BackgroundEpstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied.Methods and FindingsUsing Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate.ConclusionsOur data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus , GBS), resulting in the activation of IRF5 and production of IFNβ, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus , GBS, Streptococcus pneumonia, Pseudomonas aeruginosa , and E. coli . The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus , GBS, and S. pneumonia , the TLR8 inhibitor almost eliminated the production of IL-1β and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1β, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNβ and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1β and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.

IntroductionThe CD150 (IPO-3, SLAM) cell surface receptor is expressed on activated T and B lymphocytes, dendritic cells, and monocytes. CD150 is a member of the CD2 subfamily of the immunoglobulin (Ig) superfamily of receptors and shares homology with members of this subfamily such as CD2, CD48, CD58, CD229, CD244, BLAME, SF2001, NTB-A/SF2000, CD84, and CS1/CRACC. 1-5 CD150 has diverse functions including costimulation of T and B cells, augmentation of T-cell cytotoxicity and CD95-mediated apoptosis, and also regulation of proliferation and differentiation of B cells. 1,2,[6][7][8] Moreover, a number of morbilliviruses, including the measles virus, use CD150 as a receptor for cellular entry. 9,10 The apparently opposing functions of CD150 are linked to the unique structure of its cytoplasmic tail, which contains a paired immunoreceptor tyrosine-based switch motif (ITSM) TxYxxV/ I. 3,11 This switch motif is involved directly and/or indirectly in binding different signal transduction molecules, including the protein tyrosine phosphatase SHP-2, the inositol phosphatase SHIP, the Src-family kinases Fyn, Lyn, and Fgr, and the adaptor molecules SH2D1A and EAT-2. 7,12-14 Since SH2D1A regulates binding of different sets of SH2-containing molecules to the CD150 cytoplasmic tail, 11,13,[15][16][17] it may switch the signal transduction pathways initiated via CD150. 3 SH2D1A is encoded by a gene that is altered in most patients with X-linked lymphoproliferative disorder (XLP), as well as in a subset of patients with B-cell non-Hodgkin lymphoma (NHL), common variable immunodeficiency syndrome, and familial hemophagocytic lymphohistiocytosis. 12,14,18,19 SH2D1A was found in T lymphocytes, 18,20 natural killer (NK) cells, 21,22 and in a small subpopulation of tonsillar B cells. 11 In contrast to its limited expression in primary human B cells, some Burkitt lymphoma cell lines with germinal center phenotype, Hodgkin disease (HD) cell lines, and a few B lymphoblastoid cell lines express high level of SH2D1A mRNA and protein. 11,23,24 Since the malignant cells in NHL and HD represent normal B-cell counterparts arrested at different stages of differentiation, 25,26 we hypothesize that SH2D1A may be expressed during a restricted period of B-cell maturation, where it might function to coordinate the intracellular signaling pathways required for cell survival, proliferation, and/or differentiation.Despite our knowledge about CD150 interaction with molecules that are involved in intracellular signaling, very little is known about the mechanisms that regulate CD150-mediated signal transduction in normal or transformed B lymphocytes. To further address this question we used the DT40 B-cell line model system. Here we showed that in B cells, CD150 is linked to ERK and Akt For personal use only. on June 19, 2019. by guest www.bloodjournal.org From pathways, and signals elicited by CD150 may differ depending on concurrent SH2D1A expression. To determine whether SH2D1A and CD150 may cooperate during B-cell development, we analyzed CD150...

Epstein-Barr virus (EBV), like other DNA tumor viruses, induces an S-phase in the natural host cell, the human B lymphocyte. This is linked with blast transformation. It is believed that the EBVencoded nuclear antigen 6 (EBNA-6) is involved in the regulation of cell cycle entry. However, the possible mechanism of this regulation is not approached. In our current study, we found that EBNA-6 binds to a MRPS18-2 protein, and targets it to the nucleus. We found that MRPS18-2 binds to both hypo-and hyperphosphorylated forms of Rb protein specifically. This binding targets the small pocket of pRb, which is a site of interaction with E2F1. The MRPS18-2 competes with the binding of E2F1 to pRb, thereby raising the level of free E2F1. Our experimental data suggest that EBNA-6 may play a major role in the entry of EBV infected B cells into the S phase by binding to and raising the level of nuclear MRPS18-2, protein. This would inhibit pRb binding to E2F1 competitively and lift the block preventing S-phase entry.cell transformation ͉ cell cycle ͉ surface plasmon resonance ͉ S-phase entry

Yurchenko et al. discover that the Ig-like receptor molecule SLAMF1 enhances production of type I interferon induced by Gram-negative bacteria through modulation of MyD88-independent TLR4 signaling. This makes SLAMF1 a potential target for controlling inflammatory responses against Gram-negative bacteria.

Biosensor technologies based on optical readout are widely used in protein-protein interaction studies. Here we describe a fast and simple approach to the creation of oriented interfacial architectures for surface plasmon resonance (SPR) transducers, based on conventional biochemical procedures and custom reagents. The proposed protocol permits the oriented affinity-capture of GST fusion proteins by a specific antibody which is bound to protein A, which in turn has been immobilized on the transducer surface (after the surface has been modified by guanidine thiocyanate). The applicability of the method was demonstrated by studying the interaction between retinoblastoma tumor suppressor protein (pRb) and MRS18-2 proteins. The formation of the pRb-MRS18-2 protein complex was examined and the pRb binding site (A-box-spacer-B-box) was mapped. We have also shown that MRS18-2, which was detected as the Epstein-Barr virus-encoded EBNA-6 binding partner using the yeast two-hybrid system, binds to pRb in GST pull-down assays.

TLR8 is the major endosomal sensor of degraded RNA in human monocytes and macrophages. It has been implicated in the sensing of viruses and more recently also bacteria. We previously identified a TLR8-IFN regulatory factor 5 (IRF5) signaling pathway that mediates IFNβ and interleukin-12 (IL-12) induction by Staphylococcus aureus and is antagonized by TLR2. The relative importance of TLR8 for the sensing of various bacterial species is however still unclear. We here compared the role of TLR8 and IRF5 for the sensing of Group B Streptococcus (GBS), S. aureus, and Escherichia coli in human primary monocytes and monocyte-derived macrophages (MDM). GBS induced stronger IFNβ and TNF production as well as IRF5 nuclear translocation compared to S. aureus grown to the stationary phase, while S. aureus in exponential growth appeared similarly potent to GBS. Cytokine induction in primary human monocytes by GBS was not dependent on hemolysins, and induction of IFNβ and IL-12 as well as IRF5 activation were reduced with TLR2 ligand costimulation. Heat inactivation of GBS reduced IRF5 and NF-kB translocation, while only the viable E. coli activated IRF5. The attenuated stimulation correlated with loss of bacterial RNA integrity. The E. coli-induced IRF5 translocation was not inhibited by TLR2 costimulation, suggesting that IRF5 was activated via a TLR8-independent mechanism. Gene silencing of MDM using siRNA revealed that GBS-induced IFNβ, IL-12-p35, and TNF production was dependent on TLR8 and IRF5. In contrast, cytokine induction by E. coli was TLR8 independent but still partly dependent on IRF5. We conclude that TLR8-IRF5 signaling is more important for the sensing of GBS than for stationary grown S. aureus in human primary monocytes and MDM, likely due to reduced resistance of GBS to phagosomal degradation and to a lower production of TLR2 activating lipoproteins. TLR8 does not sense viable E. coli, while IRF5 still contributes to E. coli-induced cytokine production, possibly via a cytosolic nucleic acid sensing mechanism.

We report that the overexpression of mitochondrial ribosomal protein MRPS18 -2 (S18 -2) can immortalize primary rat embryonic fibroblasts (REFs). The immortalized cells (18IM) lose contact inhibition, form foci, and are capable of anchorage-independent growth. Concurrently, mesodermal markers, such as vimentin, smooth muscle actin, and Fut4, disappear completely. 18IM cells express embryonic stem cell markers, such as SSEA-1, Sox2, and Oct3/4. In confluent cultures, a portion of cells also express ectoderm-and endoderm-specific pan-keratin, ectoderm-specific beta-III-tubulin, mesoderm-specific MHC class II, and become stainable for fat with Oil red O. None of these changes was detected in c-myc؉Ha-ras (MR)-transformed cells. In immunodeficient mice, 18IM cells formed small transiently growing tumors that have down-regulated SSEA-1 and showed pan-keratin staining. We conclude that S18 -2 can immortalize REFs and induces them to express stem cell traits.immortalization ͉ mitochondrial ribosomal protein