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1.
Proc Natl Acad Sci U S A ; 113(42): E6382-E6390, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27698112

RESUMO

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/ß-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/ß-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of ß-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/ß-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/ß-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.


Assuntos
Diferenciação Celular , Autorrenovação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Via de Sinalização Wnt , Apoptose , Benzotiazóis/farmacologia , Biomarcadores , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Compostos Heterocíclicos com 3 Anéis/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Modelos Biológicos , Proteômica/métodos , RNA Interferente Pequeno/genética , Via de Sinalização Wnt/efeitos dos fármacos
2.
Nat Cell Biol ; 17(12): 1523-35, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26571212

RESUMO

For nearly a century developmental biologists have recognized that cells from embryos can differ in their potential to differentiate into distinct cell types. Recently, it has been recognized that embryonic stem cells derived from both mice and humans exhibit two stable yet epigenetically distinct states of pluripotency: naive and primed. We now show that nicotinamide N-methyltransferase (NNMT) and the metabolic state regulate pluripotency in human embryonic stem cells (hESCs).  Specifically, in naive hESCs, NNMT and its enzymatic product 1-methylnicotinamide are highly upregulated, and NNMT is required for low S-adenosyl methionine (SAM) levels and the H3K27me3 repressive state. NNMT consumes SAM in naive cells, making it unavailable for histone methylation that represses Wnt and activates the HIF pathway in primed hESCs. These data support the hypothesis that the metabolome regulates the epigenetic landscape of the earliest steps in human development.


Assuntos
Diferenciação Celular , Epigênese Genética/genética , Células-Tronco Embrionárias Humanas/metabolismo , Metaboloma , Animais , Western Blotting , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectrometria de Massas , Metabolômica/métodos , Metilação , Camundongos , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Nicotinamida N-Metiltransferase/genética , Nicotinamida N-Metiltransferase/metabolismo , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , S-Adenosilmetionina/metabolismo , Transdução de Sinais
3.
PLoS One ; 5(3): e9725, 2010 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-20305814

RESUMO

Activator of G protein Signaling 3 (AGS3) is a receptor-independent G protein activator that has been implicated in multiple biological events such as brain development, neuroplasticity and addiction, cardiac function, Golgi structure/function, macroautophagy and metabolism. However, how AGS3 is regulated is little known. We demonstrate here that AGS3 interacts with a ubiquitin specific protease USP9x, and this interaction is at least partially mediated through the C-terminal G protein regulatory domain of AGS3. Knockdown of USP9x causes a moderate reduction in the level of AGS3. In contrast, overexpression of either USP9x or its deubiquitinating domain UCH increases the amount of AGS3, whereas expression of the mutant UCH domain that lacks deubiquitinating activity does not have the same effect. As previously observed in AGS3 knockdown cells, the localization of several marker proteins of the late Golgi compartments is disturbed in cells depleted of USP9x. Taken together, our study suggests that USP9x can modulate the level of a subpopulation of AGS3, and this modulation plays a role in regulating the structure of the late Golgi compartments. Finally, we have found that levels of AGS3 and USP9x are co-regulated in the prefrontal cortex of rats withdrawn from repeated cocaine treatment. In conjunction with the above data, this observation indicates a potential role of USP9X in the regulation of the AGS3 level during cocaine-induced neuroplasticity.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/fisiologia , Animais , Células COS , Chlorocebus aethiops , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Masculino , Neurônios/efeitos dos fármacos , Estrutura Terciária de Proteína , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Ubiquitina/química , Ubiquitina Tiolesterase/metabolismo
4.
J Cell Biol ; 186(3): 343-53, 2009 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-19651892

RESUMO

Histone lysine methyltransferase complexes are essential for chromatin organization and gene regulation. Whether any of this machinery functions in membrane traffic is unknown. In this study, we report that mammal Dpy-30 (mDpy-30), a subunit of several histone H3 lysine 4 (H3K4) methyltransferase (H3K4MT) complexes, resides in the nucleus and at the trans-Golgi network (TGN). The TGN targeting of mDpy-30 is mediated by BIG1, a TGN-localized guanine nucleotide exchange factor for adenosine diphosphate ribosylation factor GTPases. Altering mDpy-30 levels changes the distribution of cation-independent mannose 6-phosphate receptor (CIMPR) without affecting that of TGN46 or transferrin receptor. Our experiments also indicate that mDpy-30 functions in the endosome to TGN transport of CIMPR and that its knockdown results in the enrichment of internalized CIMPR and recycling endosomes near cell protrusions. Much like mDpy-30 depletion, the knockdown of Ash2L or RbBP5, two other H3K4MT subunits, leads to a similar redistribution of CIMPR. Collectively, these results suggest that mDpy-30 and probably H3K4MT play a role in the endosomal transport of specific cargo proteins.


Assuntos
Endossomos/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Transporte Biológico , Células COS , Células Cultivadas , Chlorocebus aethiops , Células HeLa , Humanos , Ratos
5.
J Biol Chem ; 282(17): 13087-97, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17331948

RESUMO

Plasma membrane proteins such as receptors and ion channels allow a cell to communicate with its environment and regulate many intracellular activities. Thus, the proper control of the surface number of these proteins is essential for maintaining the structural and functional homeostasis of a cell. Internalization and recycling plays a key role in determining the surface density of receptors and channels. Whereas the clathrin-mediated internalization and its associated recycling have been the focus of research in this field, recent studies have revealed that an increasing number of receptors and channels enter a cell via clathrin-independent pathways. However, little is known about the trafficking motifs involved in controlling clathrin-independent internalization and various associated recycling pathways. By using a potassium channel as a model system, we identified a class of trafficking motifs that function along a clathrin-independent pathway to increase the surface density of a membrane protein by preventing its rapid internalization and/or facilitating its recycling via the ADP-ribosylation factor 6-dependent recycling pathway. Moreover our data suggest that these motifs may enhance the association of membrane proteins with the EFA6 family of guanine nucleotide exchange factors for ADP-ribosylation factor 6.


Assuntos
Clatrina , Homeostase , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP , Motivos de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Fatores de Troca do Nucleotídeo Guanina , Células HeLa , Homeostase/genética , Humanos , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Transporte Proteico/genética
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