Abstract
Recently, one of the interferon-induced transmembrane (IFITM) family proteins, IFITM3, has become an important target for the activity against influenza A (H1N1) virus infection. In this protein, a post-translational modification by fatty acids covalently attached to cysteine, termed S-palmitoylation, plays a crucial role for the antiviral activity. IFITM3 possesses three cysteine residues for the S-palmitoylation in the first transmembrane (TM1) domain and in the cytoplasmic (CP) loop. Because these cysteines are well conserved in the mammalian IFITM family proteins, the S-palmitoylation on these cysteines is significant for their functions. IFITM5 is another IFITM family protein and interacts with the FK506-binding protein 11 (FKBP11) to form a higher-order complex in osteoblast cells, which induces the expression of immunologically relevant genes. In this study, we investigated the role played by S-palmitoylation of IFITM5 in its interaction with FKBP11 in the cells, because this interaction is a key process for the gene expression. Our investigations using an established reporter, 17-octadecynoic acid (17-ODYA), and an inhibitor for the S-palmitoylation, 2-bromopalmitic acid (2BP), revealed that IFITM5 was S-palmitoylated in addition to IFITM3. Specifically, we found that cysteine residues in the TM1 domain and in the CP loop were S-palmitoylated in IFITM5. Then, we revealed by immunoprecipitation and western blot analyses that the interaction of IFITM5 with FKBP11 was inhibited in the presence of 2BP. The mutant lacking the S-palmitoylation site in the TM1 domain lost the interaction with FKBP11. These results indicate that the S-palmitoylation on IFITM5 promotes the interaction with FKBP11. Finally, we investigated bone nodule formation in osteoblast cells in the presence of 2BP, because IFITM5 was originally identified as a bone formation factor. The experiment resulted in a morphological aberration of the bone nodule. This also indicated that the S-palmitoylation contributes to bone formation.
Highlights
The interferon-induced transmembrane (IFITM) protein family is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino- and carboxyl-terminal polypeptide sequences (Figure 1-A)
Identification of S-palmitoylation on IFITM5 To identify the S-palmitoylation on IFITM5, the osteoblast cells harboring the plasmid DNA encoding IFITM5-WT were cultured in the absence and presence of 2-bromopalmitic acid (2BP), which inhibits the S-palmitoylation (Figure 2-A) [31]
These results suggested that IFITM5-WT has high and low molecular-mass forms in the absence and presence of 2BP, respectively
Summary
The interferon-induced transmembrane (IFITM) protein family ( known as the Fragilis family in mice) is a part of the dispanin family [1] and is composed of double-transmembrane α-helices connected by a cytoplasmic (CP) loop and extracellular (EC) amino- and carboxyl-terminal polypeptide sequences (Figure 1-A). Recent genomic research has revealed that there are 5 IFITM members in humans (IFITM1, 2, 3, 5 and 10) and 7 members in mice (IFITM1, 2, 3, 5, 6, 7, and 10) These proteins play roles in diverse biological processes, such as germ cell maturation during gastrulation (IFITM1-3) [3,4,5], cell-to-cell adhesion (IFITM1) [6,7,8], antiviral activity (IFITM1-3) [9,10,11,12,13,14,15,16,17], and bone formation (IFITM5) [18,19,20,21,22], the detailed functions of IFITM6, 7, and 10 are unknown at present.
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