Tethering of hexokinase 2 to mitochondria promotes resistance of liver cancer cells to natural killer cell cytotoxicity.

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance.

Pharmacological induction of the hypoxia response pathway in Huh7 hepatoma cells limits proliferation but increases resilience under metabolic stress.

The hypoxia response pathway enables adaptation to oxygen deprivation. It is mediated by hypoxia-inducible factors (HIF), which promote metabolic reprogramming, erythropoiesis, angiogenesis and tissue remodeling. This led to the successful development of HIF-inducing drugs for treating anemia and some of these molecules are now in clinic. However, elevated levels of HIFs are frequently associated with tumor growth, poor prognosis, and drug resistance in various cancers, including hepatocellular carcinoma (HCC). Consequently, there are concerns regarding the recommendation of HIF-inducing drugs in certain clinical situations. Here, we analyzed the effects of two HIF-inducing drugs, Molidustat and Roxadustat, in the well-characterized HCC cell line Huh7. These drugs increased HIF-1α and HIF-2α protein levels which both participate in inducing hypoxia response genes such as BNIP3, SERPINE1, LDHA or EPO. Combined transcriptomics, proteomics and metabolomics showed that Molidustat increased the expression of glycolytic enzymes, while the mitochondrial network was fragmented and cellular respiration decreased. This metabolic remodeling was associated with a reduced proliferation and a lower demand for pyrimidine supply, but an increased ability of cells to convert pyruvate to lactate. This was accompanied by a higher resistance to the inhibition of mitochondrial respiration by antimycin A, a phenotype confirmed in Roxadustat-treated Huh7 cells and Molidustat-treated hepatoblastoma cells (Huh6 and HepG2). Overall, this study shows that HIF-inducing drugs increase the metabolic resilience of liver cancer cells to metabolic stressors, arguing for careful monitoring of patients treated with HIF-inducing drugs, especially when they are at risk of liver cancer.

Domain 2 of Hepatitis C Virus Protein NS5A Activates Glucokinase and Induces Lipogenesis in Hepatocytes.

Hepatitis C virus (HCV) relies on cellular lipid metabolism for its replication, and actively modulates lipogenesis and lipid trafficking in infected hepatocytes. This translates into an intracellular accumulation of triglycerides leading to liver steatosis, cirrhosis and hepatocellular carcinoma, which are hallmarks of HCV pathogenesis. While the interaction of HCV with hepatocyte metabolic pathways is patent, how viral proteins are able to redirect central carbon metabolism towards lipogenesis is unclear. Here, we report that the HCV protein NS5A activates the glucokinase (GCK) isoenzyme of hexokinases through its D2 domain (NS5A-D2). GCK is the first rate-limiting enzyme of glycolysis in normal hepatocytes whose expression is replaced by the hexokinase 2 (HK2) isoenzyme in hepatocellular carcinoma cell lines. We took advantage of a unique cellular model specifically engineered to re-express GCK instead of HK2 in the Huh7 cell line to evaluate the consequences of NS5A-D2 expression on central carbon and lipid metabolism. NS5A-D2 increased glucose consumption but decreased glycogen storage. This was accompanied by an altered mitochondrial respiration, an accumulation of intracellular triglycerides and an increased production of very-low density lipoproteins. Altogether, our results show that NS5A-D2 can reprogram central carbon metabolism towards a more energetic and glycolytic phenotype compatible with HCV needs for replication.

Instability of the NS1 Glycoprotein from La Reunion 2018 Dengue 2 Virus (Cosmopolitan-1 Genotype) in Huh7 Cells Is Due to Lysine Residues on Positions 272 and 324.

La Reunion island in the South West Indian Ocean is now endemic for dengue following the introduction of dengue virus serotype 2 (DENV-2) cosmopolitan-I genotype in 2017. DENV-2 infection causes a wide spectrum of clinical manifestations ranging from flu-like disease to severe dengue. The nonstructural glycoprotein 1 (NS1) has been identified as playing a key role in dengue disease severity. The intracellular NS1 exists as a homodimer, whereas a fraction is driven towards the plasma membrane or released as a soluble hexameric protein. Here, we characterized the NS1 glycoproteins from clinical isolates DES-14 and RUN-18 that were collected during the DENV-2 epidemics in Tanzania in 2014 and La Reunion island in 2018, respectively. In relation to hepatotropism of the DENV, expression of recombinant DES-14 NS1 and RUN-18 NS1 glycoproteins was compared in human hepatoma Huh7 cells. We observed that RUN-18 NS1 was poorly stable in Huh7 cells compared to DES-14 NS1. The instability of RUN-18 NS1 leading to a low level of NS1 secretion mostly relates to lysine residues on positions 272 and 324. Our data raise the issue of the consequences of a defect in NS1 stability in human hepatocytes in relation to the major role of NS1 in the pathogenesis of the DENV-2 infection.

The current landscape of coronavirus-host protein-protein interactions.

In less than 20 years, three deadly coronaviruses, SARS-CoV, MERS-CoV and SARS-CoV-2, have emerged in human population causing hundreds to hundreds of thousands of deaths. Other coronaviruses are causing epizootic representing a significant threat for both domestic and wild animals. Members of this viral family have the longest genome of all RNA viruses, and express up to 29 proteins establishing complex interactions with the host proteome. Deciphering these interactions is essential to identify cellular pathways hijacked by these viruses to replicate and escape innate immunity. Virus-host interactions also provide key information to select targets for antiviral drug development. Here, we have manually curated the literature to assemble a unique dataset of 1311 coronavirus-host protein-protein interactions. Functional enrichment and network-based analyses showed coronavirus connections to RNA processing and translation, DNA damage and pathogen sensing, interferon production, and metabolic pathways. In particular, this global analysis pinpointed overlooked interactions with translation modulators (GIGYF2-EIF4E2), components of the nuclear pore, proteins involved in mitochondria homeostasis (PHB, PHB2, STOML2), and methylation pathways (MAT2A/B). Finally, interactome data provided a rational for the antiviral activity of some drugs inhibiting coronaviruses replication. Altogether, this work describing the current landscape of coronavirus-host interactions provides valuable hints for understanding the pathophysiology of coronavirus infections and developing effective antiviral therapies.

Toll-like Receptor 4-Induced Glycolytic Burst in Human Monocyte-Derived Dendritic Cells Results from p38-Dependent Stabilization of HIF-1α and Increased Hexokinase II Expression.

Cell metabolism now appears as an essential regulator of immune cells activation. In particular, TLR stimulation triggers metabolic reprogramming of dendritic cells (DCs) with an increased glycolytic flux, whereas inhibition of glycolysis alters their functional activation. The molecular mechanisms involved in the control of glycolysis upon TLR stimulation are poorly understood for human DCs. TLR4 activation of human monocyte-derived DCs (MoDCs) stimulated glycolysis with an increased glucose consumption and lactate production. Global hexokinase (HK) activity, controlling the initial rate-limiting step of glycolysis, was also increased. TLR4-induced glycolytic burst correlated with a differential modulation of HK isoenzymes. LPS strongly enhanced the expression of HK2, whereas HK3 was reduced, HK1 remained unchanged, and HK4 was not expressed. Expression of the other rate-limiting glycolytic enzymes was not significantly increased. Exploring the signaling pathways involved in LPS-induced glycolysis with various specific inhibitors, we observed that only the inhibitors of p38-MAPK (SB203580) and of HIF-1α DNA binding (echinomycin) reduced both the glycolytic activity and production of cytokines triggered by TLR4 stimulation. In addition, LPS-induced HK2 expression required p38-MAPK-dependent HIF-1α accumulation and transcriptional activity. TLR1/2 and TLR2/6 stimulation increased glucose consumption by MoDCs through alternate mechanisms that are independent of p38-MAPK activation. TBK1 contributed to glycolysis regulation when DCs were stimulated via TLR2/6. Therefore, our results indicate that TLR4-dependent upregulation of glycolysis in human MoDCs involves a p38-MAPK-dependent HIF-1α accumulation, leading to an increased HK activity supported by enhanced HK2 expression.

Activity of hexokinase is increased by its interaction with hepatitis C virus protein NS5A.

UNLABELLED: The study of cellular central carbon metabolism modulations induced by viruses is an emerging field. Human cytomegalovirus (HCMV), herpes simplex virus (HSV), Kaposi's sarcoma-associated herpesvirus (KSHV), and hepatitis C virus (HCV) have been shown recently to reprogram cell metabolism to support their replication. During HCV infection the global glucidolipidic metabolism of hepatocytes is highly impacted. It was suggested that HCV might modify glucose uptake and glycolysis to increase fatty acids synthesis, but underlying mechanisms have not been completely elucidated. We thus investigated how HCV may modulate glycolysis. We observed that in infected Huh7.5 cells and in subgenomic replicon-positive Huh9.13 cells, glucose consumption as well as lactate secretion was increased. Using protein complementation assays and coimmunoprecipitation, we identified a direct interaction between the HCV NS5A protein and cellular hexokinase 2 (HK2), the first rate-limiting enzyme of glycolysis. NS5A expression was sufficient to enhance glucose consumption and lactate secretion in Huh7.5 cells. Moreover, determination of HK activity in cell homogenates revealed that addition of exogenous NS5A protein, either the full-length protein or its D2 or D3, but not D1, domain, was sufficient to increase enzyme activity. Finally, determination of recombinant HK2 catalytic parameters (V(max) and K(m)) in the presence of NS5A identified this viral protein as an activator of the enzyme. In summary, this study describes a direct interaction between HCV NS5A protein and cellular HK2 which is accompanied by an increase in HK2 activity that might contribute to an increased glycolysis rate during HCV infection. IMPORTANCE: Substantial evidence indicates that viruses reprogram the central carbon metabolism of the cell to support their replication. Nevertheless, precise underlying mechanisms are poorly described. Metabolic pathways are structured as connected enzymatic cascades providing elemental biomolecular blocks necessary for cell life and viral replication. In this study, we observed an increase in glucose consumption and lactate secretion in HCV-infected cells, revealing higher glycolytic activity. We also identified an interaction between the HCV NS5A nonstructural protein and cellular hexokinase 2, the first rate-limiting enzyme of glycolysis. This interaction results in an enhancement of catalytic parameters of the enzyme, which might explain, at least in part, the aerobic glycolysis shift observed in HCV-infected cells.

Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients.

Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

Circulating RNA molecules as biomarkers in liver disease.

Liver disease is a major cause of morbidity and mortality worldwide. As in other fields of medicine, there is a stringent need for non-invasive markers to improve patient diagnostics, monitoring and prognostic ability in liver pathology. Cell-free circulating RNA molecules have been recently acknowledged as an important source of potential medical biomarkers. However, many aspects related to the biology of these molecules remain to be elucidated. In this review, we summarize current concepts related to the origin, transportation and possible functions of cell-free RNA. We outline current development of extracellular RNA-based biomarkers in the main forms of non-inherited liver disease: chronic viral hepatitis, hepatocellular carcinoma, non-alcoholic fatty liver, hepato-toxicity, and liver transplantation. Despite recent technological advances, the lack of standardization in the assessment of these markers makes their adoption into clinical practice difficult. We thus finally review the main factors influencing quantification of circulating RNA. These factors should be considered in the reporting and interpretation of current findings, as well as in the proper planning of future studies, to improve reliability and reproducibility of results.

Dengue Virus dependence on glucokinase activity and glycolysis Confers Sensitivity to NAD(H) biosynthesis inhibitors.

Viruses have developed sophisticated strategies to control metabolic activity of infected cells in order to supply replication machinery with energy and metabolites. Dengue virus (DENV), a mosquito-borne flavivirus responsible for dengue fever, is no exception. Previous reports have documented DENV interactions with metabolic pathways and shown in particular that glycolysis is increased in DENV-infected cells. However, underlying molecular mechanisms are still poorly characterized and dependence of DENV on this pathway has not been investigated in details yet. Here, we identified an interaction between the non-structural protein 3 (NS3) of DENV and glucokinase regulator protein (GCKR), a host protein that inhibits the liver-specific hexokinase GCK. NS3 expression was found to increase glucose consumption and lactate secretion in hepatic cell line expressing GCK. Interestingly, we observed that GCKR interaction with GCK decreases DENV replication, indicating the dependence of DENV to GCK activity and supporting the role of NS3 as an inhibitor of GCKR function. Accordingly, in the same cells, DENV replication both induces and depends on glycolysis. By targeting NAD(H) biosynthesis with the antimetabolite 6-Amino-Nicotinamide (6-AN), we decreased cellular glycolytic activity and inhibited DENV replication in hepatic cells. Infection of primary organotypic liver cultures (OLiC) from hamsters was also inhibited by 6-AN. Altogether, our results show that DENV has evolved strategies to control glycolysis in the liver, which could account for hepatic dysfunctions associated to infection. Besides, our findings suggest that lowering intracellular availability of NAD(H) could be a valuable therapeutic strategy to control glycolysis and inhibit DENV replication in the liver.

IRGM is a common target of RNA viruses that subvert the autophagy network.

Autophagy is a conserved degradative pathway used as a host defense mechanism against intracellular pathogens. However, several viruses can evade or subvert autophagy to insure their own replication. Nevertheless, the molecular details of viral interaction with autophagy remain largely unknown. We have determined the ability of 83 proteins of several families of RNA viruses (Paramyxoviridae, Flaviviridae, Orthomyxoviridae, Retroviridae and Togaviridae), to interact with 44 human autophagy-associated proteins using yeast two-hybrid and bioinformatic analysis. We found that the autophagy network is highly targeted by RNA viruses. Although central to autophagy, targeted proteins have also a high number of connections with proteins of other cellular functions. Interestingly, immunity-associated GTPase family M (IRGM), the most targeted protein, was found to interact with the autophagy-associated proteins ATG5, ATG10, MAP1CL3C and SH3GLB1. Strikingly, reduction of IRGM expression using small interfering RNA impairs both Measles virus (MeV), Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1)-induced autophagy and viral particle production. Moreover we found that the expression of IRGM-interacting MeV-C, HCV-NS3 or HIV-NEF proteins per se is sufficient to induce autophagy, through an IRGM dependent pathway. Our work reveals an unexpected role of IRGM in virus-induced autophagy and suggests that several different families of RNA viruses may use common strategies to manipulate autophagy to improve viral infectivity.

High plasma level of nucleocapsid-free envelope glycoprotein-positive lipoproteins in hepatitis C patients.

UNLABELLED: Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture-produced HCV (HCVcc) and ex vivo-characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid-free subviral LVPs are assembled and secreted by apoB-producing cell lines. To determine whether such subviral particles circulate in HCV-infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low-density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A-purified LVPs contained at least the envelope glycoprotein E2 and E2-specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride-rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 10(6) apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid-free, envelope glycoprotein-containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. CONCLUSION: LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family.

[Role of cellular metabolism in the control of chronic viral hepatitis]. / Rôle du métabolisme cellulaire dans le contrôle des hépatites virales chroniques.

Hepatitis viruses modify the cellular metabolism of hepatocytes by interacting with specific enzymes such as glucokinase. The metabolic changes induced by viruses can have a direct impact on the innate antiviral response. The complex interactions between viral components, innate immunity, and hepatocyte metabolism explain why chronic hepatitis infections lead to liver inflammation, progressing to cirrhosis, fibrosis, and hepatocellular carcinoma. Metabolic regulators could be used in innovative therapies to deprive viruses of key metabolites and induce an antiviral defense.

Title: Rôle du métabolisme cellulaire dans le contrôle des hépatites virales chroniques. Abstract: Les virus des hépatites modifient le métabolisme cellulaire des hépatocytes en interagissant avec des enzymes spécifiques, telles que la glucokinase. Les changements métaboliques induits par les virus peuvent avoir un impact direct sur la réponse antivirale innée. Les interactions complexes entre les composants viraux, l'immunité innée et le métabolisme des hépatocytes expliquent pourquoi les infections hépatiques chroniques conduisent à l'inflammation du foie, évoluant vers la cirrhose, la fibrose et le carcinome hépatocellulaire. Des régulateurs du métabolisme pourraient être utilisés dans des thérapies innovantes pour priver les virus de métabolites clés et induire une défense antivirale.

What role for cellular metabolism in the control of hepatitis viruses?

Hepatitis B, C and D viruses (HBV, HCV, HDV, respectively) specifically infect human hepatocytes and often establish chronic viral infections of the liver, thus escaping antiviral immunity for years. Like other viruses, hepatitis viruses rely on the cellular machinery to meet their energy and metabolite requirements for replication. Although this was initially considered passive parasitism, studies have shown that hepatitis viruses actively rewire cellular metabolism through molecular interactions with specific enzymes such as glucokinase, the first rate-limiting enzyme of glycolysis. As part of research efforts in the field of immunometabolism, it has also been shown that metabolic changes induced by viruses could have a direct impact on the innate antiviral response. Conversely, detection of viral components by innate immunity receptors not only triggers the activation of the antiviral defense but also induces in-depth metabolic reprogramming that is essential to support immunological functions. Altogether, these complex triangular interactions between viral components, innate immunity and hepatocyte metabolism may explain why chronic hepatitis infections progressively lead to liver inflammation and progression to cirrhosis, fibrosis and hepatocellular carcinoma (HCC). In this manuscript, we first present a global overview of known connections between the innate antiviral response and cellular metabolism. We then report known molecular mechanisms by which hepatitis viruses interfere with cellular metabolism in hepatocytes and discuss potential consequences on the innate immune response. Finally, we present evidence that drugs targeting hepatocyte metabolism could be used as an innovative strategy not only to deprive viruses of key metabolites, but also to restore the innate antiviral response that is necessary to clear infection.

Morphological characterization and fusion properties of triglyceride-rich lipoproteins obtained from cells transduced with hepatitis C virus glycoproteins.

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of beta-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.

A hexokinase isoenzyme switch in human liver cancer cells promotes lipogenesis and enhances innate immunity.

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). Here, we show that in HCC tumors the highest expression level of HK2 is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK+/HK2- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition.

There is an increasing interest in the field of cancer therapy for small compounds targeting pyrimidine biosynthesis, and in particular dihydroorotate dehydrogenase (DHODH), the fourth enzyme of this metabolic pathway. Three available DHODH structures, featuring three different known inhibitors, were used as templates to screen in silico an original chemical library from Erevan University. This process led to the identification of P1788, a compound chemically related to the alkaloid cerpegin, as a new class of pyrimidine biosynthesis inhibitors. In line with previous reports, we investigated the effect of P1788 on the cellular innate immune response. Here we show that pyrimidine depletion by P1788 amplifies cellular response to both type-I and type II interferons, but also induces DNA damage as assessed by γH2AX staining. Moreover, the addition of inhibitors of the DNA damage response led to the suppression of the P1788 stimulatory effects on the interferon pathway. This demonstrates that components of the DNA damage response are bridging the inhibition of pyrimidine biosynthesis by P1788 to the interferon signaling pathway. Altogether, these results provide new insights on the mode of action of novel pyrimidine biosynthesis inhibitors and their development for cancer therapies.

Transactivation of the hepatitis B virus core promoter by the nuclear receptor FXRalpha.

Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXRalpha) in modulating transcription from the HBV core promoter. FXRalpha is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXRalpha). Electrophoretic mobility shift assays demonstrated that FXRalpha-RXRalpha heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXRalpha response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXRalpha. Moreover, using a greater-than-genome-length HBV construct, we showed that FXRalpha also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXRalpha is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.

Modified lipoproteins provide lipids that modulate dendritic cell immune function.

Both physiological and pathological situations can result in biochemical changes of low-density lipoproteins (LDL). Because they can deliver signals to dendritic cells (DC), these modified lipoproteins now appear as regulators of the immune response. Among these modified lipoproteins, oxidized LDL (oxLDL) that accumulate during inflammatory conditions have been extensively studied. Numerous studies have shown that oxLDL induce the maturation of DC, enhancing their ability to activate IFNγ secretion by T cells. LDL treated by secreted phospholipase A(2) also promote DC maturation. Among the bioactive lipids generated by oxidation or phospholipase treatment of LDL, lysophosphatidylcholine (LPC) and some saturated fatty acids induce DC maturation whereas some unsaturated fatty acids or oxidized derivatives have opposite effects. Among other factors, the nuclear receptor peroxisome-proliferator activated receptor γ (PPARγ) plays a crucial role in this regulation. Non-modified lipoproteins also contribute to the regulation of DC function, suggesting that the balance between native and modified lipoproteins, as well as the biochemical nature of the LDL modifications, can regulate the activation threshold of DC. Here we discuss two pathological situations in which the impact of LDL modifications on inflammation and immunity could play an important role. During atherosclerosis, modified LDL accumulating in the arterial intima may interfere with DC maturation and function, promoting a Th1 immune response and a local inflammation favoring the development of the pathology. In patients chronically infected, the hepatitis C virus (HCV) interferes with lipoprotein metabolism resulting in the production of infectious modified lipoproteins. These lipo-viral-particles (LVP) are modified low-density lipoproteins containing viral material that can alter DC maturation and affect specific toll-like receptor signaling. In conclusion, lipoprotein modifications play an important role in the regulation of immunity by delivering signals of danger to DC and modulating their function.

High-density lipoprotein phospholipids interfere with dendritic cell Th1 functional maturation.

Lipoproteins are both lipid carriers in the blood and regulators of essential biological processes. Several studies demonstrated that lipoproteins modified during pathological conditions could alter dendritic cell (DC) maturation. Here the immune function of non-pathological lipoproteins is addressed by analysing their impact on human DC maturation triggered by TLR ligands. Upon TLR4 stimulation, low- and high-density lipoproteins (LDL and HDL) strongly inhibited the ability of DC to induce a Th1 response of T cells, characterized by high levels of IFNγ secretion, whereas the effect of very low-density lipoprotein was subject to variations. HDL also inhibited the Th1 function of DC stimulated by TLR1/2 and TLR2/6 ligands. The phospholipid fraction from HDL retained the inhibitory activity of the lipoprotein. We identified the 1-palmitoyl-2-linoleyl-phosphatidylcholine (PLPC) as one active phospholipid that inhibited the Th1 function of mature DCs whereas the dipalmitoyl-phosphatidylcholine had no significant effect. The treatment of DC by PLPC, 24h before TLR4 stimulation, resulted in reduced activation of NF-κB. This study shows that some HDL phospholipids have a direct immunoregulatory function, by modulating DC ability to activate a Th1 response of T cells.