A study on human IFIT3 modulation of IFIT1 protein stability
Interferon-induced proteins with tetratricopeptide repeats (IFITs) are expressed in both interferon-dependent and interferon-independent manners in cells after pathogen-associated molecular pattern recognition by the pathogen recognition receptors. Four IFITs (IFIT1, IFIT2, IFIT3 and IFIT5) have been identified and characterized in humans. They are cytoplasmic proteins with repetitive tetratricopeptide repeats protein motifs well-characterized to mediate protein-protein interactions. IFITs play several functions in cells and their antiviral roles are the most well-established, one of which includes the ability of IFIT1 to bind non-self cap0 mRNAs and inhibit their translation. IFIT1 interacts with IFIT2 and IFIT3 in cells while IFIT5 does not. We show that transfected IFIT1 expressed poorly on its own in cells, but its protein level was greatly enhanced when co-expressed with IFIT3. Likewise, downregulation of endogenous IFIT3 decreased the expression of IFIT1 at only the protein level without affecting IFIT1 transcript level. The stabilization was found to be dependent on the interaction of the C- terminus of IFIT3 with IFIT1 via a specific motif in both proteins, disruption of which significantly reduced IFIT1 stability. We then examined IFIT1 degradation in the absence of IFIT3 using inhibitors of either the proteasomal or the autophagy-lysosomal protein degradation pathway in both endogenous and overexpression systems. We developed a novel system using tandem mass tag (TMT)-based quantitative proteomics aimed at identifying IFIT1 binding proteins which interact in the absence of IFIT3. Overall, our findings illuminate the mechanism involved in IFIT3 modulation of the protein stability of IFIT1. These findings will contribute to a deeper understanding of the role of IFITs during immune responses and may help in identifying methods by which their function can be manipulated.