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1.
Autophagy ; 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33292048

RESUMO

Macroautophagic/autophagic degradation of nuclear components (or nuclear autophagy) is a poorly understood area in autophagy research. We previously reported the nuclear lamina protein LMNB1 (lamin B1) as a nuclear autophagy substrate in primary human cells, stimulating the investigation of nuclear autophagy in the mammalian system. We recently reported the sirtuin protein SIRT1 as a new selective substrate of nuclear autophagy in senescence and aging. Upon senescence of primary human cells, SIRT1 degradation is mediated by a direct nuclear SIRT1-LC3 interaction, followed by nucleus-to-cytoplasm shuttling of SIRT1 and autophagosome-lysosome degradation. In vivo, SIRT1 is downregulated by lysosomes in hematopoietic and immune organs upon natural aging in mice and in aged human T cells. Our study identified another substrate of nuclear autophagy and suggests a new strategy to promote SIRT1-mediated health benefits by suppressing its autophagic degradation.

2.
Nat Cell Biol ; 22(10): 1170-1179, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32989246

RESUMO

SIRT1 (Sir2) is an NAD+-dependent deacetylase that plays critical roles in a broad range of biological events, including metabolism, the immune response and ageing1-5. Although there is strong interest in stimulating SIRT1 catalytic activity, the homeostasis of SIRT1 at the protein level is poorly understood. Here we report that macroautophagy (hereafter referred to as autophagy), a catabolic membrane trafficking pathway that degrades cellular components through autophagosomes and lysosomes, mediates the downregulation of mammalian SIRT1 protein during senescence and in vivo ageing. In senescence, nuclear SIRT1 is recognized as an autophagy substrate and is subjected to cytoplasmic autophagosome-lysosome degradation, via the autophagy protein LC3. Importantly, the autophagy-lysosome pathway contributes to the loss of SIRT1 during ageing of several tissues related to the immune and haematopoietic system in mice, including the spleen, thymus, and haematopoietic stem and progenitor cells, as well as in CD8+CD28- T cells from aged human donors. Our study reveals a mechanism in the regulation of the protein homeostasis of SIRT1 and suggests a potential strategy to stabilize SIRT1 to promote productive ageing.

3.
Immunity ; 51(5): 840-855.e5, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31606264

RESUMO

TCF-1 is a key transcription factor in progenitor exhausted CD8 T cells (Tex). Moreover, this Tex cell subset mediates responses to PD-1 checkpoint pathway blockade. However, the role of the transcription factor TCF-1 in early fate decisions and initial generation of Tex cells is unclear. Single-cell RNA sequencing (scRNA-seq) and lineage tracing identified a TCF-1+Ly108+PD-1+ CD8 T cell population that seeds development of mature Tex cells early during chronic infection. TCF-1 mediated the bifurcation between divergent fates, repressing development of terminal KLRG1Hi effectors while fostering KLRG1Lo Tex precursor cells, and PD-1 stabilized this TCF-1+ Tex precursor cell pool. TCF-1 mediated a T-bet-to-Eomes transcription factor transition in Tex precursors by promoting Eomes expression and drove c-Myb expression that controlled Bcl-2 and survival. These data define a role for TCF-1 in early-fate-bifurcation-driving Tex precursor cells and also identify PD-1 as a protector of this early TCF-1 subset.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Redes Reguladoras de Genes , Fator 1 de Transcrição de Linfócitos T/metabolismo , Transcrição Genética , Animais , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Doença Crônica , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Fator 1 de Transcrição de Linfócitos T/genética , Viroses/genética , Viroses/imunologia , Viroses/virologia
4.
G3 (Bethesda) ; 7(12): 3857-3866, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29038170

RESUMO

Using a comprehensive library of histone H2A and H2B mutants, we assessed the biological function of each amino acid residue involved in various stress conditions including exposure to different DNA damage-inducing reagents, different growth temperatures, and other chemicals. H2B N- and H2A C-termini were critical for maintaining nucleosome function and mutations in these regions led to pleiotropic phenotypes. Additionally, two screens were performed using this library, monitoring heterochromatin gene silencing and genome stability, to identify residues that could compromise normal function when mutated. Many distinctive regions within the nucleosome were revealed. Furthermore, we used the barcode sequencing (bar-seq) method to profile the mutant composition of many libraries in one high-throughput sequencing experiment, greatly reducing the labor and increasing the capacity. This study not only demonstrates the applications of the versatile histone library, but also reveals many previously unknown functions of histone H2A and H2B.


Assuntos
Dano ao DNA/efeitos dos fármacos , Histonas/genética , Nucleossomos/genética , Aminoácidos/genética , Biblioteca Gênica , Inativação Gênica , Instabilidade Genômica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Mutantes/genética , Ligação Proteica , Saccharomyces cerevisiae/genética
5.
Nature ; 550(7676): 402-406, 2017 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-28976970

RESUMO

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.


Assuntos
Senescência Celular/genética , Cromatina/metabolismo , Citoplasma/genética , Imunidade Inata , Inflamação/genética , Inflamação/patologia , Neoplasias/genética , Neoplasias/imunologia , Animais , Linhagem Celular Tumoral , Cromatina/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Citoplasma/imunologia , Feminino , Humanos , Inflamação/imunologia , Fígado/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/patologia , Nucleotidiltransferases/metabolismo , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/imunologia , Radiação Ionizante
6.
J Clin Invest ; 126(5): 1834-56, 2016 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-27043285

RESUMO

Targeting multiple components of the MAPK pathway can prolong the survival of patients with BRAFV600E melanoma. This approach is not curative, as some BRAF-mutated melanoma cells are intrinsically resistant to MAPK inhibitors (MAPKi). At the systemic level, our knowledge of how signaling pathways underlie drug resistance needs to be further expanded. Here, we have shown that intrinsically resistant BRAF-mutated melanoma cells with a low basal level of mitochondrial biogenesis depend on this process to survive MAPKi. Intrinsically resistant cells exploited an integrated stress response, exhibited an increase in mitochondrial DNA content, and required oxidative phosphorylation to meet their bioenergetic needs. We determined that intrinsically resistant cells rely on the genes encoding TFAM, which controls mitochondrial genome replication and transcription, and TRAP1, which regulates mitochondrial protein folding. Therefore, we targeted mitochondrial biogenesis with a mitochondrium-targeted, small-molecule HSP90 inhibitor (Gamitrinib), which eradicated intrinsically resistant cells and augmented the efficacy of MAPKi by inducing mitochondrial dysfunction and inhibiting tumor bioenergetics. A subset of tumor biopsies from patients with disease progression despite MAPKi treatment showed increased mitochondrial biogenesis and tumor bioenergetics. A subset of acquired drug-resistant melanoma cell lines was sensitive to Gamitrinib. Our study establishes mitochondrial biogenesis, coupled with aberrant tumor bioenergetics, as a potential therapy escape mechanism and paves the way for a rationale-based combinatorial strategy to improve the efficacy of MAPKi.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Guanidinas/farmacologia , Lactamas Macrocíclicas/farmacologia , Melanoma/tratamento farmacológico , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Nature ; 527(7576): 105-9, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26524528

RESUMO

Macroautophagy (hereafter referred to as autophagy) is a catabolic membrane trafficking process that degrades a variety of cellular constituents and is associated with human diseases. Although extensive studies have focused on autophagic turnover of cytoplasmic materials, little is known about the role of autophagy in degrading nuclear components. Here we report that the autophagy machinery mediates degradation of nuclear lamina components in mammals. The autophagy protein LC3/Atg8, which is involved in autophagy membrane trafficking and substrate delivery, is present in the nucleus and directly interacts with the nuclear lamina protein lamin B1, and binds to lamin-associated domains on chromatin. This LC3-lamin B1 interaction does not downregulate lamin B1 during starvation, but mediates its degradation upon oncogenic insults, such as by activated RAS. Lamin B1 degradation is achieved by nucleus-to-cytoplasm transport that delivers lamin B1 to the lysosome. Inhibiting autophagy or the LC3-lamin B1 interaction prevents activated RAS-induced lamin B1 loss and attenuates oncogene-induced senescence in primary human cells. Our study suggests that this new function of autophagy acts as a guarding mechanism protecting cells from tumorigenesis.


Assuntos
Autofagia , Lâmina Nuclear/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Família da Proteína 8 Relacionada à Autofagia , Transformação Celular Neoplásica , Células Cultivadas , Senescência Celular , Cromatina/química , Cromatina/metabolismo , Citoplasma/metabolismo , Fibroblastos , Células HEK293 , Humanos , Lamina Tipo B/genética , Lamina Tipo B/metabolismo , Lisossomos/metabolismo , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Ligação Proteica , Proteólise
8.
J Biol Chem ; 290(28): 17546-58, 2015 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-25987563

RESUMO

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.


Assuntos
Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinase A/química , Aurora Quinase A/genética , Aurora Quinase B/química , Aurora Quinase B/genética , Compartimento Celular , Pontos de Checagem do Ciclo Celular , Centrossomo/metabolismo , Cromatina/metabolismo , Evolução Molecular , Células HeLa , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Mitose , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/metabolismo
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