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1.
Blood ; 139(23): 3439-3449, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35349664

RESUMO

We follow a patient with Diamond-Blackfan anemia (DBA) mosaic for a pathogenic RPS19 haploinsufficiency mutation with persistent transfusion-dependent anemia. Her anemia remitted on eltrombopag (EPAG), but surprisingly, mosaicism was unchanged, suggesting that both mutant and normal cells responded. When EPAG was withheld, her anemia returned. In addition to expanding hematopoietic stem/progenitor cells, EPAG aggressively chelates iron. Because DBA anemia, at least in part, results from excessive intracellular heme leading to ferroptotic cell death, we hypothesized that the excess heme accumulating in ribosomal protein-deficient erythroid precursors inhibited the growth of adjacent genetically normal precursors, and that the efficacy of EPAG reflected its ability to chelate iron, limit heme synthesis, and thus limit toxicity in both mutant and normal cells. To test this, we studied Rpl11 haploinsufficient (DBA) mice and mice chimeric for the cytoplasmic heme export protein, FLVCR. Flvcr1-deleted mice have severe anemia, resembling DBA. Mice transplanted with ratios of DBA to wild-type marrow cells of 50:50 are anemic, like our DBA patient. In contrast, mice transplanted with Flvcr1-deleted (unable to export heme) and wild-type marrow cells at ratios of 50:50 or 80:20 have normal numbers of red cells. Additional studies suggest that heme exported from DBA erythroid cells might impede the nurse cell function of central macrophages of erythroblastic islands to impair the maturation of genetically normal coadherent erythroid cells. These findings have implications for the gene therapy of DBA and may provide insights into why del(5q) myelodysplastic syndrome patients are anemic despite being mosaic for chromosome 5q deletion and loss of RPS14.


Assuntos
Anemia de Diamond-Blackfan , Anemia , Anemia/patologia , Anemia de Diamond-Blackfan/metabolismo , Animais , Deleção Cromossômica , Células Eritroides/metabolismo , Eritropoese/genética , Feminino , Heme/metabolismo , Humanos , Ferro/metabolismo , Camundongos , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
2.
EBioMedicine ; 42: 470-480, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30878599

RESUMO

BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.


Assuntos
Fator de Iniciação 2 em Eucariotos/genética , Genes Ligados ao Cromossomo X , Glucose/metabolismo , Hipopituitarismo/etiologia , Hipopituitarismo/metabolismo , Fenótipo , Substituição de Aminoácidos , Apoptose , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Pré-Escolar , Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Hipopituitarismo/diagnóstico , Hibridização In Situ , Lactente , Imageamento por Ressonância Magnética , Mutação , Linhagem , Polimorfismo de Nucleotídeo Único , Biossíntese de Proteínas
3.
Mol Cell ; 70(2): 254-264.e6, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29677493

RESUMO

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.


Assuntos
Proteínas de Transporte/biossíntese , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , Poliaminas/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Códon de Iniciação , Sequência Conservada , Células HEK293 , Humanos , Camundongos , Fases de Leitura Aberta , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A
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