A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE.

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.

Stimulation-induced down-regulation of tumor necrosis factor-alpha converting enzyme.

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.

Inhibition of endoplasmic reticulum-to-Golgi traffic by poliovirus protein 3A: genetic and ultrastructural analysis.

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.

Structure-function analysis of coxsackie B3 virus protein 2B.

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.

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

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.

Secretory pathway function, but not cytoskeletal integrity, is required in poliovirus infection.

We examined the importance of two interactions between poliovirus and its host cell: the putative association between viral proteins and a rearranged intermediate filament (IF) network and the apparent requirement for functional vesicle budding machinery within the host-cell secretory pathway. Poliovirus capsid proteins appeared to associate with reorganized IF proteins during infection. Treatment of cells with cytochalasin D and nocodazole in combination disrupted normal cytoskeletal organization and prevented the poliovirus-induced redistribution of IF proteins to a juxtanuclear location. However, this treatment had no effect on viral yields from single-cycle infections, indicating that neither cytoskeletal integrity nor a specific poliovirus-induced rearrangement of IF proteins is required in the poliovirus life cycle. In contrast, we report that the inhibition of poliovirus replication by brefeldin A (BFA), an inhibitor of secretory membrane traffic, is specific to the host cell. Polioviral yields were not affected by BFA in two BFA-resistant cell lines, demonstrating that BFA targets a host protein or process required by poliovirus. No BFA-resistant virus was detected in these experiments, further supporting the hypothesis that poliovirus replication requires secretory pathway function, perhaps for the generation of vesicles on which viral RNA replication complexes are assembled.