Identification of immune effectors and the post-translational modifications that control their activity is essential for dissecting mechanisms of immunity. Here we demonstrate that the antiviral activity of interferon-induced transmembrane protein 3 (IFITM3) is post-translationally regulated by S-palmitoylation. Large-scale profiling of palmitoylated proteins in a dendritic cell line using a chemical reporter strategy revealed over 150 lipid-modified proteins with diverse cellular functions, including innate immunity. We discovered that S-palmitoylation of IFITM3 on membrane-proximal cysteines controls its clustering in membrane compartments and its antiviral activity against influenza virus. The sites of S-palmitoylation are highly conserved among the IFITM family of proteins in vertebrates, which suggests that S-palmitoylation of these immune effectors may be an ancient post-translational modification that is crucial for host resistance to viral infections. The S-palmitoylation and clustering of IFITM3 will be important for elucidating its mechanism of action and for the design of antiviral therapeutics.Vertebrates have evolved sophisticated innate and adaptive mechanisms of immunity to combat microbial pathogens 1 . In response, viruses and pathogenic bacteria have acquired virulence factors that subvert or disarm host defenses 1 . For example, cellular membranes provide a simple barrier to infection, but viruses such as influenza virus have evolved specific proteins that fuse with membranes to allow viral replication inside host cells 2 . Alternatively, intracellular bacterial pathogens are taken up by phagocytic cells, but they then remodel cellular membranes to prevent their own degradation inside lysosomal compartments 3 . Cellular membranes are key interfaces for host resistance and prime targets for microbial virulence factors. We therefore performed large-scale profiling of palmitoylated proteins in phagocytic cells to identify membrane-associated proteins that contribute to immunity against microbial pathogens. We found that S-palmitoylation of interferon-induced transmembrane protein 3 (IFITM3) enhances its clustering in membranes * Correspondence and requests for materials should be addressed to H.C.H. hhang@rockefeller.edu. Author contributionsJ.S.Y. conceived the study, designed and performed experiments, interpreted data and co-wrote the manuscript; Y.-Y.Y. and G.C. synthesized reagents for palmitoylome profiling studies; B.M., C.B.L. and T.M.M. provided reagents and expertise on influenza virus infections; H.C.H. conceived the study, designed experiments, interpreted data and co-wrote the manuscript. Competing financial interestsThe authors declare no competing financial interests. Additional information RESULTS Proteomic analysis of palmitoylated proteins in DC2.4 cellsTo identify lipid-modified and membrane-associated proteins that may contribute to immune responses to microbial infections, we performed large-scale profiling of fatty-acylated proteins in the mouse DC line DC2.4 (ref. ...

Background: IFITM3 is a protein of the innate immune system that inhibits viral infections. Results: S-palmitoylation enhances IFITM3 membrane affinity and antiviral activity, whereas ubiquitination decreases endolysosome localization and antiviral activity. Conclusion: IFITM3 is dually and opposingly regulated by posttranslational modifications, and study of these modifications has led to an unpredicted intramembrane topology model. Significance: Understanding modes of IFITM3 regulation is critical for dissecting molecular mechanisms controlling viral inhibition.

Fatty-acylation of proteins in eukaryotes is associated with many fundamental cellular processes but has been challenging to study due to limited tools for rapid and robust detection of protein fatty-acylation in cells. The development of azido-fatty acids enabled the nonradioactive detection of fatty-acylated proteins in mammalian cells using the Staudinger ligation and biotinylated phosphine reagents. However, the visualization of protein fatty-acylation with streptavidin blotting is highly variable and not ideal for robust detection of fatty-acylated proteins. Here we report the development of alkynyl-fatty acid chemical reporters and improved bioorthogonal labeling conditions using the Cu(I)-catalyzed Huisgen [3 + 2] cycloaddition that enables specific and sensitive fluorescence detection of fatty-acylated proteins in mammalian cells. These improvements allow the rapid and robust biochemical analysis of fatty-acylated proteins expressed at endogenous levels in mammalian cells by in-gel fluorescence scanning. In addition, alkynyl-fatty acid chemical reporters enable the visualization of fatty-acylated proteins in cells by fluorescence microscopy and flow cytometry. The ability to rapidly visualize protein fatty-acylation in cells using fluorescence detection methods therefore provides new opportunities to interrogate the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.

On detecting viral RNAs, the RNA helicase retinoic acid-inducible gene I (RIG-I) activates the interferon regulatory factor 3 (IRF3) signalling pathway to induce type I interferon (IFN) gene transcription. How this antiviral signalling pathway might be negatively regulated is poorly understood. Microarray and bioinformatic analysis indicated that the expression of RIG-I and that of the tumour suppressor CYLD (cylindromatosis), a deubiquitinating enzyme that removes Lys 63-linked polyubiquitin chains, are closely correlated, suggesting a functional association between the two molecules. Ectopic expression of CYLD inhibits the IRF3 signalling pathway and IFN production triggered by RIG-I; conversely, CYLD knockdown enhances the response. CYLD removes polyubiquitin chains from RIG-I as well as from TANK binding kinase 1 (TBK1), the kinase that phosphorylates IRF3, coincident with an inhibition of the IRF3 signalling pathway. Furthermore, CYLD protein level is reduced in the presence of tumour necrosis factor and viral infection, concomitant with enhanced IFN production. These findings show that CYLD is a negative regulator of RIG-I-mediated innate antiviral response. Keywords: cylindromatosis; interferon; IRF3; RIG-I; ubiquitin EMBO reports (2008) 9, 930-936.

Fatty acylation of cysteine residues provides spatial and temporal control of protein function in cells and regulates important biological pathways in eukaryotes. Although recent methods have improved the detection and proteomic analysis of cysteine fatty (S-fatty) acylated proteins, understanding how specific sites and quantitative levels of this posttranslational modification modulate cellular pathways are still challenging. To analyze the endogenous levels of protein S-fatty acylation in cells, we developed a mass-tag labeling method based on hydroxylamine-sensitivity of thioesters and selective maleimide-modification of cysteines, termed acyl-PEG exchange (APE). We demonstrate that APE enables sensitive detection of protein S-acylation levels and is broadly applicable to different classes of S-palmitoylated membrane proteins. Using APE, we show that endogenous interferoninduced transmembrane protein 3 is S-fatty acylated on three cysteine residues and site-specific modification of highly conserved cysteines are crucial for the antiviral activity of this IFN-stimulated immune effector. APE therefore provides a general and sensitive method for analyzing the endogenous levels of protein S-fatty acylation and should facilitate quantitative studies of this regulated and dynamic lipid modification in biological systems.fatty-acylation | palmitoylation | PEGylation | influenza virus | IFITM3 P rotein S-fatty acylation describes the covalent attachment of long-chain fatty acids to cysteine (Cys) residues through a thioester bond, which alters the hydrophobicity of diverse proteins and regulates their stability, trafficking, and activity in eukaryotic cells ( Fig. 1A) (1, 2). Cys residues are predominately acylated with palmitic acid (S-palmitoylation), but can also be modified with longer chain and unsaturated fatty acids, thus more generally described as S-fatty acylation (1, 3, 4). The fatty-acylation of Cys residues is regulated by the DHHC-family of protein acyltransferases (5, 6) and different classes of thioesterases (7,8) that are associated with a variety of important physiological pathways and diseases (1). Determining the precise levels of protein S-fatty acylation is therefore crucial for understanding how this dynamic lipid modification is regulated and quantitatively controls specific cellular pathways.Recent methods to detect and enrich S-fatty acylated proteins have facilitated the characterization of key regulatory mechanisms and discovery of new S-fatty acylated proteins (1, 2). For example, alkyne-modified fatty acid chemical reporters have enabled the fluorescent detection and enrichment of metabolically labeled proteins using bioorthogonal ligation methods (Fig. S1A) (9, 10). Alternatively, exploitation of thioester sensitivity to hydroxylamine (NH 2 OH) has enabled selective capture and analysis of S-acylated proteins by acyl-biotin exchange (ABE) (Fig. S1B) (11, 12) or acyl-resin capture (acyl-RAC) (Fig. S1C) (13). However, all of these methods do not readily reveal the fraction of unmodified...