Functional Characterization of Fam26f Based on Its Subcellular Localization and Novel Interacting Partners

Abstract

Human immune system is a multifaceted entity capable of defending the human body against microbial infections by utilizing a network of cells and proteins, destructive enzymes and chemical mediators. About a decade ago, FAM26F was recognized as a protein differentially expressed in various anomalies. At present, it has gained much significance as being a critical modulator in diverse immune responses. Despite the potential role of FAM26F in immune modulation and hence its therapeutic potential, the knowledge of its subcellular localization and interacting partners which can shed light on its specific function is still lacking. The current study was aimed 1) to functionally characterize FAM26F through computational methods; 2) to determine FAM26F’s subcellular localization and its interacting partners in order to decipher the particular pathway which is regulated when FAM26F is expressed in a cell; and lastly 3) to analyze its expression with reference to its key interactor in normal and pathological condition to get an insight about its behavior and mechanism of action. Numerous advanced online computational tools were employed for the in silico characterization of FAM26F. Immuno-fluorescence was conducted on HEK293 cells transfected with GFP-tagged Human FAM26F plasmid followed by confocal laser scanning microscopy to determine the subcellular localization of FAM26F. Subsequently, co immunoprecipitation of FAM26F complex was performed using G coupled magnetic Dyna beads. The interactors of FAM26F were identified by ESI QTOF MS/MS, and its signaling pathway was then determined through Reactome Pathway Database and Ingenuity Pathway Analysis software. Finally, qPCR analysis was performed to determine the expression of FAM26F and its chief interactor in the normal individuals and HIV patients. The in silico results revealed FAM26F to be a 315 amino acid long, stable protein that has remained well-conserved throughout evolution. It is a signal peptide deprived transmembrane protein that is secreted through non-classical pathway. The presence of a single well-conserved Ca_hom_mod domain indicated FAM26F to be a cation channel involved in the transport of molecules. A potential N-glycosylation and 14 phosphorylation sites were also predicted. Moreover, the presence of an immunoglobin-like fold in FAM26F emphasized its role in immune responses. The immuno-fluorescence results revealed FAM26F to be largely localized within the Golgi apparatus of the Abstract xvi cell. However, its minor presence in endoplasmic reticulum (ER) pointed towards the probable retrograde transfer of FAM26F from Golgi to ER during adverse conditions. The co-immunoprecipitation and MS/MS results demonstrated a total of 85 proteins, 44 of which significantly co-purified with FAM26F. Interestingly, more than half of these 44 proteins were involved in innate immune system. Further characterizations showed that FAM26F largely interacts with proteins mediating calcium homeostasis of a cell, particularly with Thioredeoxin, which essentially paved the way for depicting its mechanism of action under stress/disease conditions. It is proposed that activation and inhibition of cellular immune response is essentially dependent on whether FAM26F or Thioredoxin considerably interacts with CD30R. Furthermore, the expression analysis of FAM26F along with Thioredoxin in healthy individuals and HIV patients revealed significant downregulation of FAM26F and upregulation of Thioredoxin in HIV patients as compared to controls. As initial stages of the HIV-1 life cycle considerably depend on Trx1, whereas FAM26F is a potent immune regulator and antiviral agent, new generation of anti-HIV therapeutics based on silencing of the Trx1/TrxR1 system and inciting FAM26F expression, as well as targeting the viral surface glycoproteins, may represent a promising approach for HIV treatment. The study essentially and comprehensively demonstrates the presence of FAM26F is the Golgi apparatus, and the involvement of FAM26F in regulating Ca2+ signaling of a cell. The future need to study FAM26F in other cell lines and animal models (both normal and diseased) in the context of immune dysfunction is emphasized. Moreover, knockdown studies to identify the specific Ca2+ signaling genes regulated by FAM26F expression should be explored. By highlighting the role of FAM26F as a potential therapeutic target in infections, cancers and immune diseases, it is proposed that combination therapies should be employed wile devising the treatment regime for deadly diseases, such as HIV.