[Show abstract][Hide abstract] ABSTRACT: We recently provided evidence that the ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 (HSV-1 and -2) protect cells against tumor necrosis factor alpha- and Fas ligand-induced apoptosis by interacting with caspase 8. Double-stranded RNA (dsRNA) is a viral intermediate known to initiate innate antiviral responses. Poly(I · C), a synthetic analogue of viral dsRNA, rapidly triggers caspase 8 activation and apoptosis in HeLa cells. Here, we report that HeLa cells after HSV-1 and HSV-2 infection were quickly protected from apoptosis caused by either extracellular poly(I · C) combined with cycloheximide or transfected poly(I · C). Cells infected with the HSV-1 R1 deletion mutant ICP6Δ were killed by poly(I · C), indicating that HSV-1 R1 plays a key role in antiapoptotic responses to poly(I · C). Individually expressed HSV R1s counteracted caspase 8 activation by poly(I · C). In addition to their binding to caspase 8, HSV R1s also interacted constitutively with receptor-interacting protein 1 (RIP1) when expressed either individually or with other viral proteins during HSV infection. R1(1-834)-green fluorescent protein (GFP), an HSV-2 R1 deletion mutant protein devoid of antiapoptotic activity, did not interact with caspase 8 and RIP1, suggesting that these interactions are required for protection against poly(I · C). HSV-2 R1 inhibited the interaction between the Toll/interleukin-1 receptor domain-containing adaptor-inducing beta interferon (IFN-β) (TRIF) and RIP1, an interaction that is essential for apoptosis triggered by extracellular poly(I · C) plus cycloheximide or TRIF overexpression. TRIF silencing reduced poly(I · C)-triggered caspase 8 activation in mock- and ICP6Δ-infected cells, confirming that TRIF is involved in poly(I · C)-induced apoptosis. Thus, by interacting with caspase 8 and RIP1, HSV R1s impair the apoptotic host defense mechanism prompted by dsRNA.
Preview · Article · Jun 2011 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that herpes virus ribonucleotide reductase can be inhibited by a synthetic nonapeptide whose sequence is identical to the C-terminal of the small subunit of the enzyme. This peptide is able to interfere with normal subunit association that takes place through the C-terminal of the small subunit. In this report, we illustrate that inhibition of ribonucleotide reductases by peptides corresponding to the C-terminal of subunit R2 is also observed for the enzyme isolated from Escherichia coli, hamster, and human cells. The nonapeptide corresponding to the bacterial C-terminal sequence was found to inhibit E. coli enzyme with an IC50 of 400 μM, while this peptide had no effect on mammalian ribonucleotide reductase. A corresponding synthetic peptide derived from the C-terminal of the small subunit of the human enzyme inhibited both human and hamster ribonucleotide reductases with IC50 values of 160 and 120 μM, respectively. However, this peptide had no inhibitory activity against the bacterial enzyme. Equivalent peptides derived from herpes virus ribonucleotide reductase had no effect on either the bacterial or mammalian enzymes. Thus, subunit association at the C-terminal of the small subunit appears to be a common feature of ribonucleotide reductases. In addition, the inhibitory phenomenon observed with peptides corresponding to the C-terminal appears not only to be universal, but also specific to the primary sequence of the enzyme.Key words: ribonucleotide reductase, inhibition, human, bacteria.
No preview · Article · Jan 2011 · Biochemistry and Cell Biology
[Show abstract][Hide abstract] ABSTRACT: We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein-Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.
[Show abstract][Hide abstract] ABSTRACT: Chimeric VLPs made of papaya mosaic virus (PapMV) trigger a CTL response through antigenic presentation of epitopes on MHC class I. Here, a chimeric VLP composed of malva mosaic virus (MaMV) was shown to share similar properties. We demonstrated the capacity of both VLPs to enter human APCs. The chimeric constructions were cross-presented in CD40-activated B lymphocytes leading to in vitro expansion of antigen-specific T lymphocytes. We showed that high concentrations of chimeric MaMV induced cell death, suggesting that some modifications can trigger collateral effects in vitro. Results suggest that potexvirus VLPs are an attractive vaccine platform for inducing a CTL response.
[Show abstract][Hide abstract] ABSTRACT: A newly discovered virally encoded deubiquitinating enzyme (DUB) is strictly conserved across the Herpesviridae. Epstein-Barr virus (EBV) BPLF1 encodes a tegument protein (3,149 amino acids) that exhibits deubiquitinating (DUB) activity
that is lost upon mutation of the active-site cysteine. However, targets for the herpesviral DUBs have remained elusive. To
investigate a predicted interaction between EBV BPLF1 and EBV ribonucleotide reductase (RR), a functional clone of the first
246 N-terminal amino acids of BPLF1 (BPLF1 1-246) was constructed. Immunoprecipitation verified an interaction between the
small subunit of the viral RR2 and BPLF1 proteins. In addition, the large subunit (RR1) of the RR appeared to be ubiquitinated
both in vivo and in vitro; however, ubiquitinated forms of the small subunit, RR2, were not detected. Ubiquitination of RR1
requires the expression of both subunits of the RR complex. Furthermore, coexpression of RR1 and RR2 with BPLF1 1-246 abolishes
ubiquitination of RR1. EBV RR1, RR2, and BPLF1 1-246 colocalized to the cytoplasm in HEK 293T cells. Finally, expression of
enzymatically active BPLF1 1-246 decreased RR activity, whereas a nonfunctional active-site mutant (BPLF1 C61S) had no effect.
These results indicate that the EBV deubiquitinating enzyme interacts with, deubiquitinates, and influences the activity of
the EBV RR. This is the first verified protein target of the EBV deubiquitinating enzyme.
Full-text · Article · Mar 2009 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Expansions of a (GCN)10/polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) cause autosomal dominant oculopharyngeal muscular dystrophy (OPMD). In OPMD muscles, as in models, PABPN1 accumulates in intranuclear inclusions (INIs) whereas in other diseases caused by similar polyalanine expansions, the mutated proteins have been shown to abnormally accumulate in the cytoplasm. This study presents the impact on the subcellular localization of PABPN1 produced by large expansions or deletion of its polyalanine tract. Large tracts of more than 24 alanines result in the nuclear accumulation of PABPN1 in SFRS2-positive functional speckles and a significant decline in cell survival. These large expansions do not cause INIs formation nor do they lead to cytoplasmic accumulation. Deletion of the polyalanine tract induces the formation of aggregates that are located on either side and cross the nuclear membrane, highlighting the possible role of the N-terminal polyalanine tract in PABPN1 nucleo-cytoplasmic transport. We also show that even though five other proteins with polyalanine tracts tend to aggregate when over-expressed they do not co-aggregate with PABPN1 INIs. This study presents the first experimental evidence that there may be a relative loss of function in OPMD by decreasing the availability of PABPN1 through an INI-independent mechanism.
No preview · Article · Jun 2008 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: We previously showed that exogenous oleate protects human breast cancer cells against palmitate-induced apoptosis in part by increasing esterification of this free fatty acid (FFA) into triacylglycerol (TG). Here, we studied the mechanism whereby oleate protects these cells against apoptosis induced by serum withdrawal. The metabolism of FFA, TG, and glucose, in parallel with long-term cell survival in the absence of serum, was investigated in a panel of human breast cancer cell lines and in nontransformed MCF-10A cells after treatment with exogenous oleate. Short-term (3-24 h) exposure of MDA-MB-231 human breast cancer cells to exogenous oleate resulted in a dose-dependent long-term (10 day) serum-free survival that correlated with the accumulation of TG in lipid droplets and with upregulation of lipolysis. Both effects persisted for several days after oleate removal. Rapid TG lipolysis and FFA re-esterification, supported by high rates of glycolysis that provide the glycerol backbone for TG synthesis, are consistent with the presence of very active TG-FFA cycling in human breast cancer cells. Only the cancer cell lines capable of accumulating TG showed long-term serum-free survival after oleate treatment. The results suggest that upregulation of TG-FFA cycling induced by oleate may be involved in maintenance of human breast cancer cell survival.
No preview · Article · Jul 2007 · Biochemistry and Cell Biology
[Show abstract][Hide abstract] ABSTRACT: The R1 subunit (ICP10) of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR), which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 aa, protects cells against apoptosis. As the NH(2) domain on its own is not antiapoptotic, it has been postulated that both domains of R1 or part(s) of them could be necessary for this function. Here, N- and C-terminal deletions were introduced in HSV-2 R1 to map the domain(s) involved in its antiapoptotic potential. The results showed that, whereas most of the NH(2) domain including part of the recently described putative alpha-crystallin domain is dispensable for antiapoptotic activity, it is the integrity of the structured RR domain that is required for protection. As the alpha-crystallin domain appears to play an important role in protein folding and oligomerization, the N-terminal boundary of the antiapoptotic domain could not be defined precisely. In addition, this study provided evidence that overexpression of HSV-2 R2 at levels up to 30-fold more than HSV-2 R1 did not decrease protection from tumour necrosis factor alpha, indicating that the R1 surface where R2 binds is not involved in antiapoptotic activity. Importantly, this result suggests that the co-expression of both RR subunits during the lytic cycle should not affect protection from this cytokine.
Full-text · Article · Mar 2007 · Journal of General Virology
[Show abstract][Hide abstract] ABSTRACT: Oculopharyngeal muscular dystrophy (OPMD) is caused by expansion of a (GCN)10 to a (GCN)11-17 repeat coding for a polyalanine domain at the N-terminal part of poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by the presence of intranuclear inclusions (INIs) in skeletal muscle fibers of patients. The formation of GFP-b13AlaPABPN1 INIs and their fate through the cell cycle were followed by time-lapse imaging. Our observations demonstrated that the GFP-b13AlaPABPN1 INIs are dynamic structures that can disassemble during mitosis. However, their presence in cells occasionally led to apoptosis. The length of the polyalanine tail or the overexpression of PABPN1 did not significantly affect the percentage of soluble PABPN1 in vitro. Moreover, overexpression of either the wild type (wt) or mutant (mut) forms of PABPN1 slowed down the cell proliferation. The slowing down of proliferation together with the occasional occurrence of apoptosis could contribute in vivo to the late onset of this disease.
No preview · Article · Oct 2006 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.
No preview · Article · Jan 2006 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Apoptosis of endothelial cells (EC) is appreciated as a primary pathogenic event in systemic sclerosis. Yet, how apoptosis of EC leads to fibrosis remains to be determined. We report that apoptosis of EC triggers the release of novel fibrogenic mediators. Medium conditioned by apoptotic EC (SSC) was found to inhibit apoptosis of fibroblasts, whereas medium conditioned by EC in which apoptosis was blocked (with either pan-caspase inhibition or Bcl-x(L) overexpression) did not. PI3K was activated in fibroblasts exposed to SSC. This was associated with downstream repression of Bim-EL and long-term up-regulation of Bcl-x(L) protein levels. RNA interference for Bim-EL in fibroblasts blocked apoptosis. SSC also induced PI3K-dependent myofibroblast differentiation with expression of alpha-smooth muscle actin, formation of stress fibers, and production of collagen I. A C-terminal fragment of the domain V of perlecan was identified as one of the fibrogenic mediators present in SSC. A synthetic peptide containing an EGF motif present on the perlecan fragment and chondroitin 4-sulfate, a glycosaminoglycan anchored on the domain V of perlecan, induced PI3K-dependent resistance to apoptosis in fibroblasts and myofibroblast differentiation. Human fibroblasts derived from sclerodermic skin lesions were more sensitive to the antiapoptotic activities of the synthetic peptide and chondroitin 4-sulfate than fibroblasts derived from normal controls. Hence, we propose that a chronic increase in endothelial apoptosis and/or increased sensitivity of fibroblasts to mediators produced by apoptotic EC could form the basis of a fibrotic response characterized by sustained induction of an antiapoptotic phenotype in fibroblasts and persistent myofibroblast differentiation.
Preview · Article · Jun 2005 · The Journal of Immunology
[Show abstract][Hide abstract] ABSTRACT: Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disease caused by expanded (GCN)12-17 stretches encoding the N-terminal polyalanine domain of the poly(A) binding protein nuclear 1 (PABPN1). OPMD is characterized by intranuclear inclusions (INIs) in skeletal muscle fibers, which contain PABPN1, molecular chaperones, ubiquitin, proteasome subunits, and poly(A)-mRNA. We describe an adenoviral model of PABPN1 expression that produces INIs in most cells. Microarray analysis revealed that PABPN1 overexpression reproducibly changed the expression of 202 genes. Sixty percent of upregulated genes encode nuclear proteins, including many RNA and DNA binding proteins. Immunofluorescence microscopy revealed that all tested nuclear proteins encoded by eight upregulated genes colocalize with PABPN1 within the INIs: CUGBP1, SFRS3, FKBP1A, HMG2, HNRPA1, PRC1, S100P, and HSP70. In addition, CUGBP1, SFRS3, and FKBP1A were also found in OPMD muscle INIs. This study demonstrates that a large number of nuclear proteins are sequestered in OPMD INIs, which may compromise cellular function.
No preview · Article · May 2005 · Neurobiology of Disease
[Show abstract][Hide abstract] ABSTRACT: Evidence from epidemiological studies and animal models suggests a link between high levels of dietary fat intake and risk of breast cancer. In addition, obesity, in which circulating lipids are elevated, is associated with increased risk of various cancers. Relative to this point, we previously showed that oleate stimulates the proliferation of breast cancer cells and that phosphatidylinositol 3-kinase plays a role in this process. Nonetheless, questions remain regarding the precise mechanism(s) by which oleate promotes breast cancer cell growth. Pharmacological inhibitors of the GTP-binding proteins G(i)/G(o), phospholipase C, Src, and mitogenic-extracellular signal-regulated kinase 1/2 (MEK 1/2) decreased oleate-induced [3H]thymidine incorporation in the breast cancer cell line MDA-MB-231. In addition, oleate caused a rapid and transient rise in cytosolic Ca2+ and an increase in protein kinase B phosphorylation. Overexpressing in these cells the G protein-coupled receptor GPR40, a fatty acid receptor, amplified oleate-induced proliferation, whereas silencing the GPR40 gene using RNA interference decreased it. Overexpressing GPR40 in T47D and MCF-7 breast cancer cells that are poorly responsive to oleate allowed a robust proliferative action of oleate. The data indicate that the phospholipase C, MEK 1/2, Src, and phosphatidylinositol 3-kinase/protein kinase B signaling pathways are implicated in the proliferative signal induced by oleate and that these effects are mediated at least in part via the G protein-coupled receptor GPR40. The results suggest that GPR40 is implicated in the control of breast cancer cell growth by fatty acids and that GPR40 may provide a link between fat and cancer.
Preview · Article · May 2005 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.
[Show abstract][Hide abstract] ABSTRACT: HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an alpha-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: The ORFs 5, 6 and 7, encoding for the three major structural proteins, GP5, M and N, of the IAF-Klop strain of PRRSV were cloned and expressed in 293 cells using replication-defective human type 5 adenoviral vectors (hAdVs). Although the M protein gene could be cloned into hAdVs and expressed constituvely in 293 cells under the control of the hCMV immediate early promotor/enhancer, hAdVs expressing N and GP5 proteins, which appeared to be toxic or interfered with adenovirus replication, could only be generated by inclusion of a tetracycline-regulatable promotor in the transfer vector pAdTR5. The recombinant (rec) proteins appeared similar to the authentic viral proteins in regards to their M
rs and antigenicities. However, the recGP5 apparently possesses different N-linked oligosaccharides residues. Its sensitivity to endo-β-galactosidase digestion indicates that poly-N-acetyllactosamine is present on the individually-expressed protein, but not on the authentic GP5 anchored into the virion envelope. The recGP5 apparently accumulates within the ER compartment as a glycoprotein that possesses high-mannose N-linked oligosaccharide side chains sensitive to endo-β-N-acetylglucosaminidase H treatment, by contrast to its viral counterpart for which N-linked oligosaccharide side chains are of both high-mannose and complex types. Coinfection of 293 cells with hAdVs expressing the M and GP5 did not lead to M-GP5 heterodimer formation, as demonstrated in PRRSV-infected cells. Moreover, cells infected with inducible hAdV/ORF5 showed that GP5 of the North American strain is proapoptotic. Indeed, when the expression cassette was turned-on, caspase 3 activity in hAdV/ORF5 infected cells was enhanced and DNA fragmentation could be detected by TUNEL assays. Pigs intradermally injected twice with hAdV/ORF5 developed antibody titers to the authentic viral GP5 as soon as 10 days following challenge with the homologous virulent PRRSV strain, as revealed by Western blot and virus neutralization tests, suggesting the establishment of a specific immune memory.
No preview · Article · Jun 2003 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the α-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an α-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Delta2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6Delta and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.
Full-text · Article · Dec 2002 · Journal of General Virology