The knowledge of a protein's subcellular localization and interacting partners are crucial for elucidating its cellular function and associated regulatory networks. Although FAM26F (family with sequence similarity 26, member F) has been recognized as a vital player in various infections, stimulation studies, cancer, and immune pathogenesis, the precise location and function of FAM26F are not well understood. The current study is the first to focus on functional characterization of FAM26F by analyzing its subcellular localization and identifying its novel interacting partners using advanced proteome approaches. The immunofluorescence and confocal microscopy results revealed FAM26F to be largely localized within the Golgi apparatus of the cell. However, its minor presence in endoplasmic reticulum (ER) pointed toward the probable retrograde transfer of FAM26F from Golgi to ER during adverse conditions. Moreover, co-immunoprecipitation and MS/MS results demonstrated a total of 85 proteins, 44 of which significantly copurified with FAM26F. Interestingly, out of these 44 MS/MS identified proteins, almost 52% were involved in innate immunity, 38.6% in neutrophil degranulation, and remaining 10% were either involved in phosphorylation, degradation, or regulation of apoptosis. Further characterization through Ingenuity Pathway Analysis showed that majority of these proteins was involved in maintaining calcium homeostasis of cell. Consequently, the validation of selected proteins uncovered the key interaction of FAM26F with Thioredoxin, which essentially paved the way for depicting its mechanism of action under stress or disease conditions. It is proposed that activation and inhibition of the cellular immune response is essentially dependent on whether FAM26F or Thioredoxin considerably interact with CD30R.
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