Lipid chemical reporters and bioorthogonal ligation methods developed by my laboratory have provided new opportunities to investigate the functions of lipid-modified proteins in biology. Our proteomic analysis of fatty-acylated proteins in dendritic cells using fatty acid chemical reporters and bioorthogonal chemical proteomics has revealed a new role for protein S-fatty-acylation in host defense against viral infections. We discovered that S-fatty-acylation of membrane-proximal cysteines on murine IFITM3 is crucial for its membrane localization and antiviral activity against influenza A virus. The mechanisms that control S-fatty-acylation of human IFITM proteins have not been evaluated and will be addressed in this grant proposal.
Aim 1 will evaluate the S-fatty-acylation levels, sites and function of human IFITM isoforms in different cell types/states of activation.
Aim 2 describes the characterization of fatty acids covalently attached to human IFITM3 in cells.
Aim 3 describes site-specific lipidation and reconstitution of IFITM3 in vitro for detailed biochemical and biophysical studies. Determining the mechanisms that control S-fatty-acylation IFITM3 function is crucial for understanding host immunity and may reveal new strategies for combatting virus infection in humans. The chemical approaches described in this grant should provide new reagents and methods for studying fatty-acylated proteins.

Public Health Relevance

This proposal describes chemical and biochemical approaches to characterize the roles of protein lipidation in host resistance to microbial infections. Our preliminary studies have demonstrated that S-fatty-acylation of interferon-inducible transmembrane proteins (IFITMs) is required for cellular resistance to influenza virus infection. How S-fatty-acylation controls human IFITM3 function is unclear and will be evaluated in this grant with new chemical biology tools, cell biology assays, in vitro protein biochemistry and infection studies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Nie, Zhongzhen
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Rockefeller University
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New York
United States
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Thinon, Emmanuelle; Fernandez, Joseph P; Molina, Henrik et al. (2018) Selective Enrichment and Direct Analysis of Protein S-Palmitoylation Sites. J Proteome Res 17:1907-1922
Percher, Avital; Thinon, Emmanuelle; Hang, Howard (2017) Mass-Tag Labeling Using Acyl-PEG Exchange for the Determination of Endogenous Protein S-Fatty Acylation. Curr Protoc Protein Sci 89:14.17.1-14.17.11
McMichael, Temet M; Zhang, Lizhi; Chemudupati, Mahesh et al. (2017) The palmitoyltransferase ZDHHC20 enhances interferon-induced transmembrane protein 3 (IFITM3) palmitoylation and antiviral activity. J Biol Chem 292:21517-21526
Westcott, Nathan P; Fernandez, Joseph P; Molina, Henrik et al. (2017) Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress. Nat Chem Biol 13:302-308
Thinon, Emmanuelle; Percher, Avital; Hang, Howard C (2016) Bioorthogonal Chemical Reporters for Monitoring Unsaturated Fatty-Acylated Proteins. Chembiochem 17:1800-1803
Peng, Tao; Hang, Howard C (2016) Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells. J Am Chem Soc 138:14423-14433
Percher, Avital; Ramakrishnan, Srinivasan; Thinon, Emmanuelle et al. (2016) Mass-tag labeling reveals site-specific and endogenous levels of protein S-fatty acylation. Proc Natl Acad Sci U S A 113:4302-7
Peng, Tao; Thinon, Emmanuelle; Hang, Howard C (2016) Proteomic analysis of fatty-acylated proteins. Curr Opin Chem Biol 30:77-86
Thinon, Emmanuelle; Hang, Howard C (2015) Chemical reporters for exploring protein acylation. Biochem Soc Trans 43:253-61
Moudgil, Devinderjit K; Westcott, Nathan; Famulski, Jakub K et al. (2015) A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores. J Cell Biol 208:881-96

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