Adenovirus E3-6.7K Maintains Calcium Homeostasis and Prevents Apoptosis and Arachidonic Acid Release

E3-6.7K is a small and hydrophobic membrane glycoprotein encoded by the E3 region of subgroup C adenovirus. Recently, E3-6.7K has been shown to be required for the downregulation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors by the adenovirus E3/10.4K and E3/14.5K complex of proteins. We demonstrate here that E3-6.7K has additional protective roles, independent of other virus proteins. In transfected Jurkat T-cell lymphoma cells, E3-6.7K was found to maintain endoplasmic reticulum-Ca(2+) homeostasis and inhibit the induction of apoptosis by thapsigargin. The presence of E3-6.7K also lead to a reduction in the TNF-induced release of arachidonic acid from transfected U937 human histiocytic lymphoma cells. In addition, E3-6.7K protected cells against apoptosis induced through Fas, TNF receptor, and TRAIL receptors. Therefore, E3-6.7K confers a wide range of protective effects against both Ca(2+) flux-induced and death receptor-mediated apoptosis.     

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.     

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis.

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus form a complex and function together to down-regulate the epidermal growth factor receptor.

In adenovirus-infected cells, the epidermal growth factor receptor (EGF-R) is internalized from the cell surface via endosomes and is degraded, and the E3 10,400-dalton protein (10.4K protein) is required for this effect (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now report that both the E3 10.4K and E3 14.5K proteins are required for this down-regulation of EGF-R in adenovirus-infected cells. Down-regulation of cell surface EGF-R was demonstrated by results from several methods, namely the absence of EGF-R autophosphorylation in an immune complex kinase assay, the inability to iodinate EGF-R on the cell surface, the formation of endosomes containing EGF-R as detected by immunofluorescence, and the degradation of the metabolically [35S]Met-labeled fully processed 170K species of EGF-R. No effect on the initial synthesis of EGF-R was observed. This down-regulation was ascribed to the 10.4K and 14.5K proteins through the analysis of cells infected with rec700 (wild-type), dl748 (10.4K-, 14.5K+), or dl764 (10.4K+, 14.5K-) or coinfected with dl748 plus dl764. Further evidence that the 10.4K and 14.5K proteins function in concert was obtained by demonstrating that the 10.4K protein was coimmunoprecipitated with the 14.5K protein by using three different antisera to the 14.5K protein, strongly implying that the 10.4K and 14.5K proteins exist as a complex. Together, these results indicate that the 10.4K and 14.5K proteins function as a complex to stimulate endosome-mediated internalization and degradation of EGF-R in adenovirus-infected cells.     

The adenovirus E3 14.5-kilodalton protein, which is required for down-regulation of the epidermal growth factor receptor and prevention of tumor necrosis factor cytolysis, is an integral membrane protein oriented with its C terminus in the cytoplasm.

We previously reported that the adenovirus type 5 E3 14.5-kilodalton protein (14.5K) forms a complex with E3 10.4K and that both proteins are required to down-regulate the epidermal growth factor receptor in adenovirus-infected human cells. Both proteins are also required to prevent cytolysis by tumor necrosis factor of most mouse cell lines infected by adenovirus mutants that lack E3 14.7K. The E3 14.5K amino acid sequence suggests that 14.5K is an integral membrane protein with an N-terminal signal sequence for membrane insertion. Here we show that 14.5K was found exclusively in cytoplasmic membrane fractions. Radiochemical sequencing of 14.5K indicated that the N-terminal signal sequence is cleaved predominantly between Cys-18 and Ser-19. With a mutant that does not express 10.4K, cleavage occurs predominantly between Phe-17 and Cys-18, indicating that the presence or absence of 10.4K affects the signal cleavage site. 14.5K was extracted into the detergent phase with Triton X-114, it remained associated with membranes after extraction with Na2CO3 at pH 11.5, and it was partially protected by membranes from proteinase K digestion; these observations indicate that 14.5K is an integral membrane protein. Proteinase K digestion followed by immunoprecipitation with antipeptide antisera directed against the N or C terminus of mature 14.5K indicated that 14.5K is oriented in the membrane with its N terminus in the lumen and its C terminus in the cytoplasm. Thus, 14.5K is a type I bitopic membrane protein. Previous studies indicated that 10.4K is also an integral membrane protein oriented with its C terminus in the cytoplasm. Altogether, these findings suggest that cytoplasmic membranes are the site of action when 10.4K and 14.5K down-regulate the epidermal growth factor receptor and prevent tumor necrosis factor cytolysis.     

The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.     

Tumor-derived mutations in the TRAIL receptor DR5 inhibit TRAIL signaling through the DR4 receptor by competing for ligand binding

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a cytokine that preferentially induces apoptosis in tumor cells compared with normal cells through two receptors (DR4 and DR5). Somatic mutations in these receptors have been found in different kinds of cancer; however, it is poorly understood how the mutations affect signaling. We found that point mutations (L334F, E326K, E338K, and K386N) that were identified in human tumors result in the DR5 receptor losing its ability to form a functional death-inducing signaling complex and induce apoptosis. The mutant receptors also have a "dominant negative" effect whereby they inhibit the ability of TRAIL to induce apoptosis through functional DR4 receptors. This dominant negative mechanism is achieved through competition for TRAIL binding as shown by experiments where the ability of the mutant DR5 receptor to bind with the ligand was abolished, thus restoring TRAIL signaling through DR4. The inhibitory effect on signaling through the wild-type DR4 protein can be overcome if the inhibitory mechanism is bypassed by using a DR4-agonistic antibody that is not subject to this competition. This study provides a molecular basis for the use of specific therapeutic agonists of TRAIL receptors in people whose tumors harbor somatic DR5 mutations.     

The human papillomavirus type 16 E5 protein impairs TRAIL- and FasL-mediated apoptosis in HaCaT cells by different mechanisms

The effect of the human papillomavirus type 16 (HPV-16) E5 protein on apoptosis was investigated by using the polyclonal HaCaT-cell lines stably transfected either with E5 (HaCaT/E5) or the empty vector (HaCaT/pMSG) as reference. Apoptosis was triggered either by Fas ligand (FasL) or by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and was monitored by detection of cleavage of procaspase-8 and procaspase-3, as well as their substrate poly(ADP-ribose) polymerase (PARP). In contrast to the HaCaT/pMSG control cells we found that apoptosis induced by either of the two ligands is strongly suppressed in the E5-expressing keratinocytes. Fas expression is reduced by about a factor of two in HaCaT/E5 cells, which could be part of the mechanisms that protect the cells from FasL-induced apoptosis. For the TRAIL receptors, no such downregulation was observed. Here, E5 impairs the formation of the death-inducing signaling complex triggered by TRAIL. Apparently, E5 employs different mechanisms to inhibit death receptor signaling. This effect is not restricted to HaCaT/E5 cells since we found that the mouse fibroblast cell line A31-E5 is protected from TRAIL-induced apoptosis, as well but not the E5-lacking control cells A31-Neo. However, no such protection was observed upon FasL-induced apoptosis. Presumably, some of the antiapoptotic mechanisms employed by E5 of the human pathogenic HPV-16 are cell type specific. We propose that inhibition of ligand-mediated apoptosis in human keratinocytes is a primary function of the HPV-16 E5 protein needed to prevent apoptosis at early stages of viral infection.     

Down-regulation of DcR2 sensitizes androgen-dependent prostate cancer LNCaP cells to TRAIL-induced apoptosis

Background

Dysregulation of many apoptotic related genes and androgens are critical in the development, progression, and treatment of prostate cancer. The differential sensitivity of tumour cells to TRAIL-induced apoptosis can be mediated by the modulation of surface TRAIL receptor expression related to androgen concentration. Our previous results led to the hypothesis that downregulation of TRAIL-decoy receptor DcR2 expression following androgen deprivation would leave hormone sensitive normal prostate cells vulnerable to the cell death signal generated by TRAIL via its pro-apoptotic receptors. We tested this hypothesis under pathological conditions by exploring the regulation of TRAIL-induced apoptosis related to their death and decoy receptor expression, as also to hormonal concentrations in androgen-sensitive human prostate cancer, LNCaP, cells.

Results

In contrast to androgen-insensitive PC3 cells, decoy (DcR2) and death (DR5) receptor protein expression was correlated with hormone concentrations and TRAIL-induced apoptosis in LNCaP cells. Silencing of androgen-sensitive DcR2 protein expression by siRNA led to a significant increase in TRAIL-mediated apoptosis related to androgen concentration in LNCaP cells.

Conclusions

The data support the hypothesis that hormone modulation of DcR2 expression regulates TRAIL-induced apoptosis in LNCaP cells, giving insight into cell death induction in apoptosis-resistant hormone-sensitive tumour cells from prostate cancer. TRAIL action and DcR2 expression modulation are potentially of clinical value in advanced tumour treatment.     

The 10,400- and 14,500-dalton proteins encoded by region E3 of adenovirus function together to protect many but not all mouse cell lines against lysis by tumor necrosis factor.

We have reported that the E3 14,700-dalton protein (E3 14.7K protein) protects adenovirus-infected mouse C3HA fibroblasts against lysis by tumor necrosis factor (TNF) (L. R. Gooding, L. W. Elmore, A. E. Tollefson, H. A. Brady, and W. S. M. Wold, Cell 53:341-346, 1988). We have also observed that the E1B 19K protein protects adenovirus-infected human but not mouse cells against TNF lysis (L. R. Gooding, L. Aquino, P. J. Duerksen-Hughes, D. Day, T. M. Horton, S. Yei, and W. S. M. Wold, J. Virol. 65:3083-3094, 1991). We now report that, in the absence of E3 14.7K, the E3 10.4K and E3 14.5K proteins are both required to protect C127 as well as several other mouse cell lines against TNF lysis. The 14.7K protein can also protect these cells from TNF in the absence of the 10.4K and 14.5K proteins. This protection by the 10.4K and 14.5K proteins was not observed in the C3HA cell line. These conclusions are based on 51Cr release assays of cells infected with virus E3 mutants that express the 14.7K protein alone, that express both the 10.4K and 14.5K proteins, and that delete the 14.7K in combination with either the 10.4K or 14.5K protein. The 10.4K protein was efficiently coimmunoprecipitated together with the 14.5K protein by using an antiserum to the 14.5K protein, suggesting that the 10.4K and 14.5K proteins exist as a complex in the infected mouse cells and consistent with the notion that they function in concert. Considering that three sets of proteins (E3 14.7K, E1B 19K, and E3 10.4K/14.5K proteins) exist in adenovirus to prevent TNF cytolysis of different cell types, it would appear that TNF is a major antiadenovirus defense of the host.     

The tumor suppressor RASSF1A and MAP-1 link death receptor signaling to Bax conformational change and cell death

Tumor cells typically resist programmed cell death (apoptosis) induced by death receptors. Activated death receptors evoke Bax conformational change, cytochrome c release, and cell death. We report that the tumor suppressor gene RASSF1A is required for death receptor-induced Bax conformational change and apoptosis. TNFalpha or TRAIL stimulation induced recruitment of RASSF1A and MAP-1 to receptor complexes and promoted complex formation between RASSF1A and the BH3-like protein MAP-1. Normally, MAP-1 is inhibited by an intramolecular interaction. RASSF1A/MAP-1 binding relieved this inhibitory interaction, resulting in MAP-1 association with Bax. Deletion of the RASSF1A gene or short hairpin silencing of either RASSF1A or MAP-1 expression blocked MAP-1/Bax interaction, Bax conformational change and mitochondrial membrane insertion, cytochrome c release, and apoptosis in response to death receptors. Our findings identify RASSF1A and MAP-1 as important components between death receptors and the apoptotic machinery and reveal a potential link between tumor suppression and death receptor signaling.     

Enhanced proliferation and increased IFN-gamma production in T cells by signal transduced through TNF-related apoptosis-inducing ligand

TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.     

Temperature-sensitive differential affinity of TRAIL for its receptors. DR5 is the highest affinity receptor

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 "decoy" receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the "decoy" receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 degrees C, their rank-ordered affinities are substantially different at 37 degrees C, with DR5 having the highest affinity (K(D) 相似文献