Chemoproteomics reveals Toll-like receptor fatty acylation.

Nicholas M Chesarino,James L Chen,Larry S Schlesinger,Howard C Hang,Murugesan Vs Rajaram,Balyn W Zaro,Joanne Turner,Jocelyn C Hach,Matthew R Pratt,Jacob S Yount

doi:10.1186/s12915-014-0091-3

Abstract

BackgroundPalmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.ResultsA total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands.ConclusionsThis work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. S-palmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0091-3) contains supplementary material, which is available to authorized users.

Highlights

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity
Visualization of palmitoylated proteins in murine embryonic fibroblasts (MEFs) and DC2.4 cells 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
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

Summary

Introduction

Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. Protein palmitoylation is the addition of a 16-carbon fatty acid primarily to cysteines via a thioester linkage (termed S-palmitoylation) [1]. This form of fatty acylation targets cytoplasmic proteins to membranes, but can occur on transmembrane proteins where it often affects protein localization or stability [1,2,3]. We detected novel palmitoylated proteins expressed at steady state, and validated several of these proteins, including T-lymphocyte activation antigen CD86 and Toll-like receptor 2 (TLR2) in dendritic cells (DCs), and E3 ubiquitin-protein ligase NEDD4 in MEFs. Given the complete novelty of the discovery of a lipid modification occurring on a member of the extensively studied TLR family, we chose to focus further on determining the effects of palmitoylation on TLR2

Methods

Results

Conclusion