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1.
Nat Metab ; 3(11): 1521-1535, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34799698

RESUMO

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

2.
Nat Biotechnol ; 39(7): 846-854, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33767396

RESUMO

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.


Assuntos
Proteômica/métodos , Espectrometria de Massas em Tandem , Arabidopsis/metabolismo , Biomarcadores/metabolismo , COVID-19/sangue , COVID-19/diagnóstico , Linhagem Celular , Humanos , Peptídeos/análise , Proteoma/análise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Índice de Gravidade de Doença
3.
Methods ; 164-165: 29-35, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31034882

RESUMO

Forward genetic screens are a powerful and unbiased approach for uncovering the genetic basis behind a specific phenotype. Genome-wide mutagenesis followed by phenotypic screening represents the ultimate manifestation of this method, directly linking biological phenomena to its corresponding genetic cause. Whilst this has been successful in lower organisms, deployment of genome-wide screens in mammalian systems has been hampered by both limitations of scale and inefficient bi-allelic mutagenesis. CRISPR-Cas9 technology has now largely resolved these issues, whereby delivery of genome-scale gRNA libraries in the presence of gRNA-guided Cas9 endonuclease enables the generation of mutant cell libraries; the perfect platform for performing phenotypic screens. Although the tools are now available for virtually any molecular biology laboratory to conduct such screens, many researchers are daunted by the sheer complexity and scale at which such experiments are performed. This Review will address these concerns, presenting a contextual and practical guide to deploying CRISPR-KO screens in mammalian systems. We will discuss key considerations required in all aspects of screening from initiation to conclusion, which will enable researchers to conduct screens of their own, maximising the potential of this powerful technology.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Biblioteca Genômica , Genômica/métodos , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Testes Genéticos/métodos , Humanos , RNA Guia/genética
4.
Stem Cells ; 37(7): 958-972, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30932271

RESUMO

Direct in vivo reprogramming of cardiac fibroblasts into myocytes is an attractive therapeutic intervention in resolving myogenic deterioration. Current transgene-dependent approaches can restore cardiac function, but dependence on retroviral delivery and persistent retention of transgenic sequences are significant therapeutic hurdles. Chemical reprogramming has been established as a legitimate method to generate functional cell types, including those of the cardiac lineage. Here, we have extended this approach to generate progenitor cells that can differentiate into endothelial cells and cardiomyocytes using a single inhibitor protocol. Depletion of terminally differentiated cells and enrichment for proliferative cells result in a second expandable progenitor population that can robustly give rise to myofibroblasts and smooth muscle. Deployment of a genome-wide knockout screen with clustered regularly interspaced short palindromic repeats-guide RNA library to identify novel mediators that regulate the reprogramming revealed the involvement of DNA methyltransferase 1-associated protein 1 (Dmap1). Loss of Dmap1 reduced promoter methylation, increased the expression of Nkx2-5, and enhanced the retention of self-renewal, although further differentiation is inhibited because of the sustained expression of Cdh1. Our results hence establish Dmap1 as a modulator of cardiac reprogramming and myocytic induction. Stem Cells 2019;37:958-972.


Assuntos
Benzamidas/farmacologia , Sistemas CRISPR-Cas , Reprogramação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Fibroblastos/efeitos dos fármacos , Pirazóis/farmacologia , Piridinas/farmacologia , Proteínas Repressoras/genética , Células-Tronco/efeitos dos fármacos , Animais , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes/métodos , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , Músculo Liso/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Cultura Primária de Células , RNA Guia/genética , RNA Guia/metabolismo , Proteínas Repressoras/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo
5.
Cell Rep ; 24(2): 489-502, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29996108

RESUMO

The genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Inativação de Genes , Genoma , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Autorrenovação Celular , Regulação da Expressão Gênica no Desenvolvimento , Quinase 3 da Glicogênio Sintase/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Embrionárias Murinas , Fenótipo , Transcriptoma/genética
6.
Development ; 143(17): 3050-60, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27578176

RESUMO

Phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammalian target of rapamycin (mTOR) constitute the core components of the PI3K/AKT/mTOR signalling cascade, regulating cell proliferation, survival and metabolism. Although these functions are well-defined in the context of tumorigenesis, recent studies - in particular those using pluripotent stem cells - have highlighted the importance of this pathway to development and cellular differentiation. Here, we review the recent in vitro and in vivo evidence for the role PI3K/AKT/mTOR signalling plays in the control of pluripotency and differentiation, with a particular focus on the molecular mechanisms underlying these functions.


Assuntos
Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Proliferação de Células/genética , Sobrevivência Celular/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genética
7.
Nat Commun ; 6: 7212, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25998442

RESUMO

Crosstalk between the phosphatidylinositol 3-kinase (PI3K) and the transforming growth factor-ß signalling pathways play an important role in regulating many cellular functions. However, the molecular mechanisms underpinning this crosstalk remain unclear. Here, we report that PI3K signalling antagonizes the Activin-induced definitive endoderm (DE) differentiation of human embryonic stem cells by attenuating the duration of Smad2/3 activation via the mechanistic target of rapamycin complex 2 (mTORC2). Activation of mTORC2 regulates the phosphorylation of the Smad2/3-T220/T179 linker residue independent of Akt, CDK and Erk activity. This phosphorylation primes receptor-activated Smad2/3 for recruitment of the E3 ubiquitin ligase Nedd4L, which in turn leads to their degradation. Inhibition of PI3K/mTORC2 reduces this phosphorylation and increases the duration of Smad2/3 activity, promoting a more robust mesendoderm and endoderm differentiation. These findings present a new and direct crosstalk mechanism between these two pathways in which mTORC2 functions as a novel and critical mediator.


Assuntos
Ativinas/metabolismo , Células-Tronco Embrionárias/metabolismo , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Smad Reguladas por Receptor/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Endoderma/citologia , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosforilação , Receptor Cross-Talk
8.
Stem Cells ; 32(8): 2111-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24740933

RESUMO

Telomere repeat binding factor 2 (TRF2) is a component of the shelterin complex that is known to bind and protect telomeric DNA, yet the detection of TRF2 in extra-telomeric regions of chromosomes suggests other roles for TRF2 besides telomere protection. Here, we demonstrate that TRF2 plays a critical role in antagonizing the repressive function of neuron-restrictive silencer factor, also known as repressor element-1 silencing transcription factor (REST), during the neural differentiation of human embryonic stem cells (hESCs) by enhancing the expression of a truncated REST splice isoform we term human REST4 (hREST4) due to its similarity to rodent REST4. We show that TRF2 is specifically upregulated during hESC neural differentiation concordantly with an increase in the expression of hREST4 and that both proteins are highly expressed in NPCs. Overexpression of TRF2 in hESCs increases hREST4 levels and induces their neural differentiation, whereas TRF2 knockdown in hESCs and NPCs reduces hREST4 expression, hindering their ability to differentiate to the neural lineage. Concurrently, we show that TRF2 directly interacts with the C-terminal of hREST4 through its TRF2 core binding motif [F/Y]xL, protecting hREST4 from ubiquitin-mediated proteasomal degradation and consequently furthering neural induction. Thus, the TRF2-mediated counterbalance between hREST4 and REST is vital for both the generation and maintenance of NPCs, suggesting an important role for TRF2 in both neurogenesis and function of the central nervous system.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Neurais/citologia , Neurogênese/fisiologia , Proteínas Repressoras/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismo , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Células-Tronco Neurais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética , Regulação para Cima
9.
Tissue Eng Part A ; 19(3-4): 360-7, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23003670

RESUMO

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) provide an unlimited source for the generation of human hepatocytes, owing to their indefinite self-renewal and pluripotent properties. Both hESC-/iPSC-derived hepatocytes hold great promise in treating liver diseases as potential candidates for cell replacement therapies or as an in vitro platform to conduct new drug trials. It has been previously demonstrated that the initiation of hESC differentiation in monolayer cultures increases the generation of definitive endoderm (DE) and subsequently of hepatocyte differentiation. However, monolayer culture may hinder the maturation of hESC-derived hepatocytes, since such two-dimensional (2D) conditions do not accurately reflect the complex nature of three-dimensional (3D) hepatocyte specification in vivo. Here, we report the sequential application of 2D and 3D culture systems to differentiate hESCs to hepatocytes. Human ESCs were initially differentiated in a monolayer culture to DE cells, which were then inoculated into Algimatrix scaffolds. Treatments of hESC-DE cells with a ROCK inhibitor before and after inoculation dramatically enhanced their survival and the formation of spheroids, which are distinct from HepG2 carcinoma cells. In comparison with monolayer culture alone, sequential 2D and 3D cultures significantly improved hepatocyte differentiation and function. Our results demonstrate that hESC-DE cells can be incorporated into Algimatrix 3D culture systems to enhance hepatocyte differentiation and function.


Assuntos
Técnicas de Cultura Celular por Lotes/instrumentação , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Hepatócitos/citologia , Engenharia Tecidual/instrumentação , Tecidos Suporte , Técnicas de Cultura Celular por Lotes/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Células-Tronco Embrionárias/fisiologia , Endoderma/fisiologia , Desenho de Equipamento , Hepatócitos/fisiologia , Humanos , Engenharia Tecidual/métodos
10.
PLoS One ; 7(5): e37129, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22615918

RESUMO

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.


Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Antígenos de Superfície/genética , Antígenos de Superfície/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação para Baixo , Células-Tronco Embrionárias/metabolismo , Endoderma/citologia , Endoderma/metabolismo , Endoderma/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Neurônios/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Antígenos Embrionários Estágio-Específicos/genética , Antígenos Embrionários Estágio-Específicos/metabolismo , Regulação para Cima
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