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1.
Nat Immunol ; 19(8): 828-837, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29988089

RESUMO

Memory T cells are critical for the immune response to recurring infections. Their instantaneous reactivity to pathogens is empowered by the persistent expression of cytokine-encoding mRNAs. How the translation of proteins from pre-formed cytokine-encoding mRNAs is prevented in the absence of infection has remained unclear. Here we found that protein production in memory T cells was blocked via a 3' untranslated region (3' UTR)-mediated process. Germline deletion of AU-rich elements (AREs) in the Ifng-3' UTR led to chronic cytokine production in memory T cells. This aberrant protein production did not result from increased expression and/or half-life of the mRNA. Instead, AREs blocked the recruitment of cytokine-encoding mRNA to ribosomes; this block depended on the ARE-binding protein ZFP36L2. Thus, AREs mediate repression of translation in mouse and human memory T cells by preventing undesirable protein production from pre-formed cytokine-encoding mRNAs in the absence of infection.


Assuntos
Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato/genética , Interferon gama/genética , RNA Mensageiro/genética , Linfócitos T/imunologia , Animais , Células Cultivadas , Repressão Epigenética , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Elongação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(4): e2216055120, 2023 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-36669105

RESUMO

DNA damage threatens genomic integrity and instigates stem cell failure. To bypass genotoxic lesions during replication, cells employ DNA damage tolerance (DDT), which is regulated via PCNA ubiquitination and REV1. DDT is conserved in all domains of life, yet its relevance in mammals remains unclear. Here, we show that inactivation of both PCNA-ubiquitination and REV1 results in embryonic and adult lethality, and the accumulation of DNA damage in hematopoietic stem and progenitor cells (HSPCs) that ultimately resulted in their depletion. Our results reveal the crucial relevance of DDT in the maintenance of stem cell compartments and mammalian life in unperturbed conditions.


Assuntos
Dano ao DNA , Animais , Reparo do DNA , Replicação do DNA , Células-Tronco Hematopoéticas/metabolismo , Mamíferos/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitinação
3.
RNA ; 28(2): 194-209, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34732567

RESUMO

Each day, about 1012 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.


Assuntos
Hematopoese , RNA Circular/metabolismo , RNA Mensageiro/genética , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Biossíntese de Proteínas , RNA Circular/genética , RNA Mensageiro/metabolismo , Transcriptoma
4.
Haematologica ; 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38961746

RESUMO

Differentiation of induced pluripotent stem cells (iPSCs) into hematopoietic lineages offers great therapeutic potential. During embryogenesis, hemogenic endothelium (HE) gives rise to hematopoietic stem and progenitor cells through the endothelial-to-hematopoietic transition (EHT). Understanding this process using iPSCs is key to generating functional hematopoietic stem cells (HSCs), a currently unmet challenge. In this study, we examined the role of the transcriptional factor GFI1B and its co-factor LSD1/KDM1A in EHT. To this end, we employed patient-derived iPSC lines with a dominant negative dysfunctional GFI1BQ287* and irreversible pharmacological LSD1/KDM1A inhibition in healthy iPSC lines. The formation of HE remained unaffected; however, hematopoietic output was severely reduced in both conditions. Single-cell RNA sequencing (scRNAseq) performed on the CD144+/CD31+ population derived from healthy iPSCs revealed similar expression dynamics of genes associated with in vivo EHT. Interestingly, LSD1/KDM1A inhibition in healthy lines before EHT resulted in a complete absence of hematopoietic output. However, uncommitted HE cells did not display GFI1B expression, suggesting a timed transcriptional program. To test this hypothesis, we ectopically expressed GFI1B in uncommitted HE cells, leading to downregulation of endothelial genes and upregulation of hematopoietic genes, including GATA2, KIT, RUNX1, and SPI1. Thus, we demonstrate that LSD1/KDM1A and GFI1B can function at distinct temporal points in different cellular subsets during EHT. Although GFI1B is not detected in uncommitted HE cells, its ectopic expression allows for partial hematopoietic specification. These data indicate that precisely timed expression of specific transcriptional regulators during EHT is crucial to the eventual outcome of EHT.

5.
Int J Mol Sci ; 25(3)2024 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-38338693

RESUMO

The Gárdos channel (KCNN4) and Piezo1 are the best-known ion channels in the red blood cell (RBC) membrane. Nevertheless, the quantitative electrophysiological behavior of RBCs and its heterogeneity are still not completely understood. Here, we use state-of-the-art biochemical methods to probe for the abundance of the channels in RBCs. Furthermore, we utilize automated patch clamp, based on planar chips, to compare the activity of the two channels in reticulocytes and mature RBCs. In addition to this characterization, we performed membrane potential measurements to demonstrate the effect of channel activity and interplay on the RBC properties. Both the Gárdos channel and Piezo1, albeit their average copy number of activatable channels per cell is in the single-digit range, can be detected through transcriptome analysis of reticulocytes. Proteomics analysis of reticulocytes and mature RBCs could only detect Piezo1 but not the Gárdos channel. Furthermore, they can be reliably measured in the whole-cell configuration of the patch clamp method. While for the Gárdos channel, the activity in terms of ion currents is higher in reticulocytes compared to mature RBCs, for Piezo1, the tendency is the opposite. While the interplay between Piezo1 and Gárdos channel cannot be followed using the patch clamp measurements, it could be proved based on membrane potential measurements in populations of intact RBCs. We discuss the Gárdos channel and Piezo1 abundance, interdependencies and interactions in the context of their proposed physiological and pathophysiological functions, which are the passing of small constrictions, e.g., in the spleen, and their active participation in blood clot formation and thrombosis.


Assuntos
Eritrócitos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Reticulócitos , Transporte Biológico , Cálcio/metabolismo , Eritrócitos/metabolismo , Reticulócitos/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canais Iônicos/metabolismo
6.
Biotechnol Bioeng ; 119(11): 3096-3116, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35879812

RESUMO

Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current, fully donor-dependent therapy. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55-2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2 /L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/ml without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.


Assuntos
Reatores Biológicos , Eritroblastos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Hemoglobinas , Oxigênio
8.
Haematologica ; 106(2): 464-473, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32467144

RESUMO

Haploinsufficiency for transcription factor KLF1 causes a variety of human erythroid phenotypes, such as the In(Lu) blood type, increased HbA2 levels, and hereditary persistence of fetal hemoglobin. Severe dominant congenital dyserythropoietic anemia IV (OMIM 613673) is associated with the KLF1 p.E325K variant. CDA-IV patients display ineffective erythropoiesis and hemolysis resulting in anemia, accompanied by persistent high levels of embryonic and fetal hemoglobin. The mouse Nan strain carries a variant in the orthologous residue, KLF1 p.E339D. Klf1Nan causes dominant hemolytic anemia with many similarities to CDA-IV. Here we investigated the impact of Klf1Nan on the developmental expression patterns of the endogenous beta-like and alpha-like globins, and the human beta-like globins carried on a HBB locus transgene. We observe that the switch from primitive, yolk sac-derived, erythropoiesis to definitive, fetal liver-derived, erythropoiesis is delayed in Klf1wt/Nan embryos. This is reflected in globin expression patterns measured between E12.5 and E14.5. Cultured Klf1wt/Nan E12.5 fetal liver cells display growth- and differentiation defects. These defects likely contribute to the delayed appearance of definitive erythrocytes in the circulation of Klf1wt/Nan embryos. After E14.5, expression of the embryonic/fetal globin genes is silenced rapidly. In adult Klf1wt/Nan animals, silencing of the embryonic/fetal globin genes is impeded, but only minute amounts are expressed. Thus, in contrast to human KLF1 p.E325K, mouse KLF1 p.E339D does not lead to persistent high levels of embryonic/fetal globins. Our results support the notion that KLF1 affects gene expression in a variant-specific manner, highlighting the necessity to characterize KLF1 variant-specific phenotypes of patients in detail.


Assuntos
Anemia Diseritropoética Congênita , Fatores de Transcrição Kruppel-Like , Adulto , Animais , Diferenciação Celular , Eritropoese/genética , Hemoglobinas , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos
9.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478008

RESUMO

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.


Assuntos
Sinalização do Cálcio/fisiologia , Eritroblastos/fisiologia , Estresse Mecânico , Cálcio/metabolismo , Cálcio/farmacologia , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Eritroblastos/efeitos dos fármacos , Eritropoese/efeitos dos fármacos , Eritropoese/fisiologia , Humanos , Canais Iônicos/agonistas , Canais Iônicos/fisiologia , Mecanotransdução Celular/efeitos dos fármacos , Mecanotransdução Celular/fisiologia , Pirazinas/farmacologia , Rotação , Tiadiazóis/farmacologia
10.
Am J Hum Genet ; 100(3): 506-522, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28257692

RESUMO

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.


Assuntos
Proteínas Ribossômicas/genética , Ribossomos/genética , Transtorno do Espectro Autista/genética , Proteínas de Transporte/genética , Células Cultivadas , Criança , Pré-Escolar , Códon/genética , Deficiências do Desenvolvimento/genética , Exoma , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Perda Auditiva/genética , Humanos , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Mutação , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Estresse Oxidativo , Biossíntese de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA
11.
Nucleic Acids Res ; 46(16): 8168-8180, 2018 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-30124921

RESUMO

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. CircRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.


Assuntos
Regulação da Expressão Gênica/genética , Células-Tronco Hematopoéticas/metabolismo , RNA/genética , Plaquetas/citologia , Diferenciação Celular/genética , Linhagem da Célula/genética , Eritrócitos/citologia , Transplante de Células-Tronco Hematopoéticas , Humanos , RNA/biossíntese , RNA/sangue , RNA Circular
12.
Proc Natl Acad Sci U S A ; 114(36): 9677-9682, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28835535

RESUMO

Effective T cell responses against invading pathogens require the concerted production of three key cytokines: TNF-α, IFN-γ, and IL-2. The cytokines functionally synergize, but their production kinetics widely differ. How the differential timing of expression is regulated remains, however, poorly understood. We compared the relative contribution of transcription, mRNA stability, and translation efficiency on cytokine production in murine effector and memory CD8+ T cells. We show that the immediate and ample production of TNF-α is primarily mediated by translation of preformed mRNA through protein kinase C (PKC)-induced recruitment of mRNA to polyribosomes. Also, the initial production of IFN-γ uses translation of preformed mRNA. However, the magnitude and subsequent expression of IFN-γ, and of IL-2, depends on calcium-induced de novo transcription and PKC-dependent mRNA stabilization. In conclusion, PKC signaling modulates translation efficiency and mRNA stability in a transcript-specific manner. These cytokine-specific regulatory mechanisms guarantee that T cells produce ample amounts of cytokines shortly upon activation and for a limited time.


Assuntos
Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Citocinas/genética , Proteína Quinase C/metabolismo , Animais , Sinalização do Cálcio , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-2/biossíntese , Interleucina-2/genética , Cinética , Ativação Linfocitária , Camundongos , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética
13.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32024018

RESUMO

Megakaryopoiesis is the process during which megakaryoblasts differentiate to polyploid megakaryocytes that can subsequently shed thousands of platelets in the circulation. Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to hemostasis. Mice deficient for Atxn2 did not display differences in bleeding times, but the expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), was deregulated and platelet aggregation upon specific triggers was reduced.


Assuntos
Ataxina-2/genética , Perfilação da Expressão Gênica/métodos , Células Progenitoras de Megacariócitos/citologia , Animais , Antígenos CD34/genética , Ataxina-2/metabolismo , Diferenciação Celular , Linhagem Celular , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Glicoproteína IIb da Membrana de Plaquetas/genética , Proteínas Proto-Oncogênicas/genética
14.
Hum Mol Genet ; 26(23): 4689-4698, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973399

RESUMO

The rare recessive developmental disorder Trichothiodystrophy (TTD) is characterized by brittle hair and nails. Patients also present a variable set of poorly explained additional clinical features, including ichthyosis, impaired intelligence, developmental delay and anemia. About half of TTD patients are photosensitive due to inherited defects in the DNA repair and transcription factor II H (TFIIH). The pathophysiological contributions of unrepaired DNA lesions and impaired transcription have not been dissected yet. Here, we functionally characterize the consequence of a homozygous missense mutation in the general transcription factor II E, subunit 2 (GTF2E2/TFIIEß) of two unrelated non-photosensitive TTD (NPS-TTD) families. We demonstrate that mutant TFIIEß strongly reduces the total amount of the entire TFIIE complex, with a remarkable temperature-sensitive transcription defect, which strikingly correlates with the phenotypic aggravation of key clinical symptoms after episodes of high fever. We performed induced pluripotent stem (iPS) cell reprogramming of patient fibroblasts followed by in vitro erythroid differentiation to translate the intriguing molecular defect to phenotypic expression in relevant tissue, to disclose the molecular basis for some specific TTD features. We observed a clear hematopoietic defect during late-stage differentiation associated with hemoglobin subunit imbalance. These new findings of a DNA repair-independent transcription defect and tissue-specific malfunctioning provide novel mechanistic insight into the etiology of TTD.


Assuntos
Fatores de Transcrição TFII/genética , Síndromes de Tricotiodistrofia/genética , Diferenciação Celular/genética , Reprogramação Celular/genética , DNA Helicases/genética , Reparo do DNA , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Mutação , Mutação de Sentido Incorreto , Especificidade de Órgãos , Linhagem , Fatores de Transcrição TFII/metabolismo , Transcrição Gênica , Síndromes de Tricotiodistrofia/metabolismo , Síndromes de Tricotiodistrofia/patologia
15.
Blood ; 130(12): 1441-1444, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28754683

RESUMO

Plasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.


Assuntos
Sistema do Grupo Sanguíneo Duffy/química , Sistema do Grupo Sanguíneo Duffy/metabolismo , Espaço Extracelular/química , Plasmodium vivax/fisiologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Reticulócitos/citologia , Reticulócitos/metabolismo , Tropismo/fisiologia , Especificidade de Anticorpos/imunologia , Antígenos de Protozoários/metabolismo , Diferenciação Celular , Eritrócitos/parasitologia , Humanos , Domínios Proteicos , Proteínas de Protozoários/metabolismo , Relação Estrutura-Atividade
16.
Haematologica ; 104(7): 1460-1472, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30655368

RESUMO

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 - REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.


Assuntos
Transtornos da Coagulação Sanguínea/patologia , Plaquetas/patologia , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/patologia , Megacariócitos/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Transtornos da Coagulação Sanguínea/genética , Transtornos da Coagulação Sanguínea/metabolismo , Plaquetas/metabolismo , Diferenciação Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Megacariócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Mapas de Interação de Proteínas , Proteoma/análise , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo
17.
Nucleic Acids Res ; 44(9): 4134-46, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-26762974

RESUMO

Mutations in the Shwachman-Bodian-Diamond Syndrome (SBDS) gene cause Shwachman-Diamond Syndrome (SDS), a rare congenital disease characterized by bone marrow failure with neutropenia, exocrine pancreatic dysfunction and skeletal abnormalities. The SBDS protein is important for ribosome maturation and therefore SDS belongs to the ribosomopathies. It is unknown, however, if loss of SBDS functionality affects the translation of specific mRNAs and whether this could play a role in the development of the clinical features of SDS. Here, we report that translation of the C/EBPα and -ß mRNAs, that are indispensible regulators of granulocytic differentiation, is altered by SBDS mutations or knockdown. We show that SBDS function is specifically required for efficient translation re-initiation into the protein isoforms C/EBPα-p30 and C/EBPß-LIP, which is controlled by a single cis-regulatory upstream open reading frame (uORF) in the 5' untranslated regions (5' UTRs) of both mRNAs. Furthermore, we show that as a consequence of the C/EBPα and -ß deregulation the expression of MYC is decreased with associated reduction in proliferation, suggesting that failure of progenitor proliferation contributes to the haematological phenotype of SDS. Therefore, our study provides the first indication that disturbance of specific translation by loss of SBDS function may contribute to the development of the SDS phenotype.


Assuntos
Doenças da Medula Óssea/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Insuficiência Pancreática Exócrina/metabolismo , Lipomatose/metabolismo , Proteínas/fisiologia , RNA Mensageiro/genética , Regiões 5' não Traduzidas , Doenças da Medula Óssea/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Insuficiência Pancreática Exócrina/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipomatose/genética , Neutrófilos/fisiologia , Iniciação Traducional da Cadeia Peptídica , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/metabolismo , Síndrome de Shwachman-Diamond
18.
Blood ; 125(25): 3937-48, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-25778535

RESUMO

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercc1-deficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53 is leukemogenic in this model. In addition, Ercc1-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Ercc1-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercc1-deficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuR for miR-139-3p and Prdx6, Runx1, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors.


Assuntos
Transformação Celular Neoplásica/genética , Células-Tronco Hematopoéticas/patologia , Leucemia/genética , MicroRNAs/genética , Animais , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Modelos Animais de Doenças , Endonucleases/deficiência , Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
19.
Haematologica ; 102(6): 995-1005, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28255017

RESUMO

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.


Assuntos
Quinase 6 Dependente de Ciclina/fisiologia , Citoesqueleto/ultraestrutura , Células Eritroides/ultraestrutura , Citoesqueleto de Actina , Actinas/metabolismo , Anemia , Animais , Gelsolina/metabolismo , Regulação da Expressão Gênica , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL
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