RESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease caused by accumulations of Aß peptides. Production and fibrillation of Aß are downregulated by BRI2 and BRI3, which are physiological inhibitors of amyloid precursor protein (APP) processing and Aß oligomerization. Here, we identify nuclear receptor binding protein 1 (NRBP1) as a substrate receptor of a Cullin-RING ubiquitin ligase (CRL) that targets BRI2 and BRI3 for degradation. Moreover, we demonstrate that (1) dimerized NRBP1 assembles into a functional Cul2- and Cul4A-containing heterodimeric CRL through its BC-box and an overlapping cryptic H-box, (2) both Cul2 and Cul4A contribute to NRBP1 CRL function, and (3) formation of the NRBP1 heterodimeric CRL is strongly enhanced by chaperone-like function of TSC22D3 and TSC22D4. NRBP1 knockdown in neuronal cells results in an increase in the abundance of BRI2 and BRI3 and significantly reduces Aß production. Thus, disrupting interactions between NRBP1 and its substrates BRI2 and BRI3 may provide a useful therapeutic strategy for AD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Peptídeos beta-Amiloides/biossíntese , Proteínas Culina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos ICR , Ligação Proteica , Multimerização Proteica , Receptores Citoplasmáticos e Nucleares/química , Especificidade por Substrato , Fatores de Transcrição/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/químicaRESUMO
Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.
Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alfa-Amanitina/metabolismo , Linhagem Celular , Proteínas Culina/metabolismo , Reparo do DNA , Elonguina , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Mutação , Plasmídeos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Fatores de Transcrição/genéticaRESUMO
Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription.
Assuntos
Mutagênicos/farmacologia , Estresse Oxidativo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Elonguina , Transferência Ressonante de Energia de Fluorescência , Técnica Indireta de Fluorescência para Anticorpo , Células HEK293 , Humanos , RNA Mensageiro/genética , Tretinoína/farmacologia , Raios UltravioletaRESUMO
Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.
Assuntos
RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Iniciação da Transcrição Genética/fisiologia , Fator 3 Ativador da Transcrição/biossíntese , Fator 3 Ativador da Transcrição/genética , Animais , Diclororribofuranosilbenzimidazol/farmacologia , Elonguina , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Camundongos , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/genética , Ratos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Iniciação da Transcrição Genética/efeitos dos fármacosRESUMO
Elongin A increases the rate of RNA polymerase II (pol II) transcript elongation by suppressing transient pausing by the enzyme. Elongin A also acts as a component of a cullin-RING ligase that can target stalled pol II for ubiquitylation and proteasome-dependent degradation. It is not known whether these activities of Elongin A are functionally interdependent in vivo. Here, we demonstrate that Elongin A-deficient (Elongin A(-/-)) embryos exhibit abnormalities in the formation of both cranial and spinal nerves and that Elongin A(-/-) embryonic stem cells (ESCs) show a markedly decreased capacity to differentiate into neurons. Moreover, we identify Elongin A mutations that selectively inactivate one or the other of the aforementioned activities and show that mutants that retain the elongation stimulatory, but not pol II ubiquitylation, activity of Elongin A rescue neuronal differentiation and support retinoic acid-induced upregulation of a subset of neurogenesis-related genes in Elongin A(-/-) ESCs.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Tretinoína/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Elonguina , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/citologia , RNA Polimerase II/metabolismo , Alinhamento de Sequência , Elongação da Transcrição Genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Cyclin-dependent kinase inhibitor p21Cip1 plays a crucial role in regulating cell cycle arrest and differentiation. It is known that p21Cip1 increases during terminal differentiation of cardiomyocytes, but its expression control and biological roles are not fully understood. Here, we show that the p21Cip1 protein is stabilized in cardiomyocytes after mitogenic stimulation, due to its increased CDK2 binding and inhibition of ubiquitylation. The APC/CCdc20 complex is shown to be an E3 ligase mediating ubiquitylation of p21Cip1 at the N terminus. CDK2, but not CDC2, suppressed the interaction of p21Cip1 with Cdc20, thereby leading to inhibition of anaphase-promoting complex/cyclosome and its activator Cdc20 (APC/CCdc20)-mediated p21Cip1 ubiquitylation. It was further demonstrated that p21Cip1 accumulation caused G2 arrest of cardiomyocytes that were forced to re-enter the cell cycle. Taken together, these data show that the stability of the p21Cip1 protein is actively regulated in terminally differentiated cardiomyocytes and plays a role in inhibiting their uncontrolled cell cycle progression. Our study provides a novel insight on the control of p21Cip1 by ubiquitin-mediated degradation and its implication in cell cycle arrest in terminal differentiation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação Enzimológica da Expressão Gênica , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas Cdc20 , Ciclo Celular , Diferenciação Celular , Humanos , Modelos Biológicos , Miócitos Cardíacos/citologia , Estrutura Terciária de Proteína , Processamento Pós-Transcricional do RNA , Ratos , Ratos Sprague-Dawley , Ubiquitina/químicaRESUMO
The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A-Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
Assuntos
Dano ao DNA , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas Culina/metabolismo , Elonguina , Humanos , Ligação Proteica , Ubiquitinação , Raios UltravioletaRESUMO
The tumor suppressor von Hippel-Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM). Elongin B-cerulean or cerulean-elongin B was coexpressed with elongin C-citrine or citrine-elongin C in CHO-K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B-cerulean and citrine-elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N- and C-terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.
Assuntos
Fatores de Transcrição/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Elonguina , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Conformação Proteica , Fatores de Tempo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Elongin A is the transcriptionally active subunit of the Elongin complex that strongly stimulates the rate of elongation by RNA polymerase II (pol II) by suppressing the transient pausing of the polymerase at many sites along the DNA template. We have recently shown that Elongin A-deficient mice are embryonic lethal, and mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos display not only increased apoptosis but also senescence-like phenotypes accompanied by the activation of p53. To further understand the function of Elongin A in vivo, we have carried out the structure-function analysis of Elongin A and identified sequences critical to its nuclear localization and direct interaction with pol II. Moreover, we have analyzed the replication fork movement in wild-type and Elongin A(-/-) MEFs, and shown the possibility that the genomic instability observed in Elongin A(-/-) MEFs might be caused by the replication fork collapse due to Elongin A deficiency.
Assuntos
Fatores de Transcrição/metabolismo , Animais , Apoptose , Células Cultivadas , Chlorocebus aethiops , Replicação do DNA , Elonguina , Feminino , Instabilidade Genômica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinais de Localização Nuclear , Ligação Proteica , RNA Polimerase II/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit, and two positive regulatory B and C subunits. Although the NH(2)-terminal approximately 120 amino acid region of Elongin A is dispensable for its transcriptional activity in vitro, it shares significant sequence similarity with the NH(2)-terminus of other class of transcription factors SII and CRSP70, suggesting that the NH(2)-terminus mediates interactions important for the regulation of transcription in vivo. To identify proteins that can bind to these conserved sequences, a human B cell cDNA library was screened using the NH(2)-terminus of Elongin A as bait in a yeast two-hybrid system. Here, we report on the cloning and characterization of a novel human exonuclease domain-containing protein, Elongin A-binding protein 1 (EloA-BP1). EloA-BP1 is composed of 1221 amino acids and its mRNA is ubiquitously expressed. Double immunofluorescence labeling in COS7 cells suggested that EloA-BP1 and Elongin A are colocalized to the cell nucleus. By using an in vitro binding assay, we show that EloA-BP1 is capable of binding not only the NH(2)-terminal approximately 120 amino acid region of Elongin A, but also that of SII. Although the purified EloA-BP1 had no detectable effect on the rate of transcription elongation in vitro, it may play some role in the regulation of elongation in vivo.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Exonucleases/química , Proteínas Nucleares , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Northern Blotting , Western Blotting , Células COS , DNA/metabolismo , DNA Complementar/metabolismo , Elonguina , Biblioteca Gênica , Glutationa Transferase/metabolismo , Humanos , Insetos , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA/metabolismo , RNA Polimerase II/química , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Fatores de Transcrição/metabolismo , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To investigate the function of Elongin A in vivo, the two alleles of the Elongin A gene have been disrupted by homologous recombination in murine embryonic stem (ES) cells. The Elongin A-deficient ES cells are viable, but show a slow growth phenotype because they undergo a delayed mitosis. The cDNA microarray and RNase protection assay using the wild-type and Elongin A-deficient ES cells indicate that the expression of only a small subset of genes is affected in the mutant cells. Taken together, our results suggest that Elongin A regulates transcription of a subset but not all of genes and reveal a linkage between Elongin A function and cell cycle progression.
Assuntos
Ciclo Celular/fisiologia , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Divisão Celular , Mapeamento Cromossômico , Clonagem Molecular , Ciclinas/genética , Elonguina , Vetores Genéticos , Homozigoto , Camundongos , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Células-Tronco/citologia , Células-Tronco/fisiologia , Fatores de Transcrição/deficiênciaRESUMO
The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II by suppressing the transient pausing of the polymerase at many sites along the DNA template. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, the latter binding stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. To further understand the role of Elongin A in transcriptional regulation by RNA polymerase II, we are attempting to identify Elongin A-related proteins. Here, we report on the molecular cloning, expression, and biochemical characterization of human Elongin A3, a novel transcription elongation factor that exhibits 49 and 81% identity to Elongin A and the recently identified Elongin A2, respectively. The mRNA of Elongin A3 is ubiquitously expressed, and the protein is localized to the nucleus of cells. Mechanistic studies have demonstrated that Elongin A3 possesses similar biochemical features to Elongin A2. Both stimulate the rate of transcription elongation by RNA polymerase II and are capable of forming a stable complex with Elongin BC. In contrast to Elongin A, however, their transcriptional activities are not activated by Elongin BC. Structure-function analyses using fusion proteins composed of Elongin A3 and Elongin A revealed that the COOH-terminal region of Elongin A is important for the activation by Elongin BC.