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1.
Biosci Rep ; 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38808772

RESUMO

Liver fibrosis is the excessive accumulation of extracellular matrix proteins that occurs in most  chronic liver diseases.  At the cellular level, liver fibrosis is associated with the activation of hepatic stellate cells (HSCs) which transdifferentiate into a myofibroblast-like phenotype that is contractile, proliferative and profibrogenic.  HSC transdifferentiation induces genome-wide changes in gene expression that enable the cell to adopt its profibrogenic functions. We have previously identified that the deubiquitinase ubiquitin C-terminal hydrolase 1 (UCHL1) is highly induced following HSC activation, however, the cellular targets of its deubiquitinating activity are poorly defined.     Here we describe a role for UCHL1 in regulating the levels and activity of hypoxia-inducible factor 1 (HIF1), an oxygen-sensitive transcription factor, during HSC activation and liver fibrosis.  HIF1 is elevated during HSC activation and promotes the expression of profibrotic mediator HIF target genes. Increased HIF1α expression correlated with induction of UCHL1 mRNA and protein with HSC activation. Genetic deletion or chemical inhibition of UCHL1 impaired HIF activity through reduction of HIF1α levels. Furthermore, our mechanistic studies have shown that UCHL1 elevates HIF activity through specific cleavage of degradative ubiquitin chains, elevates levels of pro-fibrotic gene expression and increases proliferation rates. As we also show that UCHL1 inhibition blunts fibrogenesis in a pre-clinical 3D human liver slice model of fibrosis, these results demonstrate how small molecule inhibitors of DUBs can exert therapeutic effects through modulation of HIF transcription factors in liver disease.  Furthermore, inhibition of HIF activity using UCHL1 inhibitors may represent a therapeutic opportunity with other HIF-related pathologies.

2.
EMBO Rep ; 24(12): e57849, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-37877678

RESUMO

Oxygen is essential for viability in mammalian organisms. However, cells are often exposed to changes in oxygen availability, due to either increased demand or reduced oxygen supply, herein called hypoxia. To be able to survive and/or adapt to hypoxia, cells activate a variety of signalling cascades resulting in changes to chromatin, gene expression, metabolism and viability. Cellular signalling is often mediated via post-translational modifications (PTMs), and this is no different in response to hypoxia. Many enzymes require oxygen for their activity and oxygen can directly influence several PTMS. Here, we review the direct impact of changes in oxygen availability on PTMs such as proline, asparagine, histidine and lysine hydroxylation, lysine and arginine methylation and cysteine dioxygenation, with a focus on mammalian systems. In addition, indirect hypoxia-dependent effects on phosphorylation, ubiquitination and sumoylation will also be discussed. Direct and indirect oxygen-regulated changes to PTMs are coordinated to achieve the cell's ultimate response to hypoxia. However, specific oxygen sensitivity and the functional relevance of some of the identified PTMs still require significant research.


Assuntos
Lisina , Oxigênio , Animais , Humanos , Oxigênio/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Cromatina , Hipóxia/metabolismo , Mamíferos/metabolismo
3.
Dev Cell ; 57(22): 2584-2598.e11, 2022 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-36413951

RESUMO

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.


Assuntos
NAD , Saccharomyces cerevisiae , Animais , Camundongos , Humanos , Sobrevivência Celular , Autofagia , Morte Celular
5.
Mol Neurobiol ; 59(10): 5987-6008, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35831557

RESUMO

The neuroblastoma cell line SH-SY5Y is widely used to study retinoic acid (RA)-induced gene expression and differentiation and as a tool to study neurodegenerative disorders. SH-SY5Y cells predominantly exhibit adrenergic neuronal properties, but they can also exist in an epigenetically interconvertible alternative state with more mesenchymal characteristics; as a result, these cells can be used to study gene regulation circuitry controlling neuroblastoma phenotype. Using a combination of pharmacological inhibition and targeted gene inactivation, we have probed the requirement for DNA topoisomerase IIB (TOP2B) in RA-induced gene expression and differentiation and in the balance between adrenergic neuronal versus mesenchymal transcription programmes. We found that expression of many, but not all genes that are rapidly induced by ATRA in SH-SY5Y cells was significantly reduced in the TOP2B null cells; these genes include BCL2, CYP26A1, CRABP2, and NTRK2. Comparing gene expression profiles in wild-type versus TOP2B null cells, we found that long genes and genes expressed at a high level in WT SH-SY5Y cells were disproportionately dependent on TOP2B. Notably, TOP2B null SH-SY5Y cells upregulated mesenchymal markers vimentin (VIM) and fibronectin (FN1) and components of the NOTCH signalling pathway. Enrichment analysis and comparison with the transcription profiles of other neuroblastoma-derived cell lines supported the conclusion that TOP2B is required to fully maintain the adrenergic neural-like transcriptional signature of SH-SY5Y cells and to suppress the alternative mesenchymal epithelial-like epigenetic state.


Assuntos
DNA Topoisomerases Tipo II , Neuroblastoma , Proteínas de Ligação a Poli-ADP-Ribose , Adrenérgicos , Diferenciação Celular , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Neuroblastoma/metabolismo , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Tretinoína/metabolismo , Tretinoína/farmacologia
6.
Sci Rep ; 12(1): 7943, 2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35562367

RESUMO

The product encoded by the X-linked inhibitor of apoptosis (XIAP) gene is a multi-functional protein which not only controls caspase-dependent cell death, but also participates in inflammatory signalling, copper homeostasis, response to hypoxia and control of cell migration. Deregulation of XIAP, either by elevated expression or inherited genetic deletion, is associated with several human disease states. Reconciling XIAP-dependent signalling pathways with its role in disease progression is essential to understand how XIAP promotes the progression of human pathologies. In this study we have created a panel of genetically modified XIAP-null cell lines using TALENs and CRISPR/Cas9 to investigate the functional outcome of XIAP deletion. Surprisingly, in our genetically modified cells XIAP deletion had no effect on programmed cell death, but instead the primary phenotype we observed was a profound increase in cell migration rates. Furthermore, we found that XIAP-dependent suppression of cell migration was dependent on XIAPdependent control of C-RAF levels, a protein kinase which controls cell signalling pathways that regulate the cytoskeleton. These results suggest that XIAP is not necessary for control of the apoptotic signalling cascade, however it does have a critical role in controlling cell migration and motility that cannot be compensated for in XIAP-knockout cells.


Assuntos
Linfócitos Nulos , Proteínas Proto-Oncogênicas c-raf , Apoptose , Caspases/metabolismo , Linfócitos Nulos/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
7.
Biochem J ; 479(3): 245-257, 2022 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-35119457

RESUMO

Hypoxia is a common denominator in the pathophysiology of a variety of human disease states. Insight into how cells detect, and respond to low oxygen is crucial to understanding the role of hypoxia in disease. Central to the hypoxic response is rapid changes in the expression of genes essential to carry out a wide range of functions to adapt the cell/tissue to decreased oxygen availability. These changes in gene expression are co-ordinated by specialised transcription factors, changes to chromatin architecture and intricate balances between protein synthesis and destruction that together establish changes to the cellular proteome. In this article, we will discuss the advances of our understanding of the cellular oxygen sensing machinery achieved through the application of 'omics-based experimental approaches.


Assuntos
Hipóxia Celular/genética , Regulação da Expressão Gênica , Oxigênio/metabolismo , Transdução de Sinais/genética , Transcriptoma/genética , Animais , Cromatina/genética , Cromatina/metabolismo , Humanos , Metaboloma/genética , Proteoma/genética , Proteoma/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
Cells ; 10(6)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34200988

RESUMO

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.


Assuntos
Hipóxia Celular , Proteínas de Ligação a DNA/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/fisiologia , Células A549 , Células HeLa , Humanos , Biossíntese de Proteínas , Transdução de Sinais
9.
Cells ; 8(2)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717305

RESUMO

Hypoxia-Inducible Factors (HIFs) play essential roles in the physiological response to low oxygen in all multicellular organisms, while their deregulation is associated with human diseases. HIF levels and activity are primarily controlled by the availability of the oxygen-sensitive HIFα subunits, which is mediated by rapid alterations to the rates of HIFα protein production and degradation. While the pathways that control HIFα degradation are understood in great detail, much less is known about the targeted control of HIFα protein synthesis and what role this has in controlling HIF activity during the hypoxic response. This review will focus on the signalling pathways and RNA binding proteins that modulate HIFα mRNA half-life and/or translation rate, and their contribution to hypoxia-associated diseases.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Oxigênio/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Hipóxia Celular/efeitos dos fármacos , Humanos , Proteínas de Ligação a RNA/metabolismo , Ubiquitina/metabolismo
10.
Nucleic Acids Res ; 46(8): 3878-3890, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29529249

RESUMO

HIF1α (hypoxia inducible factor 1α) is the central regulator of the cellular response to low oxygen and its activity is deregulated in multiple human pathologies. Consequently, given the importance of HIF signaling in disease, there is considerable interest in developing strategies to modulate HIF1α activity and down-stream signaling events. In the present study we find that under hypoxic conditions, activation of the PERK branch of the unfolded protein response (UPR) can suppress the levels and activity of HIF1α by preventing efficient HIF1α translation. Activation of PERK inhibits de novo HIF1α protein synthesis by preventing the RNA-binding protein, YB-1, from interacting with the HIF1α mRNA 5'UTR. Our data indicate that activation of the UPR can sensitise tumor cells to hypoxic stress, indicating that chemical activation of the UPR could be a strategy to target hypoxic malignant cancer cells.


Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteína 1 de Ligação a Y-Box/metabolismo , eIF-2 Quinase/metabolismo , Regiões 5' não Traduzidas , Regulação para Baixo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células PC-3 , Biossíntese de Proteínas , Estabilidade Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Tapsigargina/farmacologia , Hipóxia Tumoral/genética , Resposta a Proteínas não Dobradas , eIF-2 Quinase/antagonistas & inibidores
11.
Nat Commun ; 9(1): 256, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29343728

RESUMO

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival.


Assuntos
Autofagia , Proteostase , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo , Sequência de Aminoácidos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Células HeLa , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos Knockout , Oxidantes/farmacologia , Oxirredução , Homologia de Sequência de Aminoácidos , Proteína Sequestossoma-1/genética
12.
Nucleic Acids Res ; 45(16): 9336-9347, 2017 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-28666324

RESUMO

The cellular response to hypoxia is characterised by a switch in the transcriptional program, mediated predominantly by the hypoxia inducible factor family of transcription factors (HIF). Regulation of HIF1 is primarily controlled by post-translational modification of the HIF1α subunit, which can alter its stability and/or activity. This study identifies an unanticipated role for the X-linked inhibitor of apoptosis (XIAP) protein as a regulator of Lys63-linked polyubiquitination of HIF1α. Lys63-linked ubiquitination of HIF1α by XIAP is dependent on the activity of E2 ubiquitin conjugating enzyme Ubc13. We find that XIAP and Ubc13 dependent Lys63-linked polyubiquitination promotes HIF1α nuclear retention leading to an increase in the expression of HIF1 responsive genes. Inhibition of the Lys63-linked polyubiquitination pathway leads to reduced levels of nuclear HIF1α, promoter occupancy, HIF-dependent gene expression and cell viability. Our data reveals an additional and significant level of control of the HIF1 by XIAP, with important implications in understanding the role of HIF1 and XIAP in human disease.


Assuntos
Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lisina/metabolismo , Ubiquitinação , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Hipóxia Celular , Linhagem Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Regiões Promotoras Genéticas , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Enzimas de Conjugação de Ubiquitina/metabolismo , Regulação para Cima
13.
Cell Cycle ; 13(6): 1006-14, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24552822

RESUMO

Copper is an essential trace element that plays key roles in many metabolic processes. Homeostatic regulation of intracellular copper is normally tightly controlled, but deregulated copper levels are found in numerous metabolic and neurodegenerative diseases, as well as in a range of neoplasms. There are conflicting reports regarding the exact role of copper in the regulation of NFκB-responsive genes, specifically whether copper leads to increased activation of the NFκB pathways, or downregulation. Here we show that increased intracellular levels of copper, using the ionophore clioquinol, leads to a potent inhibition of NFκB pathways, induced by multiple distinct stimuli. Addition of copper to cells inhibits ubiquitin-mediated degradation of IκBα by preventing its phoshorylation by the upstream IKK complex. Intriguingly, copper-dependent inhibition of NFκB can be reversed by the addition of the reducing agent, N-acetylcysteine (NAC). These results suggest that the oxidative properties of excess copper prevent NFκB activation by blocking IκBα destruction, and that NFκB activity should be assessed in diseases associated with copper excess.


Assuntos
Cobre/metabolismo , NF-kappa B/metabolismo , Acetilcisteína/farmacologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Clioquinol/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Proteínas I-kappa B/metabolismo , Ionóforos/farmacologia , Camundongos , Inibidor de NF-kappaB alfa , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Transdução de Sinais , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
14.
Biochem J ; 449(1): 275-84, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23016877

RESUMO

The IKK [inhibitor of NF-κB (nuclear factor κB) kinase] complex has an essential role in the activation of the family of NF-κB transcription factors in response to a variety of stimuli. To identify novel IKK-interacting proteins, we performed an unbiased proteomics screen where we identified TfR1 (transferrin receptor 1). TfR1 is required for transferrin binding and internalization and ultimately for iron homoeostasis. TfR1 depletion does not lead to changes in IKK subunit protein levels; however, it does reduce the formation of the IKK complex, and inhibits TNFα (tumour necrosis factor α)-induced NF-κB-dependent transcription. We find that, in the absence of TfR1, NF-κB does not translocate to the nucleus efficiently, and there is a reduction in the binding to target gene promoters and consequentially less target gene activation. Significantly, depletion of TfR1 results in an increase in apoptosis in response to TNFα treatment, which is rescued by elevating the levels of RelA/NF-κB. Taken together, these results indicate a new function for TfR1 in the control of IKK and NF-κB. Our data indicate that IKK-NF-κB responds to changes in iron within the cell.


Assuntos
Antígenos CD/metabolismo , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Receptores da Transferrina/metabolismo , Transdução de Sinais/fisiologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Quinase I-kappa B/fisiologia , Ferro/química , Ferro/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/fisiologia , Ligação Proteica/fisiologia , Receptores da Transferrina/deficiência
15.
Curr Opin Cell Biol ; 24(6): 871-5, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23219152

RESUMO

The cytoprotective properties of vertebrate inhibitor of apoptosis (IAP) proteins have been the subject of much study. These proteins have, however, emerged as key signaling intermediates modulating a variety of cellular functions through their ability to act as E3 ubiquitin ligases. This review will focus on the cell death-independent roles of the IAP proteins, focusing on recent reports indicating that c-IAPs and XIAP are key molecules involved in modulating cell migration and development.


Assuntos
Movimento Celular , Proteínas Inibidoras de Apoptose/metabolismo , Animais , Apoptose , Crescimento e Desenvolvimento , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt
16.
Biochem J ; 443(2): 355-9, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22332634

RESUMO

A recent study revealed that ES (embryonic stem) cell lines derived from the 129 murine strain carry an inactivating mutation within the caspase 11 gene (Casp4) locus [Kayagaki, Warming, Lamkanfi, Vande Walle, Louie, Dong, Newton, Qu, Liu, Heldens, Zhang, Lee, Roose-Girma and Dixit (2011) Nature 479, 117-121]. Thus, if 129 ES cells are used to target genes closely linked to caspase 11, the resulting mice might also carry the caspase 11 deficiency as a passenger mutation. In the present study, we examined the genetic loci of mice targeted for the closely linked c-IAP (cellular inhibitor of apoptosis) genes, which were generated in 129 ES cells, and found that, despite extensive backcrossing into a C57BL/6 background, c-IAP1(-/-) animals are also deficient in caspase 11. Consequently, data obtained from these mice should be re-evaluated in this new context.


Assuntos
Caspases/genética , Proteínas Inibidoras de Apoptose/metabolismo , Mutação , Animais , Caspases/metabolismo , Caspases Iniciadoras , Linhagem Celular , Ativação Enzimática , Proteínas Inibidoras de Apoptose/deficiência , Camundongos , Camundongos da Linhagem 129
17.
Mol Cell ; 45(4): 541-52, 2012 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-22281053

RESUMO

Polo-like kinase Plk1 controls numerous aspects of cell-cycle progression. We show that it associates with tRNA and 5S rRNA genes and regulates their transcription by RNA polymerase III (pol III) through direct binding and phosphorylation of transcription factor Brf1. During interphase, Plk1 promotes tRNA and 5S rRNA expression by phosphorylating Brf1 directly on serine 450. However, this stimulatory modification is overridden at mitosis, when elevated Plk1 activity causes Brf1 phosphorylation on threonine 270 (T270), which prevents pol III recruitment. Thus, although Plk1 enhances net tRNA and 5S rRNA production, consistent with its proliferation-stimulating function, it also suppresses untimely transcription when cells divide. Genomic instability is apparent in cells with Brf1 T270 mutated to alanine to resist Plk1-directed inactivation, suggesting that chromosome segregation is vulnerable to inappropriate pol III activity.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , RNA Ribossômico 5S/genética , RNA de Transferência/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/genética , Instabilidade Genômica , Células HeLa , Humanos , Mitose , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase III/fisiologia , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Quinase 1 Polo-Like
18.
PLoS Genet ; 7(1): e1001285, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21298084

RESUMO

Hypoxia Inducible Factor-1 (HIF-1) is essential for mammalian development and is the principal transcription factor activated by low oxygen tensions. HIF-α subunit quantities and their associated activity are regulated in a post-translational manner, through the concerted action of a class of enzymes called Prolyl Hydroxylases (PHDs) and Factor Inhibiting HIF (FIH) respectively. However, alternative modes of HIF-α regulation such as translation or transcription are under-investigated, and their importance has not been firmly established. Here, we demonstrate that NF-κB regulates the HIF pathway in a significant and evolutionary conserved manner. We demonstrate that NF-κB directly regulates HIF-1ß mRNA and protein. In addition, we found that NF-κB-mediated changes in HIF-1ß result in modulation of HIF-2α protein. HIF-1ß overexpression can rescue HIF-2α protein levels following NF-κB depletion. Significantly, NF-κB regulates HIF-1ß (tango) and HIF-α (sima) levels and activity (Hph/fatiga, ImpL3/ldha) in Drosophila, both in normoxia and hypoxia, indicating an evolutionary conserved mode of regulation. These results reveal a novel mechanism of HIF regulation, with impact in the development of novel therapeutic strategies for HIF-related pathologies including ageing, ischemia, and cancer.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Regulação da Expressão Gênica , NF-kappa B/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fibroblastos/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Células HEK293 , Células HeLa , Humanos , Hipóxia/genética , Camundongos , NF-kappa B/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia
19.
Mol Biol Cell ; 21(21): 3630-8, 2010 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-20844082

RESUMO

The tumor suppressor adenomatous polyposis coli (APC) is mutated in the majority of colorectal cancers and is best known for its role as a scaffold in a Wnt-regulated protein complex that determines the availability of ß-catenin. Another common feature of solid tumors is the presence of hypoxia as indicated by the up-regulation of hypoxia-inducible factors (HIFs) such as HIF-1α. Here, we demonstrate a novel link between APC and hypoxia and show that APC and HIF-1α antagonize each other. Hypoxia results in reduced levels of APC mRNA and protein via a HIF-1α-dependent mechanism. HIF-1α represses the APC gene via a functional hypoxia-responsive element on the APC promoter. In contrast, APC-mediated repression of HIF-1α requires wild-type APC, low levels of ß-catenin, and nuclear factor-κB activity. These results reveal down-regulation of APC as a new mechanism that contributes to the survival advantage induced by hypoxia and also show that loss of APC mutations produces a survival advantage by mimicking hypoxic conditions.


Assuntos
Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Genes APC , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , NF-kappa B/metabolismo , Transcrição Gênica , beta Catenina/metabolismo
20.
EMBO J ; 29(17): 2966-78, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20657549

RESUMO

In response to replication stress, Claspin mediates the phosphorylation and activation of Chk1 by ATR. Claspin is not only necessary for the propagation of the DNA-damage signal, but its destruction by the ubiquitin-proteosome pathway is required to allow the cell to continue the cell cycle allowing checkpoint recovery. Here, we demonstrate that both the NF-kappaB family of transcription factors and their upstream kinase IKK can regulate Claspin levels by controlling its mRNA expression. Furthermore, we show that c-Rel directly controls Claspin gene transcription. Disruption of IKK and specific NF-kappaB members impairs ATR-mediated checkpoint function following DNA damage. Importantly, hyperactivation of IKK results in a failure to inactivate Chk1 and impairs the recovery from the DNA checkpoint. These results uncover a novel function for IKK and NF-kappaB modulating the DNA-damage checkpoint response, allowing the cell to integrate different signalling pathways with the DNA-damage response.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA , Técnicas de Inativação de Genes , Humanos , Quinase I-kappa B/genética , Mutagênese Insercional , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Transdução de Sinais , Transdução Genética
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