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Review
. 2023 Nov 28;15(12):2329.
doi: 10.3390/v15122329.

Discovery and Characterization of IFITM S-Palmitoylation

Tandrila Das  1 , Howard C Hang  2
Affiliations
Review

Discovery and Characterization of IFITM S-Palmitoylation

Tandrila Das et al. Viruses. .

Abstract

Interferon-induced transmembrane proteins (IFITM1, 2 and 3) are important host antiviral defense factors. They are active against viruses like the influenza A virus (IAV), dengue virus (DENV), Ebola virus (EBOV), Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus (SARS-CoV). In this review, we focus on IFITM3 S-palmitoylation, a reversible lipid modification, and describe its role in modulating IFITM3 antiviral activity. Our laboratory discovered S-palmitoylation of IFITMs using chemical proteomics and demonstrated the importance of highly conserved fatty acid-modified Cys residues in IFITM3 antiviral activity. Further studies showed that site-specific S-palmitoylation at Cys72 is important for IFITM3 trafficking to restricted viruses (IAV and EBOV) and membrane-sterol interactions. Thus, site-specific lipid modification of IFITM3 directly regulates its antiviral activity, cellular trafficking, and membrane-lipid interactions.

Keywords: IFITM3; S-palmitoylation; influenza A virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
S-palmitoylation of proteins. Dynamic S-palmitoylation is mediated by DHHC palmitoyl acyltransferases (DHHC-PATs) and depalmitoylases.
Figure 2
Figure 2
Discovery and analysis of IFITM3 S-palmitoylation. (a) Metabolic labeling of cells with palmitic acid reporters and further reaction with azido-modified fluorescent or affinity tags (in pink) for fluorescent visualization or affinity enrichment of S-palmitoylated proteins enabled discovery of IFITM3 S-Palmitoylation. (b) Acyl-PEG exchange (APE) involves the capping of free cysteine residues with N-ethyl maleimide (NEM) and the removal of fatty acid groups with hydroxylamine (NH2OH). Then, the exposed cysteines are reacted with mPEG-Mal. Proteins are separated via SDS-PAGE and analyzed using Western blot, enabling the detection of both unmodified and S-fatty acylated proteins. (c) S-palmitoylation analysis of endogenous proteins. Also, palmitic acid reporter modified proteins are reacted with azide-biotin (in pink) and captured on Neutravidin beads. Then, thioesters are hydrolyzed with hydroxylamine (NH2OH) and alkylated with iodoacetamide before LC-MS/MS analysis. Cys modified by iodoacetamide are marked as S-fatty-acylation sites.
Figure 3
Figure 3
Genetic code expansion enables the site-specific incorporation of an unnatural amino acid into a protein utilizing cellular translation machinery, which further enables bio-orthogonal ligation with tetrazine dyes for live cell labeling and imaging studies.
Figure 4
Figure 4
Chemical lipidation of recombinant IFITM3 and IFITM3 in cells. (a) Site-specific lipidation of recombinant IFITM3 using maleimide coupling. (b) Scheme for the site-specific lipidation of IFITM3 via genetic code expansion for unnatural amino acid incorporation and the bio-orthogonal tetrazine ligation reaction).
See this image and copyright information in PMC

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