RESUMO
Promyelocytic leukemia (PML) protein is the organizer of nuclear matrix-associated nuclear bodies (NBs), and its conjugation to the small ubiquitin-like modifier (SUMO) is required for the formation of these structures. Several alternatively spliced PML transcripts from a single PML gene lead to the production of seven PML isoforms (PML isoform I [PMLI] to VII [PMLVII]), which all share a N-terminal region that includes the RBCC (RING, B boxes, and a α-helical coiled-coil) motif but differ in the C-terminal region. This diversity of PML isoforms determines the specific functions of each isoform. There is increasing evidence implicating PML in host antiviral defense and suggesting various strategies involving PML to counteract viral production. We reported that mouse embryonic fibroblasts derived from PML knockout mice are more sensitive than wild-type cells to infection with encephalomyocarditis virus (EMCV). Here, we show that stable expression of PMLIV or PMLIVa inhibited viral replication and protein synthesis, leading to a substantial reduction of EMCV multiplication. This protective effect required PMLIV SUMOylation and was not observed with other nuclear PML isoforms (I, II, III, V, and VI) or with the cytoplasmic PMLVII. We demonstrated that only PMLIV interacted with EMCV 3D polymerase (3Dpol) and sequestered it within PML NBs. The C-terminal region specific to PMLIV was required for both interaction with 3Dpol and the antiviral properties. Also, depletion of PMLIV by RNA interference significantly boosted EMCV production in interferon-treated cells. These findings indicate the mechanism by which PML confers resistance to EMCV. They also reveal a new pathway mediating the antiviral activity of interferon against EMCV.
Assuntos
RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Encefalomiocardite/enzimologia , Vírus da Encefalomiocardite/imunologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , Cricetinae , Humanos , Camundongos , Proteína da Leucemia Promielocítica , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/metabolismoRESUMO
Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.
Assuntos
Apoptose/fisiologia , Linfócitos B/imunologia , Fator de Transcrição STAT1/fisiologia , Western Blotting , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Type I interferon (IFN) enhances the transcription of the tumor suppressor gene p53. To elucidate the molecular mechanism mediating IFN-induced apoptosis, we analysed programmed cell death in response to type I (IFNalpha) or type II (IFNgamma) treatment in relation to p53 status. In two cell lines (MCF-7, SKNSH), IFNalpha, but not IFNgamma, enhanced apoptosis in a p53-dependent manner. Furthermore, only IFNalpha upregulated p53 as well as p53 target genes (Noxa, Mdm2 and CD95). The apoptotic response to IFNalpha decreased in the presence of ZB4, an anti-CD95 antibody, suggesting that CD95 is involved in this process. When p53 was inactivated by the E6 viral protein or the expression of a p53 mutant, IFNalpha-induced apoptosis and p53 target genes upregulation were abrogated. Altogether these results demonstrate that p53 plays a pivotal role in the IFNalpha-induced apoptotic response. IFNalpha-induced PML was unable to recruit p53 into nuclear bodies and its downregulation by siRNA did not alter CD95 expression. In contrast, IFNgamma-induced apoptosis is p53-independent. CD95 and IFN-regulatory factor 1 (IRF1) are directly upregulated by this cytokine. Apoptotic response to IFNgamma is decreased in the presence of ZB4 and strongly diminished by IRF1 siRNA, implicating both CD95 and IRF1 in IFNgamma-induced apoptotic response. Taken together, these results show that in two different cell lines, IFNalpha and IFNgamma, induce p53-dependent -independent apoptosis, respectively.
Assuntos
Apoptose/efeitos dos fármacos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Fator Regulador 1 de Interferon , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteína da Leucemia Promielocítica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2 , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor , Regulação para Cima/efeitos dos fármacos , Receptor fas/genética , Receptor fas/metabolismoRESUMO
Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms. The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, ...) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis. Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics). This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.
Assuntos
Apoptose/fisiologia , Leveduras/citologia , Leveduras/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
PML nuclear bodies (NBs) are dynamic intranuclear structures harboring numerous transiently or permanently localized proteins. PML, the NBs' organizer, is directly induced by interferon, and its expression is critical for antiviral host defense. We describe herein the molecular events following poliovirus infection that lead to PML-dependent p53 activation and protection against virus infection. Poliovirus infection induces PML phosphorylation through the extracellular signal-regulated kinase pathway, increases PML SUMOylation, and induces its transfer from the nucleoplasm to the nuclear matrix. These events result in the recruitment of p53 to PML NBs, p53 phosphorylation on Ser15, and activation of p53 target genes leading to the induction of apoptosis. Moreover, the knock-down of p53 by small interfering RNA results in higher poliovirus replication, suggesting that p53 participates in antiviral defense. This effect, which requires the presence of PML, is transient since poliovirus targets p53 by inducing its degradation in a proteasome- and MDM2-dependent manner. Our results provide evidence of how poliovirus counteracts p53 antiviral activity by regulating PML and NBs, thus leading to p53 degradation.