Identification and characterization of a tumor necrosis factor receptor like protein encoded by Singapore grouper iridovirus

Virus encoded tumor necrosis factor receptors (TNFRs) have been demonstrated to facilitate virus to escape from apoptosis or other host immune response for viral replication. Singapore grouper iridovirus (SGIV), a large DNA virus which belongs to genus Ranavirus, is a major pathogen resulting in heavy economic losses to grouper aquaculture. Here, SGIV ORF096 (VP96) encoding a putative homolog of TNFR was identified and characterized. Multiple sequence alignment indicated that SGIV-VP96 contained two extracellular cysteine-rich domains (CRDs) with conserved four or six cysteine residues, but lacked the transmembrane domain at the C-terminus. SGIV-VP96 was identified as an early (E) gene and localized in the cytoplasm in transfected or infected cells. Overexpression of SGIV-VP96 in vitro enhanced cell proliferation, and improved cell survival against SGIV infection. Furthermore, virus infection induced apoptosis and caspase-3 activity were inhibited in SGIV-VP96 expressing FHM cells compared to the control cells. Taken together, our results suggested that SGIV might utilize virus encoded TNFR like genes to modulate the host apoptotic response for effective virus replication.     

Light- and drug-activated G-protein-coupled receptors to control intracellular signalling

G-protein-coupled receptors (GPCRs) integrate extracellular cues into intracellular signals to modulate the cellular state. Owing to their diverse modulatory functions, GPCRs represent one of the major drug targets of the pharmaceutical industry. Until now, the characterization and control of GPCRs and their intracellular signalling cascades have mainly relied on chemical compounds, which either activate or inhibit GPCR pathways, albeit with limited receptor and cell-type specificity. Recently, new approaches have been developed to control signalling cascades in cell- and receptor-type-specific ways. The chemical approach focuses on GPCR design and activation by an inert chemical compound, whereas the physical approach uses designer GPCRs and activation by physical stimuli, such as light.     

肝细胞生长因子拮抗放线菌素D诱导的肝细胞凋亡及机制探讨

目的： 探讨肝细胞生长因子（HGF）对放线菌素D (ActD)诱导HL7702肝细胞凋亡的拮抗作用及可能机制。 方法： 本实验采用HL7702正常人肝细胞株，MTT法检测ActD对肝细胞存活力的影响；Hoechst33342进行凋亡形态学染色；DNA凝胶电泳及流式细胞仪检测细胞凋亡数量；Western blotting方法检测细胞总Akt及磷酸化Akt蛋白的表达。 结果： ActD可以诱导HL7702肝细胞凋亡，其浓度在0.25-8 mg/L范围内呈现剂量效应关系；PI3K特异性抑制剂wortmannin能够增强ActD诱导的肝细胞凋亡作用；肝细胞生长因子(HGF)对ActD诱导的肝细胞凋亡有拮抗作用，在一定剂量范围内呈现剂量效应关系；并且HGF能够激活PI3K/Akt信号转导途径；进一步用wortmannin阻断PI3K/Akt信号途径后，HGF的拮抗凋亡作用被抑制。 结论： 一定剂量的ActD可以诱导肝细胞凋亡；wortmannin能够增强ActD诱导肝细胞凋亡的作用；HGF对ActD诱导的这种细胞凋亡有明显的拮抗作用，并且HGF的抗凋亡作用与其激活细胞内PI3K/Akt信号转导通路有关。     

Regulation of G protein-coupled receptor trafficking

G protein-coupled receptors (GPCRs) mediate cellular responses to diverse extracellular stimuli to play a vital role in the control of physiology and behaviour. GPCR trafficking is of fundamental importance for the regulation of GPCRs signaling. In this mini review, we will discuss some of the recent findings on the mechanisms that regulate GPCR trafficking, which include (i) large dense-core vesicle (LDCV)-associated GPCR delivery which could be a general cell biological mechanism for rapid modulation of membrane receptors in response to certain stimuli; (ii) lateral diffusion of GPCRs in the plasma membrane for rapid change of the number of neurotransmitter receptors during synaptic plasticity and (iii) constitutive internalization of GPCRs, that contributes to receptor resensitization and distribution, including axonal polarization.     

Cellular FLICE/caspase-8-inhibitory protein as a principal regulator of cell death and survival in human hepatocellular carcinoma

Human hepatocellular carcinomas (HCCs) show resistance to apoptosis mediated by several death receptors. Because cellular FLICE/caspase-8-inhibitory protein (cFLIP) is a recently identified intracellular inhibitor of caspase-8 activation that potently inhibits death signaling mediated by all known death receptors, including Fas, TNF-receptor (TNF-R), and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs), we investigated the expression and function of cFLIP in human HCCs. We found that cFLIP is constitutively expressed in all human HCC cell lines and is expressed more in human HCC tissues than in nontumor liver tissues. Metabolic inhibitors, actinomycin D (ActD) or cycloheximide (CHX), dramatically rendered HCC cells sensitive to Fas-mediated apoptosis. Neither caspase-8 nor caspase-3 was activated by agonistic anti-Fas antibody alone, but both caspases were activated by Fas stimulation in the presence of ActD or CHX, indicating the importance of caspase-8 inhibitors that are sensitive to metabolic inhibitors. Actually, cFLIP expression was decreased in ActD or CHX treatment. cFLIP down-regulation induced by cFLIP antisense oligodeoxynucleotides sensitized HLE cells to Fas, TNF-R, and TRAIL-R-mediated apoptosis. Furthermore, cFLIP over-expression activated nuclear factor (NF)-kappaB and cFLIP down-regulation attenuated NF-kappaB activation induced by TNF-alpha or TRAIL. Pretreatment with pan-caspase-inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD-fmk), restored NF-kappaB activity attenuated by cFLIP down-regulation. cFLIP expression was increased by TNF-alpha, TRAIL, or vascular endothelial growth factor but decreased by wortmannin, indicating that cFLIP expression is regulated by both the NF-kappaB and phosphatidylinostiol-3 kinase (PI-3)/Akt pathways. These results suggest that cFLIP plays an important role in cell survival not simply by inhibiting death-receptor-mediated apoptosis but also by regulating NF-kappaB activation in human HCCs.     

Induction of apoptosis in frog virus 3-infected cells

The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins.     

Human herpesvirus 8 viral FLICE-inhibitory protein inhibits Fas-mediated apoptosis through binding and prevention of procaspase-8 maturation.

Viral FLICE-inhibitory proteins (v-FLIPs) encoded by several herpesviruses and poxviruses share the ability to inhibit apoptosis after engagement of death receptors. In the current article, we provide insights into the mechanisms by which the v-FLIP of human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated virus) protects cells from apoptosis after Fas-induced signaling. Using v-FLIP expression vectors, our results clearly show that HHV-8 v-FLIP reduces the cleavage of procaspase-8 into its active p18 and p10 protease subunits upon Fas-induced cell death. These results were confirmed by lower caspase-8 and caspase-3 protease activities in extracts of HeLa cells expressing HHV-8 v-FLIP. Coimmunoprecipitation studies further indicate that HHV-8 v-FLIP physically interacts with procaspase-8, but not with Fas-associated protein with death domain in the cellular cytoplasm. These results suggest that binding of HHV-8 v-FLIP to procaspase-8 affects the recruitment and the activation of the latter at the death-induced signaling complex, resulting in diminished apoptotic cascade initiation. Because cellular FLIP was recently reported to modulate promoter containing NF-kappaB motifs and that both HHV-8 and human immunodeficiency virus type 1 (HWV-1) can infect monocytes, we studied the effects of v-FLIP on HIV-1 gene expression. Cotransfection experiments indicated that v-FLIP expression is associated with activation of HIV long terminal repeats: events that were strictly dependent on the presence of NF-kappaB consensus elements. In conclusion, HHV-8 v-FLIP can possibly contribute to the pathogenesis of both HHV-8 and HIV-1 through impaired Fas-dependent killing of infected cells by cytotoxic T cells and through activation of HIV gene expression.     

G protein coupled pattern recognition receptors expressed in neutrophils: Recognition,activation/modulation,signaling and receptor regulated functions

Neutrophils, the most abundant white blood cell in human blood, express receptors that recognize damage/microbial associated pattern molecules of importance for cell recruitment to sites of inflammation. Many of these receptors belong to the family of G protein coupled receptors (GPCRs). These receptor-proteins span the plasma membrane in expressing cells seven times and the down-stream signaling rely in most cases on an activation of heterotrimeric G proteins. The GPCRs expressed in neutrophils recognize a number of structurally diverse ligands (activating agonists, allosteric modulators, and inhibiting antagonists) and share significant sequence homologies. Studies of receptor structure and function have during the last 40 years generated important information on GPCR biology in general; this knowledge aids in the overall understanding of general pharmacological principles, governing regulation of neutrophil function and inflammatory processes, including novel leukocyte receptor activities related to ligand recognition, biased/functional selective signaling, allosteric modulation, desensitization, and reactivation mechanisms as well as communication (receptor transactivation/cross-talk) between GPCRs. This review summarizes the recent discoveries and pharmacological hallmarks with focus on some of the neutrophil expressed pattern recognition GPCRs. In addition, unmet challenges, including recognition by the receptors of diverse ligands and how biased signaling mediate different biological effects are described/discussed.     

Sphingosine 1-phosphate and its type 1 G protein-coupled receptor: trophic support and functional regulation of T lymphocytes

The lysophospholipid (LPL) growth factors sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are generated by macrophages, dendritic cells, mast cells, and platelets, which leads to lymph and plasma concentrations of 0.1-1 microM. Distinctive profiles of G protein-coupled receptors (GPCRs) for S1P and LPA are expressed by each type of immune cell and are regulated by cellular activation. At 1-100 nM, S1P signals T cells through their principal S1P(1) GPCRs with consequent protection from apoptosis, enhancement of chemotaxis, and facilitation of optimal regulatory activity of CD4(+)25(+) T cells. At 0.3-3 microM, S1P inhibits T cell chemotaxis and to a lesser extent other functions. These S1P-S1P(1) GPCR signals suppress homing of blood and spleen T cells to secondary lymphoid tissues. S1P(1) GPCR antagonists evoke lymphopenia by permitting blood T cells to enter lymph nodes and blocking S1P(1) GPCR-dependent T cell efflux from lymph nodes. Inversely, there is a decrease in lymphoid tissue traffic of T cells in transgenic mice, which overexpress lymphocyte S1P(1) GPCRs. The immunotherapeutic activity of S1P(1) GPCR antagonists, which limits T cell access to organ grafts and autoimmune antigens, does not reduce other functional capabilities of T cells. LPLs and their GPCRs thus constitute an immunoregulatory system of sufficient prominence for pharmacological targeting in transplantation, autoimmunity, and immunodeficiency.     

The pseudorabies virus US3 protein kinase possesses anti-apoptotic activity that protects cells from apoptosis during infection and after treatment with sorbitol or staurosporine

Most large DNA viruses, like herpesviruses, encode anti-apoptotic proteins to interfere with the apoptotic cellular response to infection. Previous studies have shown that the US3 protein kinase of herpes simplex virus, in contrast to US3 of bovine herpes virus 1, is very potent in protecting cells from apoptosis induced by the virus itself or by a broad range of exogenous apoptotic stimuli. Here, we demonstrate that US3 of the swine alphaherpesvirus pseudorabies virus (PRV) suppresses PRV-induced apoptosis in swine-testicle (ST) cells at late stages in infection, and that it protects ST cells from apoptosis induced by either sorbitol or staurosporine. Interestingly, PRV US3 encodes a short and a long isoform, the latter of which contains a functional mitochondrial localization sequence. Transient transfections showed that the PRV US3 long isoform is more efficient in protecting ST cells from PRV- or staurosporine-induced apoptosis, suggesting a potential advantage for the mitochondrial localization of PRV US3 in implementing its anti-apoptotic function.     

G-protein-coupled receptor expression, function, and signaling in macrophages

G-protein-coupled receptors (GPCRs) are widely targeted in drug discovery. As macrophages are key cellular mediators of acute and chronic inflammation, we review here the role of GPCRs in regulating macrophage function, with a focus on contribution to disease pathology and potential therapeutic applications. Within this analysis, we highlight novel GPCRs with a macrophage-restricted expression profile, which provide avenues for further exploration. We also review an emerging literature, which documents novel roles for GPCR signaling components in GPCR-independent signaling in macrophages. In particular, we examine the crosstalk between GPCR and TLR signaling pathways and highlight GPCR signaling molecules which are likely to have uncharacterized functions in this cell lineage.     

G protein-coupled receptors and the modification of FcepsilonRI-mediated mast cell activation

By releasing multiple pro-inflammatory mediators upon activation, mast cells are critical effector cells in the pathogenesis of allergic inflammation. The traditional viewpoint of antigen-dependent mast cell activation is that of a Th(2)-driven process whereby antigen-specific IgE molecules are produced by B cells followed by binding of the IgE to high affinity IgE receptors (FcepsilonRI) expressed on mast cells. Subsequent antigen-dependent aggregation of the FcepsilonRI initiates an intracellular signalling cascade that culminates in mediator release. Mast cell responses, including cell growth, survival, chemotaxis, and cell adhesion, however, can also be regulated by other receptors expressed on mast cells. Furthermore, FcepsilonRI-mediated mast cell mediator release can be significantly modified by ligation of specific classes of these receptors. One such class of receptors is the G protein-coupled receptors (GPCR). In this review, we describe how sub-populations of GPCRs can either enhance or inhibit FcepsilonRI-mediated mast cell activation depending on the particular G protein utilized for relaying signalling. Furthermore, we discuss the potential mechanisms whereby the signalling responses utilized by the FcepsilonRI for mast cell activation are influenced by those initiated by GPCRs to produce these diverse responses.     

Cytomegalovirus-encoded homologs of G protein-coupled receptors and chemokines.

BACKGROUND: Cytomegaloviruses (CMVs) have developed various sophisticated strategies to manipulate and evade the defense mechanisms of their hosts. Among the CMV genes that are predicted to be involved in these strategies are genes that encode mimics of cellular proteins, such as G protein-coupled receptors (GPCRs) and chemokines (CKs). These genes may have been pirated from the host genome during the long co-evolution of virus and host. OBJECTIVES: In this report, the putative functions of the CMV-encoded homologs of GPCRs and CKs in the pathogenesis of infection will be discussed. STUDY DESIGN: In order to present an overview of the current state of knowledge, the literature on the CMV-encoded homologs of GPCRs and CKs was reviewed. RESULTS: The GPCR and CK homologs that are encoded by the CMVs represent immunomodulatory proteins with crucial roles in the pathogenesis of infection. CONCLUSIONS: In light of their function as well as accessibility on the cell surface, the CMV-encoded GPCR homologs are attractive targets for the development of new anti-viral therapies.     

Human gammaherpesvirus cytokines and chemokine receptors.

Most herpesviruses of the beta and gamma subfamilies encode homologues of cytokines and chemokine receptor- related G protein-coupled receptors (GPCRs). The roles of these proteins during normal virus replication in the infected host have not been defined in most cases, but the available data and extrapolation from what is known about the properties and functions of their cellular counterparts indicate that they play primary roles in immune evasion or in activating cellular signaling cascades that enhance virus productive replication. Cytokines and chemokine receptors specified by the two human gammaherpesviruses, human herpesvirus 8 (HHV-8) and Epstein-Barr virus (EBV), are the subject of this review. HHV-8 encodes three chemokines, a homologue of interleukin-6, and a CXCR2-related chemokine receptor, while EBV encodes a distinct GPCR and a homologue of interleukin-10. While these viral cytokines and chemokine receptors no doubt contribute to virus biology, their properties indicate that they may also be involved in virus-induced neoplasia. This review discusses the properties, functions, and likely roles of HHV-8 and EBV cytokines and chemokine receptors in relation to both virus biology and virus-associated disease.     

Tango knock-ins visualize endogenous activity of G protein-coupled receptors in Drosophila

G protein-coupled receptors (GPCRs) represent a family of seven-pass transmembrane protein receptors whose ligands include neuropeptides and small-molecule neuromodulators such as dopamine and serotonin. These neurotransmitters act at long distances and are proposed to define the ground state of the nervous system. The Drosophila genome encodes approximately 50 neuropeptides and their functions in physiology and behavior are now under intensive studies. Key information currently lacking in the field is the spatiotemporal activation patterns of endogenous GPCRs. Here we report application of the Tango system, a reporter assay to detect GPCR activity, to endogenous GPCRs in the fly genome. We developed a method to integrate the sensor component of the Tango system to the C-terminus of endogenous genes by using genome editing techniques. We demonstrate that Tango sensors in the Sex-peptide receptor (SPR) locus allow sensitive detection of mating-dependent SPR activity in the female reproductive organ. The method is easily applicable to any GPCR and will provide a way to systematically characterize GPCRs in the fly brain.     

The 3C protease activity of enterovirus 71 induces human neural cell apoptosis

The human glioblastoma SF268 cell line was used to investigate the induction of apoptosis by the 3C protease of enterovirus 71 (EV71). Transient expression in these cells of the wild-type 3C protein encoded by EV71 induced morphological alterations typical of apoptosis, including generation of apoptotic bodies. Degradation of cellular DNA in nucleosomes was also observed. When two of the amino acids in the catalytic motif of 3C were changed by mutagenesis, the 3C protein not only lost its proteolytic activity, but also its ability to induce apoptosis in the SF268 cells. Twenty-four hours after 3C transfection, poly(ADP-ribose) polymerase, a DNA repair enzyme, was cleaved, indicating that caspases were activated by the expression of EV71 3C. The 3C-induced apoptosis was blocked by the caspase inhibitors DEVD-fmk and VAD-fmk. Our findings suggest that the proteolytic activity of 3C triggers apoptosis in the SF268 cells through a mechanism involving caspase activation and that this apoptotic pathway may play an important role in the pathogenesis of EV71 infection.     

Modulation of cell death sensitivity by mutant p53 in HCV core-expressing cells

The hepatitis C virus (HCV) core protein plays important roles in hepatocarcinogenesis through modulation of cellular proliferation, apoptosis, and immunological responses. The roles of core protein in apoptosis have been conflicting; both proapoptotic and anti-apoptotic roles have been reported from different experimental conditions. Nonetheless, the overcoming apoptosis is a key molecular event to development of hepatocellular carcinoma. We investigated whether the HCV core-expressing cells are susceptible to apoptosis after cellular stress. Furthermore, we focused on the possibility that the presence of mutant p53 can render cells resistant to apoptosis. Our data clearly indicated that core-expressing cells showed increased apoptotic cell death through caspase-3 activation pathways after genotoxic stress without modulation of Bcl-2 family proteins. However, core-expressing cells, when transiently transfected with mutant p53, showed markedly increased resistance upon apoptosis after genotoxic stress. Thus, our data suggest that even though HCV core-expressing cells are susceptible to apoptosis after genotoxic stress, cells are resistant to apoptosis under mutant p53, implying a functional abnormality of p53 giving a chance to overcome apoptosis and ultimately cells develop into hepatocellular cell carcinoma.     

姜黄素对放线菌素D/TNF-α协同诱导PC12细胞凋亡的保护作用及机制研究

目的:观察中药单体姜黄素对ActD/TNF-α协同诱导PC12细胞凋亡的影响,并探讨其机制。方法:采用MTT法确定实验药物的最佳浓度;Hoechst33258荧光染色法观察PC12细胞的凋亡;JC-1荧光分子探针检测线粒体膜电位;Real Time PCR检测凋亡基因Bcl-2/Bax的表达。结果:ActD/TNF-α协同作用可导致PC12细胞的活力降低(P<0.05);出现核固缩、核碎裂现象的细胞增多,细胞凋亡率增高(P<0.05);细胞线粒体膜电位下降;细胞内抗凋亡基因Bcl-2的表达降低(P<0.05)。经姜黄素(5μmol/L)处理后,PC12细胞的活力增强(P<0.05);细胞核固缩、核碎裂现象减少,细胞凋亡率下降(P<0.05);细胞线粒体膜电位上升;细胞内抗凋亡基因Bcl-2的表达增强(P<0.05)。结论:姜黄素可拮抗ActD/TNF-α引起的PC12细胞凋亡,可能与升高线粒体膜电位,促进抗凋亡基因Bcl-2的表达有关。