RESUMEN
The Notch1 receptor is presented at the cell membrane as a heterodimer after constitutive processing by a furin-like convertase. Ligand binding induces the proteolytic release of Notch intracellular domain by a gamma-secretase-like activity. This domain translocates to the nucleus and interacts with the DNA-binding protein CSL, resulting in transcriptional activation of target genes. Here we show that an additional processing event occurs in the extracellular part of the receptor, preceding cleavage by the gamma-secretase-like activity. Purification of the activity accounting for this cleavage in vitro shows that it is due to TACE (TNFalpha-converting enzyme), a member of the ADAM (a disintegrin and metalloprotease domain) family of metalloproteases. Furthermore, experiments carried out on TACE-/- bone marrow-derived monocytic precursor cells suggest that this metalloprotease plays a prominent role in the activation of the Notch pathway.
Asunto(s)
Desintegrinas/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas , Receptores de Superficie Celular/metabolismo , Factores de Transcripción , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Proteínas de Unión al Calcio , Diferenciación Celular , Péptidos y Proteínas de Señalización Intercelular , Ratones , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/efectos de los fármacos , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Receptor Notch1 , Proteínas Serrate-Jagged , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The extracellular domains of many proteins, including growth factors, cytokines, receptors, and adhesion molecules, are proteolytically released from cells, a process termed "shedding." Tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17) is a metalloprotease-disintegrin that sheds tumor necrosis factor-alpha and other proteins. To study the regulation of TACE-mediated shedding, we examined the effects of stimulation of cells on TACE localization and expression. Immunofluorescence microscopy revealed a punctate distribution of TACE on the surface of untreated cells, and stimulation of monocytic cells with lipopolysaccharide did not affect TACE staining. Phorbol 12-myristate 13-acetate (PMA), a potent inducer of shedding, decreased cell-surface staining for TACE. Surface biotinylation experiments confirmed and extended this observation; PMA decreased the half-life of surface-biotinylated TACE without increasing the turnover of total cell-surface proteins. Soluble fragments of TACE were not detected in the medium of cells that had down-regulated TACE, and TACE was not down-regulated when endocytosis was inhibited. Antibody uptake experiments suggested that cell-surface TACE was internalized in response to PMA. Surprisingly, a metalloprotease inhibitor prevented the PMA-induced turnover of TACE. Thus, PMA activates shedding and causes the down-regulation of a major "sheddase," suggesting that induced shedding may be regulated by a mechanism that decreases the amount of active TACE on the cell surface.
Asunto(s)
Regulación hacia Abajo , Metaloendopeptidasas/metabolismo , Proteínas ADAM , Proteína ADAM17 , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Endocitosis , Retroalimentación , Humanos , Lipopolisacáridos/farmacología , Metaloendopeptidasas/inmunología , Microscopía Fluorescente , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/enzimología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Poliovirus protein 3A, only 87 amino acids in length, is a potent inhibitor of protein secretion in mammalian cells, blocking anterograde protein traffic from the endoplasmic reticulum (ER) to the Golgi complex. The function of viral protein 3A in blocking protein secretion is extremely sensitive to mutations near the N terminus of the protein. Deletion of the first 10 amino acids or insertion of a single amino acid between amino acids 15 and 16, a mutation that causes a cold-sensitive defect in poliovirus RNA replication, abrogates the inhibition of protein secretion although wild-type amounts of the mutant proteins are expressed. Immunofluorescence light microscopy and immunoelectron microscopy demonstrate that 3A protein, expressed in the absence of other viral proteins, colocalizes with membranes derived from the ER. The precise topology of 3A with respect to ER membranes is not known, but it is likely to be associated with the cytosolic surface of the ER. Although the glycosylation of 3A in translation extracts has been reported, we show that tunicamycin, under conditions in which glycosylation of cellular proteins is inhibited, has no effect on poliovirus growth. Therefore, glycosylation of 3A plays no functional role in the viral replicative cycle. Electron microscopy reveals that the ER dilates dramatically in the presence of 3A protein. The absence of accumulated vesicles and the swelling of the ER-derived membranes argues that ER-to-Golgi traffic is inhibited at the step of vesicle formation or budding from the ER.
Asunto(s)
Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Poliovirus/fisiología , Proteínas del Núcleo Viral/fisiología , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células COS , Glicosilación , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Poliovirus/efectos de los fármacos , Proteínas/metabolismo , Relación Estructura-Actividad , Tunicamicina/farmacología , Proteínas del Núcleo Viral/genética , Replicación Viral/efectos de los fármacos , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Expression of poliovirus protein 2B in mammalian cells inhibits protein secretion and increases the susceptibility of the cells to hygromycin B, consistent with the increase in plasma membrane permeability seen during poliovirus infection (J. R. Doedens and K. Kirkegaard, EMBO J. 14, 894-907, 1995). We report here that expression of protein 2B of the closely related coxsackie B3 virus (CBV3) leads to the same biochemical alterations. Analysis of several mutant CBV3 2B proteins that contain mutations in a predicted cationic amphipathic alpha-helix (F. J. M. van Kuppeveld, J. M. D. Galama, J. Zoll, P. J. J. C. van den Hurk, and W. J. G. Melchers, J. Virol. 70, 3876-3886, 1996) demonstrated that the integrity of this domain is crucial for both biochemical functions of 2B. Mutations in a second hydrophobic domain (F. J. M. van Kuppeveld, J. M. D. Galama, J. Zoll, and W. J. G. Melchers, J. Virol. 69, 7782-7790, 1995), on the other hand, are more disruptive to the ability of CBV3 2B to inhibit protein secretion than to increase membrane permeability. Therefore, inhibition of protein secretion is not merely a consequence of the membrane changes that increase uptake of hygromycin B. The existence of mutations that interfere with virus growth but do not impair the ability of 2B to inhibit protein secretion or increase membrane permeability argues for additional functions of protein 2B.
Asunto(s)
Enterovirus Humano B/fisiología , Proteínas Virales/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Células COS , Farmacorresistencia Microbiana , Enterovirus Humano B/efectos de los fármacos , Enterovirus Humano B/genética , Higromicina B/farmacología , Datos de Secuencia Molecular , Mutación , Biosíntesis de Proteínas/efectos de los fármacos , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/genética , alfa 1-Antitripsina/metabolismoRESUMEN
Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.