iProXProteomicsFei GuoHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0011430000guoafei@hotmail.comNational Institute of Pathogen Biology, Chinese Academy of Medical SciencesiProXThe Membrane-Associated RING-CH (MARCH) family of E3 ubiquitin ligases modulates membrane protein stability and contributes to host antiviral immune responses. Their functions largely depend on the nature of protein substrates. In this study, we identified interferon-induced transmembrane protein 3 (IFITM3), a critical antiviral effector protein, as a novel substrate of MARCH8 by coimmunoprecipitation coupled with LC-MS/MS analysis. Mechanistically, MARCH8 promotes the lysosomal degradation of IFITM3 primarily through K63-linked polyubiquitination at lysine 24, which facilitates its trafficking and turnover from the plasma membrane to endosomes and lysosomes. MARCH8-KO cells exhibit plasma membrane accumulation of IFITM3 compared with WT controls after interferon treatment. Functionally, MARCH8 expression attenuated the IFITM3-mediated restriction of vesicular stomatitis virus and influenza A virus entry, thereby increasing cell susceptibility to viral infection. Together, these findings establish a novel regulatory mechanism whereby MARCH8 modulates innate immunity through regulating the trafficking and turnover of antiviral protein IFITM3.MARCH8-mediated ubiquitination regulates expression of the antiviral protein IFITM3.Wei Liang L, Zhao Fei F, Liu Xiaoman X, Mei Shan S, Huang Yu Y, Xie Yu Y, Hu Yamei Y, Wang Liming L, Wang Lingwa L, Gao Zhao Z, Chen Chen C, Shi Yueyue Y, He Yurong Y, Wang Jiaxun J, Xue Tiffany T, Xu Fengwen F, Fang Jugao J, Guo Fei FfalseProteomic Profiling in HeLa CellsThis study combined co-immunoprecipitation (Co-IP) and LC-MS/MS proteomics in MARCH8-overexpressing HeLa cells to identify potential substrates of the E3 ubiquitin ligase MARCH8. Using empty vector-transfected cells as controls, we generated a comprehensive protein interaction dataset to elucidate MARCH8’s regulatory mechanisms in membrane protein trafficking and degradation, providing a resource for exploring its roles in viral defense, immune modulation, and related pathologies.Sun Mar 23 00:00:00 GMT 2025PXD062157960641197719