Abstract:
FAM26F (family with sequence similarity 26, member F) is a tetraspanin-like membrane
glycoprotein involved in calcium homeostasis and in immune modulation. There is
substantial evidence supporting FAM26F's potential role in immunity, but the protein's
complete modulatory signalling pathways have yet to be investigated. The current study aims
to explore the expression of FAM26F in vitro in human embryonic kidney cell line (HEK 293), and how its inhibition or overexpression affects the downstream signalling cascade
proteins. This was investigated through two ways: 1) siRNA based knockdown study carried
out by transfecting cells with FAM26F siRNA to assess the expression of FAM26F. 2)
Overexpression achieved in the HEK293 cell line through plasmid-mediated delivery of the
FAM26F gene. The transfection was confirmed through one-step qPCR and western blotting
of transfected cells, and then MS/MS analysis was performed to determine the proteins
affected by inhibition and overexpression of FAM26F. MS/MS analysis revealed that among
the overexpressed cells, there were 780 proteins, 85 of which were up-regulated and 695
were down-regulated. In the knockdown group, however, only two proteins were up regulated and twenty were down-regulated. Out of these, all of knockdown proteins, and top
50 hits from overexpressed category were selected and analysed using STRING software to
predict their protein-protein interaction, and Reactome pathway browser for pathway
analysis. STRING detected no interactions between FAM26F and any of the other proteins.
However, it identified interactions among other various proteins. Reactome pathway
analysis revealed proteins to be primarily involved in gene expression, immune system,
protein metabolism (primarily translation), infectious disease (HIV and SARS CoV2), RNA
metabolism, and vesicle-mediated transport pathways. Validating the findings of previous
research, this study demonstrates that FAM26F plays a role in immunity and various
anomalies by either regulating differential gene expression or protein metabolism. The exact
relation of FAM26F with these proteins can be validated in future by employing real time
PCR technique for gene expression analysis and western blotting for proteomics analysis.
