , Receptores Notch/genética , Receptores Notch/imunologia , Análise de Célula Única , Fatores de Transcrição/imunologia
11.
Bioelectrochemistry ; 82(1): 22-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21600857

RESUMO

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158±5mV); [4Fe-4S](+2/+1) (E(0')=-474±5mV) and [3Fe-4S](0/-2) (E(0')=-660±5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1±0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.


Assuntos
Proteínas de Bactérias/química , Desulfovibrio/química , Ferredoxinas/química , Proteínas de Bactérias/isolamento & purificação , Técnicas Eletroquímicas , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Ferredoxinas/isolamento & purificação , Oxirredução
12.
Eur J Biochem ; 271(7): 1329-38, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15030483

RESUMO

This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.


Assuntos
Aldeído Oxirredutases/química , Desulfovibrio gigas/enzimologia , Eletroquímica/métodos , Aldeídos , Benzaldeídos/química , Carbono/química , Catálise , Transporte de Elétrons , Ouro , Modelos Moleculares , Oxirredução , Eletricidade Estática
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