CHIP promotes CAD ubiquitination and degradation to suppress the proliferation and colony formation of glioblastoma cells.

PURPOSE: Cancer cells are characterized as the uncontrolled proliferation, which demands high levels of nucleotides that are building blocks for DNA synthesis and replication. CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase) is a trifunctional enzyme that initiates the de novo pyrimidine synthesis, which is normally enhanced in cancer cells to preserve the pyrimidine pool for cell division. Glioma, representing most brain cancer, is highly addicted to nucleotides like pyrimidine to sustain the abnormal growth and proliferation of cells. CAD is previously reported to be dysregulated in glioma, but the underlying mechanism remains unclear. METHODS: The expression of CAD and CHIP (carboxyl terminus of Hsc70-interacting protein) protein in normal brain cells and three glioblastoma (GBM) cell lines were measured by immunoblots. Lentiviruses-mediated expression of target proteins or shRNAs were used to specifically overexpress or knock down CAD and CHIP. Cell counting, colony formation, apoptosis and cell cycle assays were used to assess the roles of CAD and CHIP in GBM cell proliferation and survival. Co-immunoprecipitation and ubiquitination assays were used to examine the interaction of CHIP with CAD and the ubiquitination of CAD. The correlation of CAD and CHIP expression with GBM patients' survival was obtained by analyzing the GlioVis database. RESULTS: In this study, we showed that the expression of CAD was upregulated in glioma, which was positively correlated with the tumor grade and survival of glioma patients. Knockdown of CAD robustly inhibited the cell proliferation and colony formation of GBM cells, indicating the essential role of CAD in the pathogenesis of GBM. Mechanistically, we firstly identified that CAD was modified by the K29-linked polyubiquitination, which was mediated by the E3 ubiquitin ligase CHIP. By interacting with and ubiquitinating CAD, CHIP enhanced its proteasomal and lysosomal degradation, which accounted for the anti-proliferative role of CHIP in GBM cells. To sustain the expression of CAD, CHIP is significantly downregulated, which is correlated with the poor prognosis and survival of GBM patients. Notably, the low level of CHIP and high level of CAD overall predict the short survival of GBM patients. CONCLUSION: Altogether, these results illustrated the essential role of CAD in GBM and revealed a novel therapeutic strategy for CAD-positive and CHIP-negative cancer.

Targeting FoxO proteins induces lytic reactivation of KSHV for treating herpesviral primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus consisting of both latent and lytic life cycles. Primary effusion lymphoma (PEL) is an aggressive B-cell lineage lymphoma, dominantly latently infected by KSHV. The latent infection of KSHV is persistent and poses an obstacle to killing tumor cells. Like the "shock and kill" strategy designed to eliminate latent HIV reservoir, methods that induce viral lytic reactivation in tumor latently infected by viruses represent a unique antineoplastic strategy, as it could potentially increase the specificity of cytotoxicity in cancer. Inspired by this conception, we proposed that the induction of KSHV lytic reactivation from latency could be a potential therapeutic stratagem for KSHV-associated cancers. Oxidative stress, the clinical hallmark of PEL, is one of the most prominent inducers for KSHV reactivation. Paradoxically, we found that hydrogen peroxide (H2O2) triggers robust cytotoxic effects on KSHV-negative rather than KSHV-positive B lymphoma cells in a dose-dependent manner. Mechanistically, we identified forkhead box protein O1 (FoxO1) and FoxO3 as irrevocable antioxidant defense genes and both of them are upregulated by KSHV latent infection, which is essential for the promoted ROS scavenging in KSHV-positive B lymphoma cells. Pharmacological inhibition or functional knockdown of either FoxO1 or FoxO3 is sufficient to ablate the antioxidant ability and therefore increases the intracellular ROS level that further reverses KSHV from latency to active lytic replication in PEL cells, resulting in tremendous cell death both in vitro and in vivo. Additionally, the elevated level of ROS by inhibiting FoxO proteins further sensitizes PEL cells to ROS-induced apoptosis. Our study therefore demonstrated that the lytic reactivation of KSHV by inhibiting FoxO proteins is a promising therapeutic approach for PEL, which could be further extended to other virus-associated diseases.

Tannic acid attenuates intestinal oxidative damage by improving antioxidant capacity and intestinal barrier in weaned piglets and IPEC-J2 cells.

Tannic acid (TA) has received widespread attention for its beneficial biological function with antioxidant capacity. This study investigated the protective role of TA on the intestinal antioxidant capacity and intestinal barrier in weaned piglets and porcine intestinal epithelial cells (IPEC-J2). A total of 18 weaned piglets were randomly allocated into two groups (n = 9) and fed with a basal diet (control, CON) and a basal diet containing 1,000 mg/kg TA for two weeks. The in vivo results showed that treatment with TA increased both glutathione peroxidase (GSH-PX) activity and the protein expression of ZO-1 in the jejunum of weaned piglets, and reduced the level of malondialdehyde (MDA) in the serum and the mRNA and protein expression of Keap1 in the jejunum of weaned piglets. Furthermore, in vitro results indicated that TA treatment effectively alleviated tert-butyl hydroperoxide (TBH)-induced oxidative stress in IPEC-J2 cells, improved the antioxidant capacity by elevating the cell redox state and activating the Nrf2 pathway, and improved the intestinal barrier by upregulating the mRNA and protein expression of intestinal tight junction proteins and increasing the transepithelial electrical resistance (TEER) value. In conclusion, these results confirmed that TA relieves oxidative injury and improves intestinal barrier function and intestinal antioxidant capacity by activating the Nrf2 signaling pathway. These findings suggest that TA has the potential application in alleviating oxidative stress in the intestine of weaned piglets.

The Central Role of mTORC1 in Amino Acid Sensing.

The mTOR is a master regulator of cell growth that controls cell homeostasis in response to nutrients, growth factors, and other environmental cues. Recent studies have emphasized the importance of lysosomes as a hub for nutrient sensing, especially amino acid sensing by mTORC1. This review highlights recent advances in understanding the amino acid-mTORC1 signaling axis and the role of mTORC1 in cancer.

Honokiol ameliorates cisplatin-induced acute kidney injury via inhibition of mitochondrial fission.

BACKGROUND AND PURPOSE: Mitochondrial damage and oxidative stress are crucial contributors to the tubular cell injury and death in acute kidney injury. Novel therapeutic strategies targeting mitochondria protection and halting the progression of acute kidney injury are urgently needed. Honokiol is a small-molecule polyphenol that exhibits extraordinary cytoprotective effects, such as anti-inflammatory and anti-oxidative. Thus, we investigated whether honokiol could ameliorate cisplatin-induced acute kidney injury via preventing mitochondrial dysfunction. EXPERIMENTAL APPROACH: Acute kidney injury was induced by cisplatin administration. Biochemical and histological analysis were used to determine kidney injury. The effect of honokiol on mitochondrial function and morphology were determined using immunohistochemistry, transmission electron microscopy, immunoblot and immunofluorescence. To investigate the mechanism by which honokiol alters mitochondrial dynamics, remodelling and resistance to apoptosis, we used transfection experiments, immunoblotting, immunoprecipitation and flow cytometry assay. KEY RESULTS: We demonstrated that the prominent mitochondrial fragmentation occurred in experimental models of cisplatin-induced nephrotoxicity, which was coupled to radical oxygen species (ROS) overproduction, deterioration of mitochondrial function, release of apoptogenic factors and the consequent apoptosis. Honokiol treatment caused notable reno-protection and attenuated of these cisplatin-induced changes. Mechanistically, honokiol treatment recovered the expression of SIRT3 and improved AMPK activity in tubular cells exposure to cisplatin, which preserved the Drp1 phosphorylation at Ser637 and blocked its translocation in mitochondria, consequently preventing mitochondrial fragmentation and subsequent cell injury and death. CONCLUSION AND IMPLICATIONS: Our results indicate that honokiol may protect against cisplatin-induced acute kidney injury by preserving mitochondrial integrity and function by SIRT3/AMPK-dependent mitochondrial dynamics remodelling.

Effects of Dietary Chlorogenic Acid Supplementation Derived from Lonicera macranthoides Hand-Mazz on Growth Performance, Free Amino Acid Profile, and Muscle Protein Synthesis in a Finishing Pig Model.

Chlorogenic acid (CGA), as one of the richest polyphenol compounds in nature, has broad applications in many fields due to its various biological properties. However, initial data on the effects of dietary CGA on protein synthesis and related basal metabolic activity has rarely been reported. The current study is aimed at (1) determining whether dietary CGA supplementation improves the growth performance and carcass traits, (2) assessing whether dietary CGA alters the free amino acid profile, and (3) verifying whether dietary CGA promotes muscle protein synthesis in finishing pigs. Thirty-two (Large × White × Landrace) finishing barrows with an average initial body weight of 71.89 ± 0.92 kg were randomly allotted to 4 groups and fed diets supplemented with 0, 0.02%, 0.04%, and 0.08% CGA, respectively. The results indicated that, compared with the control group, dietary supplementation with 0.04% CGA slightly stimulated the growth performance of pigs, whereas no significant correlation was noted between the dietary CGA levels and animal growth (P > 0.05). Furthermore, the carcass traits of pigs were improved by 0.04% dietary CGA (P < 0.01). In addition, dietary CGA significantly improved the serum free amino acid profiles of pigs (P < 0.01), while 0.04% dietary CGA promoted more amino acids to translocate to skeletal muscles (P < 0.05). The relative mRNA expression levels of SNAT2 in both longissimus dorsi (LD) and biceps femoris (BF) muscles were augmented in the 0.02% and 0.04% groups (P < 0.05), and the LAT1 mRNA expression in the BF muscle was elevated in the 0.02% group (P < 0.05). We also found that dietary CGA supplementation at the levels of 0.04% or 0.08% promoted the expression of p-Akt and activated the mTOR-S6K1-4EBP1 axis in the LD muscle (P < 0.05). Besides, the MAFbx mRNA abundance in the 0.02% and 0.04% groups was significantly lower (P < 0.05). Our results revealed that dietary supplementation with CGA of 0.04% improved the free amino acid profile and enhanced muscle protein biosynthesis in the LD muscle in finishing pigs.

Pyrimidine Biosynthetic Enzyme CAD: Its Function, Regulation, and Diagnostic Potential.

CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Over the past two decades, extensive investigations have been conducted to unmask CAD as a central player for the synthesis of nucleic acids, active intermediates, and cell membranes. Meanwhile, the important role of CAD in various physiopathological processes has also been emphasized. Deregulation of CAD-related pathways or CAD mutations cause cancer, neurological disorders, and inherited metabolic diseases. Here, we review the structure, function, and regulation of CAD in mammalian physiology as well as human diseases, and provide insights into the potential to target CAD in future clinical applications.

Dietary Supplementation With Chlorogenic Acid Derived From Lonicera macranthoides Hand-Mazz Improves Meat Quality and Muscle Fiber Characteristics of Finishing Pigs via Enhancement of Antioxidant Capacity.

Chlorogenic acid (CGA), one of the most abundant polyphenol compounds in nature, is regarded as a potential feed additive to promote animal health and enhance the meat products' quality via its various biological properties. The current study aims: (1) to determine whether dietary CGA supplementation improves meat quality and muscle fiber characteristics, and (2) to ascertain whether the corresponding improvement is associated with enhancing the antioxidant capacity of the finishing pigs. Thirty-two (Large × White × Landrace) finishing pigs with an average initial body weight of 71.89 ± 0.92 kg were allotted to 4 groups, and each was fed diets supplemented with 0, 0.02, 0.04, or 0.08% (weight/weight) of CGA. The meat quality traits, muscle fiber characteristics, and the serum and muscle antioxidant capacity were assessed. Results suggested that, compared with the control group, dietary CGA supplementation at a level of 0.04% significantly decreased the b∗ value and distinctly increased the inosinic acid content of longissimus dorsi (LD) and biceps femoris (BF) muscles (P < 0.01). Moreover, dietary supplementation with 0.04% of CGA markedly improved the amino acid composition of LD and BF muscles, as well as augmented the mRNA abundance of Nrf-2, GPX-1, MyoD, MyoG, and oxidative muscle fiber (I and IIa) in LD muscle (P < 0.05). This result indicates that a diet supplemented with 0.04% of CGA promotes myogenesis and induces a transformation toward more oxidative muscle fibers in LD muscle, subsequently improving meat quality. Besides, dietary supplementation with 0.02% and 0.04% of CGA notably enhanced the serum GSH-PX level (P < 0.01). Considering all these effects are closely related to the alteration of antioxidant activities of the finishing pigs, the underlying metabolism is likely connected to the boosting of their antioxidant capacity induced by dietary CGA.

Resveratrol Attenuates Oxidative Stress-Induced Intestinal Barrier Injury through PI3K/Akt-Mediated Nrf2 Signaling Pathway.

Oxidative stress is implicated in a wide range of intestinal disorders and closely associated with their pathological processes. Resveratrol (RSV), a plant extract, plays a vital role in protecting various organs in vitro and in vivo. However, the benefits of RSV are controversial, and underlying mechanisms for its antioxidant effects on intestinal epithelial cells remain unclear. In this study, we evaluated the effects of RSV on oxidative stress induced by H2O2 in IPEC-J2 cells. We found that pretreatment with RSV significantly increased cell viability; increased expression levels of tight junction (TJ) proteins (claudin-1, occludin, and ZO-1); improved activities of superoxide dismutase-1 (SOD-1), catalase (CAT), and glutathione peroxidase (GSH-Px); and decreased intracellular reactive oxygen species (ROS) levels and apoptosis induced by H2O2 (P < 0.05). In addition, RSV upregulated Akt phosphorylation, Nrf2 phosphorylation, and expression levels of antioxidant genes HO-1, SOD-1, and CAT in a dose-dependent manner (P < 0.05) under oxidative stress. Knockdown of Nrf2 by short-hairpin RNA (shRNA) abrogated RSV-mediated protection against H2O2-induced apoptosis, RSV-induced increase of TJ protein levels, and antioxidant gene expression (SOD-1, CAT, and GSH-Px) (P < 0.05). Consistent with Nrf2 knockdown, the PI3K/Akt inhibitor LY294002 significantly suppressed RSV-induced Nrf2 phosphorylation and RSV-induced increase of TJ protein levels and antioxidant gene expression under H2O2 treatment (P < 0.05). Collectively, these results demonstrate that RSV can directly protect IPEC-J2 cells against oxidative stress through the PI3K/Akt-mediated Nrf2 signaling pathway, suggesting that RSV may be an effective feed additive against intestinal damage in livestock production.

Dietary vitamin E affects small intestinal histomorphology, digestive enzyme activity, and the expression of nutrient transporters by inhibiting proliferation of intestinal epithelial cells within jejunum in weaned piglets1.

Vitamin E (VE) is an indispensable vitamin in piglet feed formula. Among other things, it affects tissues including small intestine tissues and in particular its major unit intestinal epithelial cells. Previously, limited in vivo experiments have focused on the effect of VE on the intestine, particularly digestion and absorption. VE has been shown to inhibit proliferation of some types of cells. This experiment was conducted to test the hypothesis that VE affects intestinal functions by influencing the intestinal epithelial cell proliferation. Thirty 21-d old weaned [(Yorkshire × Landrace) × Duroc] piglets with BWs of 6.36 ± 0.55 kg were randomly divided into five VE-containing feeding formula groups. The treatments were (i) 0 IU (control), (ii) 16 IU, (iii) 32 IU, (iv) 4. 80 IU, and (v) 5. 160 IU. The treatments lasted 14 d. At the end of the experiment, all subjects were sacrificed to obtain blood and tissue samples. The results suggest that VE did not affect the growth performance. VE did tend to decrease jejunal crypt depth (linear, P = 0.056) and villus width (linear, P < 0.05). Sucrase activity significantly decreased in the adding 80 IU VE compared with the control (P < 0.05). Jejunal crypt, cell proliferation in 80 IU group significantly decreased compared with the control group (P < 0.05). This study suggests that dietary VE may affect intestinal morphology and functions by inhibiting weaned piglet jejunal epithelial cell proliferation.

Tannic acid modulates intestinal barrier functions associated with intestinal morphology, antioxidative activity, and intestinal tight junction in a diquat-induced mouse model.

Oxidative stress is more likely to occur in the intestine compared to other organs because it is located at the interface between an organism and its luminal environment. Tannic acid (TA) is reported to serve as an antioxidant, antimicrobial, anticarcinogenic and antimutagenic agent in various models. In the present study, we evaluated the effects of TA on body weight, intestinal morphology, antioxidative activity, and intestinal barrier in diquat-induced oxidative stress mouse model. The results showed that TA had failed to affect antioxidative enzymes in diquat-challenged mice, while the concentration of 2.5 mg kg-1 to 10 mg kg-1 TA had no negative effect on body weight and enhanced the colon length in mice. The dose of 2.5 mg kg-1 TA ameliorated the morphological damage in the jejunum by increasing the villus height and crypt depth, activated the antioxidative pathway by decreasing jejunal protein expression of Kelch like-ECH-associated protein 1 (KEAP1) and increasing protein expression of Nuclear factor erythroid 2-related factor 2 (NRF2), and affected the intestinal barrier by inhibiting the jejunal mRNA expression of claudin and promoting mRNA expression of zonula occludens (zo-1). In conclusion, the pretreatment of TA in a mouse model of oxidative stress failed to change the antioxidative enzymes but modulated the jejunal morphology, colon length, antioxidative pathway and intestinal barrier in the diquat oxidative model.

Down-regulation of PKM2 decreases FASN expression in bladder cancer cells through AKT/mTOR/SREBP-1c axis.

Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.

Species-Specific Deamidation of cGAS by Herpes Simplex Virus UL37 Protein Facilitates Viral Replication.

Herpes simplex virus 1 (HSV-1) establishes infections in humans and mice, but some non-human primates exhibit resistance via unknown mechanisms. Innate immune recognition pathways are highly conserved but are pivotal in determining susceptibility to DNA virus infections. We report that variation of a single amino acid residue in the innate immune sensor cGAS determines species-specific inactivation by HSV-1. The HSV-1 UL37 tegument protein deamidates human and mouse cGAS. Deamidation impairs the ability of cGAS to catalyze cGAMP synthesis, which activates innate immunity. HSV-1 with deamidase-deficient UL37 promotes robust antiviral responses and is attenuated in mice in a cGAS- and STING-dependent manner. Mutational analyses identified a single asparagine in human and mouse cGAS that is not conserved in many non-human primates. This residue underpins UL37-mediated cGAS deamidation and species permissiveness of HSV-1. Thus, HSV-1 mediates cGAS deamidation for immune evasion and exploits species sequence variation to disarm host defenses.

Suckling Piglet Intestinal Enterocyte Nutrient Metabolism Changes.

BACKGROUND/AIMS: Intestinal morphology and the types of enterocytes are changed in piglets during the suckling period, but it is unclear whether these changes are associated with metabolic changes in epithelium. The present study was conducted to test the hypothesis that glucose, fatty acids, and amino acid metabolism in differentiated piglet enterocytes changed during suckling. METHODS: Twenty-four piglets (Duroc × [Landrace × Yorkshire]) from 8 litters (3 piglets/litter) were selected. A single piglet from each litter was randomly selected and euthanized at days 7, 14, and 21. Differentiated enterocytes (DE) were isolated from their mid-jejunum. Isobaric tags for relative and absolute quantification and subsequent liquid chromatography-tandem mass spectrometry were used to identify and measure protein synthesis. RESULTS: The results showed that various activities, including: cellular processes; metabolic processes; biological regulation; pigmentation; and, localization, in DEs changed during suckling. Metabolic process analyses revealed that protein expression related to glycolysis and citrate cycle was decreased from day 7 to day 14. The number of differentiated enterocytes of 21 d piglets increased compared to 7 d piglets. Most of the proteins involved in fatty acid and amino acids metabolism had decreased DE expression between day 7 and day 14. Some, but not all, detected proteins down-regulated in DEs of 21 day piglets compared to 7 day piglets. CONCLUSION: These results indicate that glucose, fatty acids, and amino acids metabolism changed during suckling. This may provide useful information for designing feed formulas and regulating piglet intestinal growth and development.

Down-regulation of PKM2 enhances anticancer efficiency of THP on bladder cancer.

Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels that are correlated with the sensitivity of anticancer chemotherapeutic drugs. THP is one of the major drugs used in non-muscle-invasive bladder cancer instillation chemotherapy. However, low response ratio of THP (19.7%) treatment to human genitourinary tumours using collagen gel matrix has been observed. This study aims to investigate the effect of down-regulation of PKM2 on THP efficiency. Via inhibitor or siRNA, the effects of reduced PKM2 on the efficiency of THP were determined in 2 human and 1 murine bladder cancer cell lines, using MTT, cologenic and fluorescence approaches. Molecular mechanisms of PKM2 on THP sensitization were explored by probing p-AMPK and p-STAT3 levels via WB. Syngeneic orthotopic bladder tumour model was applied to evaluate this efficiency in vivo, analysed by Kaplan-Meier survival curves, body and bladder weights plus immunohistochemistric tumour biomarkers. PKM2 was overexpressed in bladder cancer cells and tissues, and down-regulation of PKM2 enhanced the sensitivity of THP in vitro. Activation of AMPK is essential for THP to exert anti-bladder cancer activities. On the other hand, down-regulating PKM2 activates AMPK and inhibits STAT3, correlated with THP sensitivity. Compared with THP alone (400 µmol L-1 , 50 µL), the combination with metformin (60 mmol L-1 , 50 µL) stopped growth of bladder cancer completely in vivo (combination group VS normal group P = .078). Down-regulating the expression of PKM2 enhances the anticancer efficiency of THP. This study provides a new insight for improving the chemotherapeutic effect of THP.

Recent advances on viral manipulation of NF-κB signaling pathway.

NF-κB transcription factors regulate the expression of hundreds of genes primarily involved in immune responses. Signaling events leading to NF-κB activation constitute a major antiviral immune pathway. To replicate and persist within their hosts, viruses have evolved diverse strategies to evade and exploit cellular NF-κB immune signaling cascades for their benefit. We summarize recent studies concerning viral manipulation of the NF-κB signaling pathway downstream of pattern recognition receptors. Signal transduction mediated by pattern recognition receptors is a research frontier for both infectious disease and innate immunology.

Herpesviral G protein-coupled receptors activate NFAT to induce tumor formation via inhibiting the SERCA calcium ATPase.

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi's sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of "constitutive" NFAT activation by viral GPCRs.

Viral pseudo-enzymes activate RIG-I via deamidation to evade cytokine production.

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here, we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologs of phosphoribosylformylglycinamidine synthetase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homolog thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.

Identification of a bHLH-type G-box binding factor and its regulation activity with G-box and Box I elements of the PsCHS1 promoter.

With the use of in vivo recombination theory, the screening time of yeast one-hybrid system was decreased in the present study. A basic helix-loop-helix (bHLH) protein PsGBF was successfully obtained from a glutathione (GSH)-induced pea cDNA library using the G-box cis-element of the PsCHS1 promoter as a bait. Electrophoretic mobility shift assay (EMSA) and beta-galactosidase assay results suggested that PsGBF possesses both G-box-specific binding and transcription-activating activities. The specific interaction of PsGBF with G-box was further confirmed by in vivo transient expression assays in tobacco. The current study examined the combination effect of G-box with Box I elements in the interaction with PsGBF or OsMYC. The results indicated that PsGBF bound with the G-box, but not the Box I element. Moreover, this combination effect of G-box and Box I only associated with PsGBF but not with other bHLH-type proteins such as OsMYC.

Identification of a new AT-rich-element binding factor PsATF1 and its combined effect with PsGBF on the activation of PsCHS1 promoter.

Chaltone synthase (CHS) is a key speed-limiting enzyme in the phenylpropanoid pathway which plays an important role in plant defense response against pathogens. In the PsCHS1 promoter, there is an AT-rich element (ATRE) which is required for the maximal elicitor-mediated activation. However, the transcription activator of the ATRE and its regulation mechanism in pea keep unclear. In this paper, a new ATRE-binding factor was isolated from an elicitor-induced pea cDNA expression library and was designated as PsATF1. Electrophoretic mobility shift assay (EMSA) indicated the ATRE-specific binding activity of PsATF1. Beta-galactosidase assays in yeast cells suggested that PsATF1 possessed transcription-activating activity because PsATF1 activated the expression of the reporter gene even without the GAL4 activation domain (AD). The current study also examined the co-activation effects of PsATF1 with another transcription factor PsGBF on ATRE or PsCHS1 promoter through a transient expression system. The present work reports that PsATF1 acts as a complete transcription activator and first indicates that there are combined effects of PsATF1 with PsGBF on the activation of PsCHS1 promoter. These results provide theoretical basis to the plant defense gene expression mechanism regulated by multiple activators.