Palmitoylation on Conserved and Nonconserved Cysteines of Murine IFITM1 Regulates Its Stability and Anti-Influenza A Virus Activity

Abstract

The interferon-induced transmembrane proteins (IFITMs) restrict infection by numerous viruses, yet the importance and regulation of individual isoforms remains unclear. Here, we report that murine IFITM1 (mIFITM1) is palmitoylated on one nonconserved cysteine and three conserved cysteines that are required for anti-influenza A virus activity. Additionally, palmitoylation of mIFITM1 regulates protein stability by preventing proteasomal degradation, and modification of the nonconserved cysteine at the mIFITM1 C terminus supports an intramembrane topology with mechanistic implications.T he interferon-induced transmembrane proteins (IFITMs) have been described to inhibit infection by a wide range of viruses, including important human pathogens such as influenza virus, West Nile virus, severe acute respiratory syndrome (SARS) coronavirus, human immunodeficiency virus, and dengue virus (1-6). The viruses that are restricted share the common ability to enter cells through endocytosis, and the restriction is indeed dependent upon the glycoproteins used for cellular entry (1, 2). Inhibition by IFITM3 occurs prior to influenza virus fusion with the endosome (7), which is a primary site of IFITM3 localization (2,7,8). IFITM3 appears to promote acidification or merging of endosomes with lysosomes, thereby exposing viral particles to a degradative environment (7-9). Consistent with this model, IFITM3 has recently been shown to reduce membrane fluidity and alter the curvature of membranes in such a way that virus hemifusion from within the endosome is inhibited while the fusion of opposing membranes possessing IFITMs may be enhanced (10).The mechanism by which IFITMs change membrane curvature and the endolysosomal compartment is unknown. Addressing this question, we have recently proposed that the predicted dual transmembrane topology for IFITM3 is incorrect and that the active protein instead contains intramembrane domains that do not fully span the lipid bilayer ( Fig. 1A and B) (8). Partial insertion of intramembrane domains into the membrane bilayer is well understood to promote curvature by virtue of the bilayer couple effect (Fig. 1B) (11). Evidence for this topology emerges largely from analysis of posttranslational modifications of IFITM3. Mass spectrometry and mutational analysis identified ubiquitination of lysine 24 (K24) on the IFITM3 N terminus (8). If the predicted topology is adopted, this residue would reside in the endoplasmic reticulum (ER) or endosomal lumen. However, known ubiquitin ligases are cytoplasmic, suggesting that the IFITM3 N terminus is localized in the cytoplasm. This is also supported by the discovery that phosphorylation of IFITM3 on tyrosine 20 (Y20) by the cytoplasmic kinase Fyn is required for IFITM3 antiviral activity (12). We further tested this alternative topology hypothesis by demonstrating that neither the N nor the C terminus nor the central loop domain of IFITM3 is exposed to the ER lumen glycosylation machinery but an antivirally active IFITM3 mutant engineered to possess myristoy...

Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation

“…S5 and S6). Our results are consistent with previous IFITM mutagenesis and antiviral activity studies (21,22,27,32,35), but demonstrates that antiviral activity is directly correlated to S-fatty acylation levels and that dually S-fatty acylated IFITM3 at Cys72 and Cys105 is likely the most active isoform in mammalian cells (Fig. S9).…”

Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites

“…Digestion with chymotrypsin yielded 137 modified peptides from 44 proteins, 19 of which were identified in the alk-16 + NH 2 OH (SI Table S4). Some known S-fatty-acylation sites were identified, including sites for IFITM3, 10 FLOT1, 43 Caveolin-1, 44 STOM, 45 and IFITM1 46 (SI Figure S17, SI Table S6). As expected, both enzymes produced distinct list of peptides due to different peptide length and solubility.…”

Chemoproteomics reveals Toll-like receptor fatty acylation

“…Having made the previous discovery of the critical role of palmitoylation of IFITMs in the innate antiviral immune response [7,14,15], we sought to determine whether any additional IFN-induced proteins are regulated by palmitoylation. For our experiments, we chose to use murine antigen presenting cells (DC2.4) and MEFs because these cell lines are responsive to type I IFNs [14], are amenable to labeling with the alk-16 reporter of protein palmitoylation [6,14] and serve as a control for one another in that type I IFN should induce a similar set of proteins in both cell types.…”

“…Azido-rhodamine (az-rho) is a detection tag that can be used for visualization, while azido-azo-biotin (az-biotin) allows selective retrieval of alk-16-labeled proteins using streptavidincoated agarose [7,8,12]. We, and others, have previously used, and continue to apply these tools for enhancing our understanding of regulation of immune responses by lipid post-translational modifications (PTMs) [7,11,[13][14][15][16][17].…”