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FLN29 was identified as an interferon (IFN)-inducible gene, and it has been shown to suppress Toll-like receptor 4-mediated NF-κB activation by binding to TRAF6. To elucidate the physiological roles of FLN29, we generated FLN29-deficient mice. FLN29 deficiency resulted in hyper-response to LPS both in vivo and in vitro, demonstrating the negative regulatory role of FLN29 in TLR4 signaling. Furthermore, we found that FLN29–/– mice exhibited increased susceptibility to poly(I:C)-induced septic shock compared with WT mice. FLN29–/– fibroblasts were highly resistant to vesicular stomatitis virus infection, and these cells produced more IFN-β than WT cells did in response to not only intracellular poly(I:C) but also overexpression of IPS-1. Forced expression of FLN29 inhibited the IPS-1-dependent activation of both NF-κB and IRF3. We also found that FLN29 could interact with TRIF, IPS-1, TRAF3, and TRAF6. Together, these results suggest that FLN29, in addition to playing a negative regulatory role in the TLR4 signaling pathway, negatively regulates the RIG-I-like helicase signaling pathway at the level of IPS-1/TRAF6 and IPS-1/TRAF3 complexes.

Lipopolysaccharide (LPS) activates macrophages through toll-like receptor (TLR) 4. Although the mechanism of the TLR signaling pathway has been well documented, the mechanism of the negative regulation in response to LPS, particularly LPS tolerance, is still poorly understood. In this study we identified and characterized a novel interferon- and LPS-inducible gene, FLN29, which contains a TRAF6-related zinc finger motif and TRAF family member-associated NF-kappaB activator-related sequences. The induction of FLN29 was dependent on STAT1. The forced expression of FLN29 in macrophage-like RAW cells resulted in the suppression of TLR-mediated NF-kappaB and mitogen-activated protein kinase activation, while a reduced expression of FLN29 by small interfering RNA partly cancelled the down-regulation of LPS signaling. Furthermore, we demonstrated that NF-kappaB activation induced by TRAF6 and TAB2 was impaired by co-expression of FLN29, suggesting FLN29 may regulate the downstream of TRAF6. Taken together, FLN29 is a new negative feedback regulator of TLR signaling.

Histidine-rich protein-2 from Plasmodium falciparum (PfHRP2) binds up to 50 molecules of ferri-protoporphyrin IX (FePPIX) (Choi, C. Y., Cerda, J. F., Chu, H. A., Babcock, G. T., and Marletta, M. A. (1999) Biochemistry 38, 16916-16924). We reasoned that the PfHRP2-FePPIX complex has antioxidant properties that could be beneficial to the parasite. Therefore, we examined whether binding to PfHRP2 modulated the redox properties of FePPIX. We observed that PfHRP2 completely inhibited the auto-oxidation of ascorbate mediated by free FePPIX. We also investigated the peroxidase activity of PfHRP2-FePPIX using 13-hydroperoxy-9,11-octadienoate (18:2-OOH) as substrate. Reaction of PfHRP2-FePPIX with 18:2-OOH in the presence of added reducing agents gave 13-hydroxy-9,11-octadienoate (18:2-OH) as a major product and 13-keto-9,11-octadienoate (18:2=O) and 9,12,13-trihydroxy-10-octadecaenoate as minor products. Binding of FePPIX to PfHRP2 lowered the rate of decomposition of 18:2-OOH and increased the 18:2-OH to 18:2=O ratio. Similar to other authentic peroxidases, phenols, amines, and biological reductants like ascorbate promoted 18:2-OH production, and NaCN inhibited 18:2-OH production. Thioanisole also acted as a reductant and was converted to thioanisole sulfoxide, suggesting formation of compound I during the reaction. These data show that PfHRP2 modulates the redox activity of FePPIX and that the PfHRP2-FePPIX complex may have previously unrecognized antioxidant properties.