RESUMO
Liu and Mertz (Genes Dev. 9:1766-1780, 1995) previously identified a 119-nt pre-mRNA processing enhancer (PPE) element within the herpes simplex virus type 1 thymidine kinase gene that enables intron-independent gene expression in higher eukaryotes by binding heterogeneous nuclear ribonucleoprotein L (hnRNP L). Here, we identify a 49-nt subelement within this PPE that enhanced stability, polyadenylation, and cytoplasmic accumulation of transcripts synthesized in CV-1 cells from an intronless variant of the human beta-globin gene when present in two or more tandem copies. This 2xTK49 PPE also enhanced (i) the efficiency of polyadenylation of intronless beta-globin RNA in a cell-free polyadenylation system and (ii) the kinetics of nucleocytoplasmic export of an intronless variant of adenovirus major late leader region RNA in Xenopus oocytes. This 2xTK49 PPE bound only hnRNP L. Analysis of 2xTK49 PPE mutants showed a strong positive correlation existed between binding hnRNP L and enhancement of intronless beta-globin gene expression. hnRNP L was found to associate with both the mRNA export factor TAP and the exon-exon junction complex protein Aly/REF. Thus, we conclude that hnRNP L plays roles in enhancing stability, polyadenylation, and nucleocytoplasmic export; it does so, at least in part, by directly recruiting to intronless PPE-containing RNAs cofactors normally recruited to intron-containing RNAs.
Assuntos
Elementos Facilitadores Genéticos/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Poliadenilação/fisiologia , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Animais , Transporte Biológico/genética , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Globinas/genética , Humanos , Ligação Proteica/genética , Fatores de TempoRESUMO
Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-ß unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteassoma , Proteólise/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Ubiquitina/metabolismo , Neoplasias do Colo do Útero/patologia , Antineoplásicos/química , Biocatálise/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cloroquina/farmacologia , Ciclina D1/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Papillomaviridae/efeitos dos fármacos , Papillomaviridae/genética , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos , Neoplasias do Colo do Útero/virologiaRESUMO
Endoplasmic reticulum-associated degradation (ERAD) mediates the turnover of short-lived and misfolded proteins in the ER membrane or lumen. In spite of its important role, only subtle growth phenotypes have been associated with defects in ERAD. We have discovered that the ERAD proteins Ubc7 (Qri8), Cue1, and Doa10 (Ssm4) are required for growth of yeast that express high levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). Interestingly, the observed growth defect was exacerbated at low temperatures, producing an HMGR-dependent cold sensitivity. Yeast strains lacking UBC7, CUE1, or DOA10 also assembled aberrant karmellae (ordered arrays of membranes surrounding the nucleus that assemble when HMGR is expressed at high levels). However, rather than reflecting the accumulation of abnormal karmellae, the cold sensitivity of these ERAD mutants was due to increased HMGR catalytic activity. Mutations that compromise proteasomal function also resulted in cold-sensitive growth of yeast with elevated HMGR, suggesting that improper degradation of ERAD targets might be responsible for the observed cold-sensitive phenotype. However, the essential ERAD targets were not the yeast HMGR enzymes themselves. The sterol metabolite profile of ubc7Delta cells was altered relative to that of wild-type cells. Since sterol levels are known to regulate membrane fluidity, the viability of ERAD mutants expressing normal levels of HMGR was examined at low temperatures. Cells lacking UBC7, CUE1, or DOA10 were cold sensitive, suggesting that these ERAD proteins have a role in cold adaptation, perhaps through effects on sterol biosynthesis.
Assuntos
Aclimatação/fisiologia , Proteínas de Transporte/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Esteróis/biossíntese , Enzimas de Conjugação de Ubiquitina/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas de Transporte/genética , Temperatura Baixa , Deleção de Genes , Proteínas de Membrana/genética , Fosfoproteínas Fosfatases/genética , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esteróis/análise , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Regulação para CimaRESUMO
One of the earliest functional responses of T lymphocytes to extracellular signals that activate the Ag-specific CD3/TCR complex is a rapid, but reversible, increase in the functional activity of integrin adhesion receptors. Previous studies have implicated the tyrosine kinase zeta-associated protein of 70 kDa (ZAP-70) and the lipid kinase phosphatidylinositol 3-kinase, in the activation of beta(1) integrins by the CD3/TCR complex. In this report, we use human ZAP-70-deficient Jurkat T cells to demonstrate that the kinase activity of ZAP-70 is required for CD3/TCR-mediated increases in beta(1) integrin-mediated adhesion and activation of phosphatidylinositol 3-kinase. A tyrosine to phenylalanine substitution at position 315 in the interdomain B of ZAP-70 inhibits these responses, whereas a similar substitution at position 292 enhances these downstream signals. These mutations in the ZAP-70 interdomain B region also specifically affect CD3/TCR-mediated tyrosine phosphorylation of residues 171 and 191 in the cytoplasmic domain of the linker for activation of T cells (LAT) adapter protein. CD3/TCR signaling to beta(1) integrins is defective in LAT-deficient Jurkat T cells, and can be restored with expression of wild-type LAT. Mutant LAT constructs with tyrosine to phenylalanine substitutions at position 171 and/or position 191 do not restore CD3/TCR-mediated activation of beta(1) integrins in LAT-deficient T cells. Thus, these studies demonstrate that the interdomain B region of ZAP-70 regulates beta(1) integrin activation by the CD3/TCR via control of tyrosine phosphorylation of tyrosine residues 171 and 191 in the LAT cytoplasmic domain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Proteínas de Transporte/fisiologia , Integrina beta1/metabolismo , Proteínas de Membrana/fisiologia , Fosfoproteínas/fisiologia , Proteínas Tirosina Quinases/fisiologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/fisiologia , Linfócitos T/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Adesão Celular/genética , Adesão Celular/imunologia , Células Clonais , Citoplasma/genética , Citoplasma/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Fibronectinas/metabolismo , Humanos , Integrina beta1/fisiologia , Células Jurkat , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/antagonistas & inibidores , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Linfócitos T/metabolismo , Fosfolipases Tipo C/fisiologia , Tirosina/metabolismo , Regulação para Cima/imunologia , Proteína-Tirosina Quinase ZAP-70RESUMO
Ab stimulation of the TCR rapidly enhances the functional activity of the LFA-1 integrin. Although TCR-mediated changes in LFA-1 activity are thought to promote T cell-APC interactions, the Ag specificity and sensitivity of TCR-mediated triggering of LFA-1 is not clear. We demonstrate that peptide/MHC (pMHC) tetramers rapidly enhance LFA-1-dependent adhesion of OT-I TCR transgenic CD8(+) T cells to purified ICAM-1. Inhibition of src family tyrosine kinase or PI3K activity blocked pMHC tetramer- and anti-CD3-stimulated adhesion. These effects are highly specific because partial agonist and antagonist pMHC tetramers are unable to stimulate OT-I T cell adhesion to ICAM-1. The Ag thresholds required for T cell adhesion to ICAM-1 resemble those of early T cell activation events, because optimal LFA-1 activation occurs at tetramer concentrations that fail to induce maximal T cell proliferation. Thus, TCR signaling to LFA-1 is highly Ag specific and sensitive to low concentrations of Ag.