Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon- production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.

Non-liganded retinoic acid receptors (RARs) repress transcription of target genes by recruiting the histone deacetylase complex through a class of silencing mediators termed SMRT or N-CoR. Mutant forms of RARalpha, created by chromosomal translocations with either the PML (for promyelocytic leukaemia) or the PLZF (for promyelocytic leukaemia zinc finger) locus, are oncogenic and result in human acute promyelocytic leukaemia (APL). PML-RARalpha APL patients achieve complete remission following treatments with pharmacological doses of retinoic acids (RA); in contrast, PLZF-RARalpha patients respond very poorly, if at all. Here we report that the association of these two chimaeric receptors with the histone deacetylase (HDAC) complex helps to determine both the development of APL and the ability of patients to respond to retinoids. Consistent with these observations, inhibitors of histone deacetylase dramatically potentiate retinoid-induced differentiation of RA-sensitive, and restore retinoid responses of RA-resistant, APL cell lines. Our findings suggest that oncogenic RARs mediate leukaemogenesis through aberrant chromatin acetylation, and that pharmacological manipulation of nuclear receptor co-factors may be a useful approach in the treatment of human disease.

SUMMARY TRIM25 mediates Lys 63-linked ubiquitination of the N-terminal CARDs of the viral RNA sensor RIG-I, leading to type I interferon (IFN) production. Here, we report that the influenza A virus non-structural protein 1 (NS1) specifically inhibits TRIM25-mediated RIG-I CARD ubiquitination, thereby suppressing RIG-I signal transduction. A novel domain in NS1 comprising E96/E97 residues mediates its interaction with the coiled-coil domain of TRIM25, thus blocking TRIM25 multimerization and RIG-I CARD ubiquitination. Furthermore, a recombinant influenza A virus expressing an E96A/E97A NS1 mutant is defective in blocking TRIM25-mediated anti-viral IFN response and loses virulence in mice. Our findings reveal a novel mechanism of influenza virus to inhibit host IFN response and also emphasize the vital role of TRIM25 in modulating viral infections.

Although accurate prediction of survival is essential for palliative care, few clinical methods of determining how long a patient is likely to live have been established. To develop a validated scoring system for survival prediction, a retrospective cohort study was performed with a training-testing procedure on two independent series of terminally ill cancer patients. Performance status (PS) and clinical symptoms were assessed prospectively. In the training set (355 assessments on 150 patients) the Palliative Prognostic Index (PPI) was defined by PS, oral intake, edema, dyspnea at rest, and delirium. In the testing sample (233 assessments on 95 patients) the predictive values of this scoring system were examined. In the testing set, patients were classified into three groups: group A (PPI< or =2.0), group B (2.04.0). Group B survived significantly longer than group C, and group A survived significantly longer than either of the others. Also, when a PPI of more than 6 was adopted as a cut-off point, 3 weeks' survival was predicted with a sensitivity of 80% and a specificity of 85%. When a PPI of more than 4 was used as a cutoff point, 6 weeks' survival was predicted with a sensitivity of 80% and a specificity of 77%. In conclusion, whether patients live longer than 3 or 6 weeks can be acceptably predicted by PPI.

Silk fibroin produced by the silkworm Bombyx mori consists of a heavy chain, a light chain, and a glycoprotein, P25. The heavy and light chains are linked by a disulfide bond, and P25 associates with disulfide-linked heavy and light chains by noncovalent interactions. Quantitative enzyme-linked immunosorbent assay revealed that molar ratios of the heavy chain, light chain, and P25 were 6:6:1, both in cocoons and in fibroin secreted into the lumen of posterior silk gland. Trace amounts of fibroin produced by three "naked pupa" mutants of B. mori lacked the light chain, but the molar ratio of heavy chain and P25 was also 6:1. Gel filtration chromatography and sedimentation equilibrium analysis demonstrated that a large protein complex of approximately 2.3 MDa, designated an elementary unit of fibroin having 6:6:1 molar ratios of the heavy chain, light chain, and P25, existed in posterior silk gland cells. Inaccessibility of biotinylated concanavalin A to the native elementary unit and partial dissociation of the elementary unit after incubation with excess N-glycosidase F or endoglycosidase H suggest that a single molecule of P25 is located internally and plays an important role in maintaining integrity of the complex.A vast amount of silk fibroin is synthesized within the cells of a pair of PSGs 1 of the silkworm, Bombyx mori, during the larval fifth instar, secreted into the lumen of PSG, and transported through the middle silk gland, where heterogeneous molecules of sericin are added, and further toward the anterior part of the silk gland, where the silk fiber is formed and spun. Both the efficient secretion of the vast amount of fibroin from PSG cells into the lumen and the maintenance of solubility of fibroin during the luminal transport have been speculated to be facilitated by the formation of a molecular complex consisting of fibroin heavy chain (H-chain) of 350 kDa and two lower molecular mass protein components: fibroin light chain (Lchain) of 26 kDa (1) and P25, which is a glycoprotein of about 30 kDa (2).H-chain and L-chain are linked by a single disulfide bond between Cys-172 of L-chain and Cys-c20 (twentieth residue from the C terminus) of H-chain (3). The H-L linkage is essential for the secretion of a vast amount of fibroin, because the silkworm carrying the Nd-s or Nd-s D mutation of the L-chain gene (fibL) could not form the disulfide linkage with H-chain, and less than 1% of the normal level fibroin is secreted (4).This tremendous reduction of fibroin secretion has been suggested to be caused by the appearance of a free sulfhydryl group of Cys-c20 on H-chain, which prevents the transport of H-chain from ER to Golgi (4).On the other hand, P25 has been shown to associate with the H-L complex by noncovalent interactions, i.e. mainly by hydrophobic interactions with the H-chain moiety (2, 5). However, the role of P25 in the formation of the fibroin molecular complex has not been elucidated, mainly because a silkworm carrying a mutation of the P25 gene has not been available.In the present study, a m...

Controversy over the role of antioxidants in cancer has persisted for decades. Here, we demonstrate that synthesis of the antioxidant glutathione (GSH), driven by GCLM, is required for cancer initiation. Genetic loss of Gclm prevents a tumor's ability to drive malignant transformation. Intriguingly, these findings can be replicated using an inhibitor of GSH synthesis, but only if delivered prior to cancer onset, suggesting that at later stages of tumor progression GSH becomes dispensable potentially due to compensation from alternative antioxidant pathways. Remarkably, combined inhibition of GSH and thioredoxin antioxidant pathways leads to a synergistic cancer cell death in vitro and in vivo, demonstrating the importance of these two antioxidants to tumor progression and as potential targets for therapeutic intervention.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

The human estrogen receptor (hER) exists as two subtypes, hER a a and hER b b, that differ in the C-terminal ligand-binding domain and in the N-terminal transactivation domain. In this study, we investigated the estrogenic activities of soy isoflavones after digestion with enteric bacteria in competition binding assays with hER a a or hER b b protein, and in a gene expression assay using a yeast system. The estrogenic activities of these isoflavones were also investigated by the growth of MCF-7 breast cancer cells.Isoflavone glycoside binds weakly to both receptors and estrogen receptor-dependent transcriptional expression is poor. The aglycones bind more strongly to hER b b than to hER a a. The binding affinities of genistein, dihydrogenistein and equol are comparable to the binding affinity of 17 b b-estradiol. Equol induces transcription most strongly with hER a a and hER b b. The concentration required for maximal gene expression is much higher than expected from the binding affinities of the compounds, and the maximal activity induced by these compounds is about half the activity of 17 b b-estradiol. Although genistin binds more weakly to the receptors and induces transcription less than does genistein, it stimulates the growth of MCF-7 cells more strongly than does genistein.