Abstract Rationale FBXO5, a member of the F-box protein family, is an essential receptor-like component of an E3 ligase involved in protein degradation. While multiple studies suggest FBXO5 role in carcinogenesis and inflammation, direct studies linking FBXO5 to lung inflammation are very limited. Methods siRNA library against the human Fbox genes was used for screening and identification of Fbox genes involved in the induction of pro-inflammatory cytokine. qPCR, ELISA and RNAi were used to study gene expression by silencing specific genes. Immunoprecipitation and mass spectrometry was used to identify the interacting partners of FBXO5 protein. Results To elucidate the function and specificity of FBXO5, we performed siRNA library screening for different Fbox genes. Through this screening using bronchial epithelial cells we identified FBXO5 as key suppressor of inflammation. The FBXO5-depleted cells show elevated expression of the pro-inflammatory cytokine IL-6 after induction with Pam3CSK4, a potent immune activator thereby stimulating the production of pro-inflammatory cytokines. Further, FBXO5 depletion and its rescue experiments in different cell lines, including BEAS-2B, human primary and macrophage-like (THP-1) cells suggest its direct role in suppression of inflammation by modulating pro-inflammatory cytokine secretion. Further, the FBXO5 interacting partners or its probable substrates were identified by Mass Spectrometry using co-immunoprecipitation samples purified from cells stably expressing dual-tagged FBXO5. The preliminary validation experiments of selected mass spec hits suggest that the depletion of KCTD2 expression induces the pro-inflammatory cytokine (IL-6) secretion similar to the levels of FBXO5. Hence, KCTD2 may act as a probable FBXO5 substrate involved in inflammatory response. Conclusions The mechanistic insight involved in FBXO5 mediated suppression of lung inflammation is poorly understood. Using the RNAi, gene expression and mass spectrometry here we present the molecular mechanism of FBXO5 in immune suppression, potentially providing new insights into treating chronic inflammatory lung diseases. This abstract is funded by: This work is funded by: NIH grants; P01HL114453, R01HL096376, R01HL081784, R01HL097376 to RM
Pradhan et al. (Fri,) studied this question.