N4BP3 Regulates RIG-I-Like Receptor Antiviral Signaling Positively by Targeting Mitochondrial Antiviral Signaling Protein

Mitochondrial antiviral signaling protein (MAVS), an adaptor protein, is activated by RIG-I, which is critical for an effective innate immune response to infection by various RNA viruses. Viral infection causes the RIG-I-like receptor (RLR) to recognize pathogen-derived dsRNA and then becomes activated to promote prion-like aggregation and activation of MAVS. Subsequently, through the recruitment of TRAF proteins, MAVS activates two signaling pathways mediated by TBK1-IRF3 and IKK- NF-κb, respectively, and turns on type I interferon and proinflammatory cytokines. This study discovered that NEDD4 binding protein 3 (N4BP3) is a positive regulator of the RLR signaling pathway by targeting MAVS. Overexpression of N4BP3 promoted virus-induced activation of the interferon-β (IFN-β) promoter and interferon-stimulated response element (ISRE). Further experiments showed that knockdown or knockout N4BP3 impaired RIG-I-like receptor (RLR)-mediated innate immune response, induction of downstream antiviral genes, and cellular antiviral responses. We also detected that N4BP3 could accelerate the interaction between MAVS and TRAF2. Related experiments revealed that N4BP3 could facilitate the ubiquitination modification of MAVS. These findings suggest that N4BP3 is a critical component of the RIG-I-like receptor (RLR)-mediated innate immune response by targeting MAVS, which also provided insight into the mechanisms of innate antiviral responses.

MiR-144-3p Regulates Lipopolysaccharide-Stimulated Chondrocyte Biological Behavior via Wingless-Related Integration Site/Beta-Catenin

Osteoarthritis (OA) gradually affects all joint tissues. Chondrocytes participate in osteoarthritis. However, the role and mechanism of MiR-144-3p on chondrocytes during the development of OA has not been elucidated. OA patients and normal bone and articular cartilage tissues were collected to measure MiR-144-3p level by Real-time PCR. Chondrocytes were divided into control group, LPS group (1 μg/ml lipopolysaccharide (LPS) was added to establish an osteoarthritis (OA) stimulation model, and MiR-144-3p inhibitor group which was transfected with MiR-144-3p inhibitor followed by analysis of cell proliferation by MTT, Caspase 3 activity, Wnt/β-catenin signaling protein expression by Western blot and TNF-α and IL-6 secretion by ELISA. MiR-144-3p was significantly upregulated in OA patients (P <0.05). In LPS group, MiR-144-3p was significantly upregulated, chondrocyte proliferation decreased, Caspase 3 activity increased, Wnt/β-catenin signaling protein decreased, and TNF-α and IL-6 secretion increased (P <0.05). MiR-144-3p inhibitor transfection can significantly down-regulate MiR-144-3p, promote cell proliferation, reduce Caspase 3 activity, increase Wnt/β-catenin signaling protein expression, and reduce TNF-α and IL-6 secretion (P <0.05). MiR-144-3p is upregulated in osteoarthritis cartilage tissue. Inhibition of MiR-144-3p can inhibit articular chondrocytes apoptosis under inflammatory condition, promote cell proliferation, and alleviate joint inflammation by regulating Wnt/β-catenin signaling pathway.

Fructose appetition in "taste-blind" P2X2/P2X3 double knockout mice

Inbred mouse strains differ in their postoral appetite stimulating response (appetition) to glucose and fructose. For example, C57BL/6J (B6) and FVB strains learn strong preferences for a flavor (CS+, e.g., cherry) paired with intragastric (IG) glucose infusions, but only FVB mice learned to prefer a CS+ paired with IG fructose infusions. Consistent with these findings, "tasteless" B6 knockout (KO) mice missing the taste signaling protein TRPM5 learn strong preferences for a CS+ added to glucose solution as well as for unflavored glucose but weak or no preferences for a fructose-paired CS+ or unflavored fructose. The present experiment reports that "tasteless" P2X2/P2X3 double-knockout (P2X2/3 DKO) mice, unlike TRPM5 KO mice, learned strong preferences for a CS+ mixed with fructose as well as for unflavored fructose. Whether differences in genetic backgrounds or other factors account for the fructose appetition displayed by P2X2/3 DKO mice but not TRPM5 KO mice remains to be determined.

Ensemble epistasis: thermodynamic origins of non-additivity between mutations

Epistasis—when mutations combine non-additively—is a profoundly important aspect of biology. It is often difficult to understand its mechanistic origins. Here we show that epistasis can arise from the thermodynamic ensemble, or the set of interchanging conformations a protein adopts. Ensemble epistasis occurs because mutations can have different effects on different conformations of the same protein, leading to non-additive effects on its average, observable properties. Using a simple analytical model, we found that ensemble epistasis arises when two conditions are met: 1) a protein populates at least three conformations and 2) mutations have differential effects on at least two conformations. To explore the relative magnitude of ensemble epistasis, we performed a virtual deep-mutational scan of the allosteric signaling protein S100A4. We found that 47% of mutation pairs exhibited ensemble epistasis with a magnitude on the order of thermal fluctuations. We observed many forms of epistasis: magnitude, sign, and reciprocal sign epistasis. The same mutation pair could even exhibit different forms of epistasis under different environmental conditions. The ubiquity of thermodynamic ensembles in biology and the pervasiveness of ensemble epistasis in our dataset suggests that it may be a common mechanism of epistasis in proteins and other macromolecules.

ZomB is essential for chemotaxis of Vibrio alginolyticus by the rotational direction control of the polar flagellar motor

Bacteria exhibit chemotaxis by controlling flagellar rotation to move toward preferred places or away from non-preferred places. The change in rotation is triggered by the binding of the chemotaxis signaling protein CheY to the C-ring in the flagellar motor. Some specific bacteria, including Vibrio spp. and Shewanella spp. have a single transmembrane protein called ZomB. ZomB is essential for controlling the flagellar rotational direction in Shewanella putrefaciens and Vibrio parahaemolyticus. In this study, we confirmed that the zomB deletion results only in the counterclockwise (CCW) rotation of the motor in Vibrio alginolyticus as previously reported in other bacteria. We found that ZomB is not required for the clockwise (CW) rotation-fixing phenotype caused by mutations in fliG and fliM, and that ZomB is essential for CW rotation induced by overproduction of CheY. Purified ZomB proteins form multimers, indicating that ZomB functions as a complex. ZomB may interact with a protein involved in the flagellar rotation, stator proteins or rotor proteins. We found that ZomB is a new player in chemotaxis and is required for the rotational control in addition to CheY in Vibrio alginolyticus.

Activation of the Anti-Oxidative Stress Response Reactivates Latent HIV-1 Through the Mitochondrial Antiviral Signaling Protein Isoform MiniMAVS

The mitochondrial antiviral signaling protein (MAVS) is part of the cell’s innate immune mechanism of defense. MAVS mRNA is bicistronic and can give rise to a full length-MAVS and a shorter isoform termed miniMAVS. In response to viral infections, viral RNA can be sensed by the cytosolic RNA sensors retinoic acid-inducible gene I (RIG-I) and/or melanoma differentiation-associated protein 5 (MDA5) and activate NF-κB through interaction with MAVS. MAVS can also sense cellular stress and activate an anti-oxidative stress (AOS) response through the activation of NF-κB. Because NF-κB is a main cellular transcription factor for HIV-1, we wanted to address what role MAVS plays in HIV-1 reactivation from latency in CD4 T cells. Our results indicate that RIG-I agonists required full length-MAVS whereas the AOS response induced by Dynasore through its catechol group can reactivate latent HIV-1 in a MAVS dependent manner through miniMAVS isoform. Furthermore, we uncover that PKC agonists, a class of latency-reversing agents, induce an AOS response in CD4 T cells and require miniMAVS to fully reactivate latent HIV-1. Our results indicate that the AOS response, through miniMAVS, can induce HIV-1 transcription in response to cellular stress and targeting this pathway adds to the repertoire of approaches to reactivate latent HIV-1 in ‘shock-and-kill’ strategies.