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1.
Cell ; 177(2): 299-314.e16, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929899

RESUMO

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.


Assuntos
Envelhecimento/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Membranas Mitocondriais/fisiologia , Animais , Autofagia/fisiologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Restrição Calórica , Células HEK293 , Humanos , Longevidade/fisiologia , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Permeabilidade , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais
2.
Cell ; 178(1): 160-175.e27, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31155233

RESUMO

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Animais , Proliferação de Células , Técnicas de Cocultura , Transição Epitelial-Mesenquimal , Feminino , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Quinases Ativadas por Mitógeno/metabolismo , RNA-Seq , Fator de Transcrição STAT3/metabolismo , Células Estromais/metabolismo , Transfecção
3.
Cell ; 170(1): 86-101.e16, 2017 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-28666128

RESUMO

Through an integration of genomic and proteomic approaches to advance understanding of long noncoding RNAs, we investigate the function of the telomeric transcript, TERRA. By identifying thousands of TERRA target sites in the mouse genome, we demonstrate that TERRA can bind both in cis to telomeres and in trans to genic targets. We then define a large network of interacting proteins, including epigenetic factors, telomeric proteins, and the RNA helicase, ATRX. TERRA and ATRX share hundreds of target genes and are functionally antagonistic at these loci: whereas TERRA activates, ATRX represses gene expression. At telomeres, TERRA competes with telomeric DNA for ATRX binding, suppresses ATRX localization, and ensures telomeric stability. Depleting TERRA increases telomerase activity and induces telomeric pathologies, including formation of telomere-induced DNA damage foci and loss or duplication of telomeric sequences. We conclude that TERRA functions as an epigenomic modulator in trans and as an essential regulator of telomeres in cis.


Assuntos
DNA Helicases/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , RNA Longo não Codificante/metabolismo , Telômero/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Camundongos , Motivos de Nucleotídeos , Células-Tronco/metabolismo , Telomerase/metabolismo , Proteína Nuclear Ligada ao X
4.
Mol Cell ; 77(1): 51-66.e8, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31784357

RESUMO

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.


Assuntos
Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Cromatina/metabolismo , Heterocromatina/metabolismo , DNA Ribossômico/metabolismo , Epigênese Genética/fisiologia , Eucromatina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteômica/métodos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Gênica/fisiologia
5.
Mol Cell ; 73(5): 985-1000.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30711375

RESUMO

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.


Assuntos
Neoplasias da Mama/metabolismo , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Mutação , Fosforilação Oxidativa , Fosforilação , Ligação Proteica , Proteômica/métodos , Proteína do Retinoblastoma/genética , Transdução de Sinais/genética , Transcrição Gênica
6.
Genes Dev ; 33(1-2): 75-89, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30567999

RESUMO

Numerous DNA repair and signaling proteins function at DNA damage sites to protect the genome. Here, we show that fusion of the promiscuous biotin ligase BirAR118G with RAD18 leads to localized protein biotinylation at DNA damage sites, allowing identification of ZPET (zinc finger protein proximal to RAD eighteen)/ZNF280C as a potential DNA damage response (DDR) protein. ZPET binds ssDNA and localizes to DNA double-strand breaks (DSBs) and stalled replication forks. In vitro, ZPET inhibits MRE11 binding to ssDNA. In cells, ZPET delays MRE11 binding to chromatin after DSB formation and slows DNA end resection through binding ssDNA. ZPET hinders resection independently of 53BP1 and HELB. Cells lacking ZPET displayed enhanced homologous recombination (HR), accelerated replication forks under stress, and increased resistance to DSBs and PARP inhibition. These results not only reveal ZPET as an HR repressor but also suggest that localized protein biotinylation at DNA damage sites is a useful strategy to identify DDR proteins.


Assuntos
Biotinilação/métodos , Dano ao DNA , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Fatores de Transcrição/metabolismo , Carbono-Nitrogênio Ligases/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Técnicas de Silenciamento de Genes , Humanos , Proteína Homóloga a MRE11/metabolismo , Ligação Proteica , Transporte Proteico/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
7.
Cell ; 143(7): 1174-89, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-21183079

RESUMO

Although most tissues in an organism are genetically identical, the biochemistry of each is optimized to fulfill its unique physiological roles, with important consequences for human health and disease. Each tissue's unique physiology requires tightly regulated gene and protein expression coordinated by specialized, phosphorylation-dependent intracellular signaling. To better understand the role of phosphorylation in maintenance of physiological differences among tissues, we performed proteomic and phosphoproteomic characterizations of nine mouse tissues. We identified 12,039 proteins, including 6296 phosphoproteins harboring nearly 36,000 phosphorylation sites. Comparing protein abundances and phosphorylation levels revealed specialized, interconnected phosphorylation networks within each tissue while suggesting that many proteins are regulated by phosphorylation independently of their expression. Our data suggest that the "typical" phosphoprotein is widely expressed yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue. We offer this dataset as an online resource for the biological research community.


Assuntos
Perfilação da Expressão Gênica , Camundongos/genética , Especificidade de Órgãos , Fosforilação , Proteínas/metabolismo , Animais , Camundongos/metabolismo , Proteínas Quinases/genética , Proteômica
9.
Mol Ther ; 32(5): 1373-1386, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38504517

RESUMO

Epidemiological studies show that individuals who carry the relatively uncommon APOE ε2 allele rarely develop Alzheimer disease, and if they do, they have a later age of onset, milder clinical course, and less severe neuropathological findings than people without this allele. The contrast is especially stark when compared with the major genetic risk factor for Alzheimer disease, APOE ε4, which has an age of onset several decades earlier, a more aggressive clinical course and more severe neuropathological findings, especially in terms of the amount of amyloid deposition. Here, we demonstrate that brain exposure to APOE ε2 via a gene therapy approach, which bathes the entire cortical mantle in the gene product after transduction of the ependyma, reduces Aß plaque deposition, neurodegenerative synaptic loss, and, remarkably, reduces microglial activation in an APP/PS1 mouse model despite continued expression of human APOE ε4. This result suggests a promising protective effect of exogenous APOE ε2 and reveals a cell nonautonomous effect of the protein on microglial activation, which we show is similar to plaque-associated microglia in the brain of Alzheimer disease patients who inherit APOE ε2. These data increase the potential that an APOE ε2 therapeutic could be effective in Alzheimer disease, even in individuals born with the risky ε4 allele.


Assuntos
Doença de Alzheimer , Apolipoproteína E2 , Modelos Animais de Doenças , Terapia Genética , Camundongos Transgênicos , Microglia , Placa Amiloide , Animais , Doença de Alzheimer/terapia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/etiologia , Camundongos , Terapia Genética/métodos , Humanos , Apolipoproteína E2/genética , Apolipoproteína E2/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Microglia/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Doenças Neuroinflamatórias/etiologia , Doenças Neuroinflamatórias/terapia , Doenças Neuroinflamatórias/metabolismo , Peptídeos beta-Amiloides/metabolismo , Biomarcadores
10.
Mol Cell ; 65(2): 336-346, 2017 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-28089683

RESUMO

ATR is a key regulator of cell-cycle checkpoints and homologous recombination (HR). Paradoxically, ATR inhibits CDKs during checkpoint responses, but CDK activity is required for efficient HR. Here, we show that ATR promotes HR after CDK-driven DNA end resection. ATR stimulates the BRCA1-PALB2 interaction after DNA damage and promotes PALB2 localization to DNA damage sites. ATR enhances BRCA1-PALB2 binding at least in part by inhibiting CDKs. The optimal interaction of BRCA1 and PALB2 requires phosphorylation of PALB2 at S59, an ATR site, and hypo-phosphorylation of S64, a CDK site. The PALB2-S59A/S64E mutant is defective for localization to DNA damage sites and HR, whereas the PALB2-S59E/S64A mutant partially bypasses ATR for its localization. Thus, HR is a biphasic process requiring both high-CDK and low-CDK periods. As exemplified by the regulation of PALB2 by ATR, ATR promotes HR by orchestrating a "CDK-to-ATR switch" post-resection, directly coupling the checkpoint to HR.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
11.
Mol Cell ; 61(5): 760-773, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26942679

RESUMO

MicroRNAs predominantly decrease gene expression; however, specific mRNAs are translationally upregulated in quiescent (G0) mammalian cells and immature Xenopus laevis oocytes by an FXR1a-associated microRNA-protein complex (microRNP) that lacks the microRNP repressor, GW182. Their mechanism in these conditions of decreased mTOR signaling, and therefore reduced canonical (cap-and-poly(A)-tail-mediated) translation, remains undiscovered. Our data reveal that mTOR inhibition in human THP1 cells enables microRNA-mediated activation. Activation requires shortened/no poly(A)-tail targets; polyadenylated mRNAs are partially activated upon PAIP2 overexpression, which interferes with poly(A)-bound PABP, precluding PABP-enhanced microRNA-mediated inhibition and canonical translation. Consistently, inhibition of PARN deadenylase prevents activation. P97/DAP5, a homolog of canonical translation factor, eIF4G, which lacks PABP- and cap binding complex-interacting domains, is required for activation, and thereby for the oocyte immature state. P97 interacts with 3' UTR-binding FXR1a-associated microRNPs and with PARN, which binds mRNA 5' caps, forming a specialized complex to translate recruited mRNAs in these altered canonical translation conditions.


Assuntos
Senescência Celular , MicroRNAs/metabolismo , Oócitos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , Linhagem Celular , Fator de Iniciação Eucariótico 4G/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , MicroRNAs/genética , Proteômica/métodos , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Transfecção , Xenopus laevis
12.
Mol Cell ; 63(6): 1006-20, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27635760

RESUMO

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.


Assuntos
Acetil-CoA Carboxilase/metabolismo , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Leucemia Mieloide Aguda/metabolismo , Prolina/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos NOD , Modelos Moleculares , Transplante de Neoplasias , Oxirredução , Prolina/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Análise de Sobrevida
13.
Genes Dev ; 29(17): 1875-89, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26314710

RESUMO

The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.


Assuntos
Mitocôndrias/metabolismo , Fosforilação Oxidativa , Proteína do Retinoblastoma/genética , Animais , Células Cultivadas , Colo/fisiopatologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Pulmão/fisiopatologia , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteômica , Proteína do Retinoblastoma/metabolismo , Estresse Fisiológico/genética , Transcriptoma
14.
FASEB J ; 35(12): e22019, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34792819

RESUMO

Exogenously applied mature naïve B220+ /CD19+ /IgM+ /IgD+ B cells are strongly protective in the context of tissue injury. However, the mechanisms by which B cells detect tissue injury and aid repair remain elusive. Here, we show in distinct models of skin and brain injury that MyD88-dependent toll-like receptor (TLR) signaling through TLR2/6 and TLR4 is essential for the protective benefit of B cells in vivo, while B cell-specific deletion of MyD88 abrogated this effect. The B cell response to injury was multi-modal with simultaneous production of both regulatory cytokines, such as IL-10, IL-35, and transforming growth factor beta (TGFß), and inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), IL-6, and interferon gamma. Cytometry analysis showed that this response was time and environment-dependent in vivo, with 20%-30% of applied B cells adopting an immune modulatory phenotype with high co-expression of anti- and pro-inflammatory cytokines after 18-48 h at the injury site. B cell treatment reduced the expression of TNFα and increased IL-10 and TGFß in infiltrating immune cells and fibroblasts at the injury site. Proteomic analysis further showed that B cells have a complex time-dependent homeostatic effect on the injured microenvironment, reducing the expression of inflammation-associated proteins, and increasing proteins associated with proliferation, tissue remodeling, and protection from oxidative stress. These findings chart and validate a first mechanistic understanding of the effects of B cells as an immunomodulatory cell therapy in the context of tissue injury.


Assuntos
Linfócitos B/fisiologia , Lesões Encefálicas/prevenção & controle , Citocinas/metabolismo , Fator 88 de Diferenciação Mieloide/fisiologia , Pele/imunologia , Cicatrização , Animais , Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Interleucina-10/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Transdução de Sinais , Pele/lesões , Pele/metabolismo , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
15.
Nature ; 539(7629): 390-395, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27799657

RESUMO

Intermediary metabolism generates substrates for chromatin modification, enabling the potential coupling of metabolic and epigenetic states. Here we identify a network linking metabolic and epigenetic alterations that is central to oncogenic transformation downstream of the liver kinase B1 (LKB1, also known as STK11) tumour suppressor, an integrator of nutrient availability, metabolism and growth. By developing genetically engineered mouse models and primary pancreatic epithelial cells, and employing transcriptional, proteomics, and metabolic analyses, we find that oncogenic cooperation between LKB1 loss and KRAS activation is fuelled by pronounced mTOR-dependent induction of the serine-glycine-one-carbon pathway coupled to S-adenosylmethionine generation. At the same time, DNA methyltransferases are upregulated, leading to elevation in DNA methylation with particular enrichment at retrotransposon elements associated with their transcriptional silencing. Correspondingly, LKB1 deficiency sensitizes cells and tumours to inhibition of serine biosynthesis and DNA methylation. Thus, we define a hypermetabolic state that incites changes in the epigenetic landscape to support tumorigenic growth of LKB1-mutant cells, while resulting in potential therapeutic vulnerabilities.


Assuntos
Transformação Celular Neoplásica , Metilação de DNA , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Inativação Gênica , Genes Supressores de Tumor , Glicina/metabolismo , Glicólise , Humanos , Camundongos , Ductos Pancreáticos/citologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Retroelementos/genética , S-Adenosilmetionina/metabolismo , Serina/biossíntese , Serina-Treonina Quinases TOR/metabolismo , Transaminases/metabolismo
16.
Nature ; 537(7618): 102-106, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27556950

RESUMO

Circulating tumour cells in women with advanced oestrogen-receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer acquire a HER2-positive subpopulation after multiple courses of therapy. In contrast to HER2-amplified primary breast cancer, which is highly sensitive to HER2-targeted therapy, the clinical significance of acquired HER2 heterogeneity during the evolution of metastatic breast cancer is unknown. Here we analyse circulating tumour cells from 19 women with ER+/HER2- primary tumours, 84% of whom had acquired circulating tumour cells expressing HER2. Cultured circulating tumour cells maintain discrete HER2+ and HER2- subpopulations: HER2+ circulating tumour cells are more proliferative but not addicted to HER2, consistent with activation of multiple signalling pathways; HER2- circulating tumour cells show activation of Notch and DNA damage pathways, exhibiting resistance to cytotoxic chemotherapy, but sensitivity to Notch inhibition. HER2+ and HER2- circulating tumour cells interconvert spontaneously, with cells of one phenotype producing daughters of the opposite within four cell doublings. Although HER2+ and HER2- circulating tumour cells have comparable tumour initiating potential, differential proliferation favours the HER2+ state, while oxidative stress or cytotoxic chemotherapy enhances transition to the HER2- phenotype. Simultaneous treatment with paclitaxel and Notch inhibitors achieves sustained suppression of tumorigenesis in orthotopic circulating tumour cell-derived tumour models. Together, these results point to distinct yet interconverting phenotypes within patient-derived circulating tumour cells, contributing to progression of breast cancer and acquisition of drug resistance.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Receptor ErbB-2/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Células Neoplásicas Circulantes/efeitos dos fármacos , Fenótipo , Receptor ErbB-2/deficiência , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Transdução de Sinais
18.
Nature ; 524(7565): 361-5, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26168401

RESUMO

Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy, a conserved self-degradative process. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. Here we show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family of transcription factors. In human PDA cells, the MiT/TFE proteins--MITF, TFE3 and TFEB--are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosome activation is specifically required to maintain intracellular amino acid pools. These results identify the MiT/TFE proteins as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate that transcriptional activation of clearance pathways converging on the lysosome is a novel hallmark of aggressive malignancy.


Assuntos
Autofagia/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Regulação Neoplásica da Expressão Gênica , Lisossomos/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Metabolismo Energético , Feminino , Xenoenxertos , Homeostase , Humanos , Lisossomos/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/metabolismo , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Transcrição Gênica
19.
Proc Natl Acad Sci U S A ; 115(36): E8403-E8412, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127002

RESUMO

Defects in cilia have been associated with an expanding human disease spectrum known as ciliopathies. Regulatory Factor X 3 (RFX3) is one of the major transcription factors required for ciliogenesis and cilia functions. In addition, RFX3 regulates pancreatic islet cell differentiation and mature ß-cell functions. However, how RFX3 protein is regulated at the posttranslational level remains poorly understood. Using chemical reporters of protein fatty acylation and mass spectrometry analysis, here we show that RFX3 transcriptional activity is regulated by S-fatty acylation at a highly conserved cysteine residue in the dimerization domain. Surprisingly, RFX3 undergoes enzyme-independent, "self-catalyzed" auto-fatty acylation and displays preferences for 18-carbon stearic acid and oleic acid. The fatty acylation-deficient mutant of RFX3 shows decreased homodimerization; fails to promote ciliary gene expression, ciliogenesis, and elongation; and impairs Hedgehog signaling. Our findings reveal a regulation of RFX3 transcription factor and link fatty acid metabolism and protein lipidation to the regulation of ciliogenesis.


Assuntos
Lipoilação , Ácido Oleico/metabolismo , Fatores de Transcrição de Fator Regulador X/metabolismo , Ácidos Esteáricos/metabolismo , Acilação , Animais , Cílios/genética , Cílios/metabolismo , Ciliopatias/genética , Ciliopatias/metabolismo , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Fatores de Transcrição de Fator Regulador X/genética
20.
PLoS Genet ; 14(10): e1007643, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30335751

RESUMO

The Fanconi Anemia (FA) pathway is important for repairing interstrand crosslinks (ICLs) between the Watson-Crick strands of the DNA double helix. An initial and essential stage in the repair process is the detection of the ICL. Here, we report the identification of UHRF2, a paralogue of UHRF1, as an ICL sensor protein. UHRF2 is recruited to ICLs in the genome within seconds of their appearance. We show that UHRF2 cooperates with UHRF1, to ensure recruitment of FANCD2 to ICLs. A direct protein-protein interaction is formed between UHRF1 and UHRF2, and between either UHRF1 and UHRF2, and FANCD2. Importantly, we demonstrate that the essential monoubiquitination of FANCD2 is stimulated by UHRF1/UHRF2. The stimulation is mediating by a retention of FANCD2 on chromatin, allowing for its monoubiquitination by the FA core complex. Taken together, we uncover a mechanism of ICL sensing by UHRF2, leading to FANCD2 recruitment and retention at ICLs, in turn facilitating activation of FANCD2 by monoubiquitination.


Assuntos
Reparo do DNA/fisiologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Sequência de Aminoácidos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA/fisiologia , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Células HEK293 , Células HeLa , Humanos , Domínios e Motivos de Interação entre Proteínas , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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