Bioorthogonal proteomics of 15-hexadecynyloxyacetic acid chemical reporter reveals preferential targeting of fatty acid modified proteins and biosynthetic enzymes

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Abstract

Chemical reporters are powerful tools for the detection and discovery of protein modifications following cellular labeling. The metabolism of alkyne- or azide-functionalized chemical reporters in cells can influence the efficiency and specificity of protein targeting. To evaluate the effect of degradation of chemical reporters of protein fatty acylation, we synthesized 15-hexadecynyloxyacetic acid (HDYOA), a reporter that was designed to be resistant to β-oxidation, and compared its ability to label palmitoylated proteins with an established reporter, 17-octadecynoic acid (ODYA). HDYOA was able to label known candidate S-palmitoylated proteins similarly to ODYA. Accordingly, bioorthogonal proteomic analysis demonstrated that 70% of proteins labeled with ODYA were also labeled with HDYOA. However, the proteins observed differentially in our proteomic studies suggested that a portion of ODYA protein labeling is a result of β-oxidation. In contrast, downstream enzymes involved in β-oxidation of fatty acids were not targeted by HDYOA. Since HDYOA can label S-palmitoylated proteins and is not utilized by downstream β-oxidation pathways, this fatty acid chemical reporter may be particularly useful for bioorthogonal proteomic studies in cell types metabolically skewed toward fatty acid breakdown.

Introduction

Chemical proteomics using bioorthogonal ligation methods has revealed new posttranslational modifications on proteins as well as targets of small molecules.1, 2, 3, 4, 5 The administration of azide- or alkyne-functionalized metabolites or drugs to cell lysates or living cells has enabled covalent labeling of protein targets that can be visualized or identified via copper catalyzed azide-alkyne cycloaddition (CuAAC) with fluorescent or affinity detection tags (Fig. 1A).1, 2, 3, 4, 5 For protein lipidation, our laboratory and others have developed azido- or alkynyl lipid analogs to detect and identify fatty-acylated6, 7, 8, 9 (Fig. 1B), prenylated10, 11, 12, 13 and cholesterylated14 proteins in mammalian cells as well as lipoproteins in bacteria.15 These lipid chemical reporters are facilitating the discovery of new lipidated proteins and the characterization of their regulatory mechanisms in diverse biological contexts.

Since protein S-palmitoylation is dynamic and difficult to predict, fatty acid chemical reporters have been particularly useful for evaluating this class of protein lipidation. Protein S-palmitoylation is a posttranslational, covalent addition of a 16-carbon saturated fatty acid chain on cysteine residues via a thioester bond (Fig. 1C). This modification not only increases hydrophobicity and thus membrane affinity, but also can affect protein stability, clustering, trafficking, and activity.16, 17 For example, the oncogenic H/N-Ras small GTPases localize to the plasma membrane or the Golgi apparatus depending on their palmitoylation state, and signal through distinct pathways at each location.18, 19 In addition to H/N-Ras, understanding the dynamic S-palmitoylation of other proteins will be important. For these studies alkynyl-fatty acid reporters in conjunction with azido-fluorescent dyes allow sensitive in-gel fluorescent visualization of S-palmitoylated proteins within minutes.6 Coupled with amino acid reporters like azido-homoalanine, dual pulse-chase labeling studies of mammalian cells with alkynyl-fatty acid reporters enables tandem imaging of S-palmitoylation and protein turnover.20 In addition to visualizing S-palmitoylation of known proteins, fatty acid reporters and cleavable affinity tags such as azido-azo-biotin21 allow the discovery of new S-palmitoylated proteins using bioorthogonal chemical proteomic strategies.7, 22, 23 For example, our bioorthogonal profiling studies of dendritic cells with an alkynyl-palmitic acid reporter revealed S-palmitoylation of interferon-induced transmembrane protein 3 (IFITM3) is crucial for its antiviral activity against influenza virus.7 Chemical proteomics with acyl biotin exchange provides a complementary method and has also allowed the identification of new S-palmitoylated proteins such as a brain-specific Cdc42 isoform.24 These chemical methods are providing opportunities to discover new S-palmitoylated proteins and characterize their regulatory mechanisms.

Alkynyl-fatty acid chemical reporters label enzymes associated with fatty acid synthesis and metabolism in addition to fatty-acylated proteins.7, 22, 23 Based upon previous studies indicating that polar fatty acid analogs can be tolerated by N-myristoyltransferase,25, 26 we evaluated whether an oxygen-bearing analog of 17-octadecynoic acid (ODYA also termed alk-16) could differentially target fatty-acylated proteins compared to metabolic enzymes. Here, we report the synthesis and characterization of 15-hexadecynyloxyacetic acid (HDYOA), an oxy-alkynyl-fatty acid chemical reporter. The analysis of HDYOA labeling of full-length IFITM3 and a triple cysteine mutant demonstrated that this fatty acid reporter specifically labels S-palmitoylated proteins in mammalian cells. Comparative bioorthogonal proteomic analysis with ODYA revealed HDYOA preferentially labels S-palmitoylated proteins and enzymes associated with fatty acid biosynthesis but not downstream enzymes involved in the degradation of fatty acids.

Section snippets

Synthesis and characterization of HDYOA

Proteomic studies from our laboratory revealed that fatty acid chemical reporters also label metabolic enzymes,7, 22 particularly in the β-oxidation pathway of fatty acid breakdown such as acyl-CoA dehydrogenase and mitochondrial trifunctional enzyme subunit β, suggesting that these reporters may be substrates for metabolic degradation. The first enzymatic step of β-oxidation of a fatty acid-CoA molecule is the reduction of the bond between the α and β carbons by acyl-CoA dehydrogenase (Fig. 2

Discussion

Fatty acid chemical reporters provide powerful reagents to analyze the specificity of protein fatty-acylation. Previous work has demonstrated that N-myristoyl transferase and palmitoyl transferases have unique preferences for utilizing alkynyl-fatty acids of different lengths.6, 22 These reporters also serve as substrates for metabolic degradation as indicated by labeling and selective retrieval of mitochondrial β-oxidation enzymes seen in various proteomic studies.7, 22, 23 We now demonstrate

Chemicals

Synthesis of ODYA,6 azido-rhodamine,6 and azido-azo-biotin21 have been described previously. All chemicals for synthesis were obtained either from Sigma–Aldrich, MP Biomedicals, Alfa Aesar, TCI, Fluka or Acros and were used as received unless otherwise noted. The silica gel used in flash column chromatography was Fisher S704 (60–200 mesh, Chromatographic Grade). Analytical thin layer chromatography (TLC) was conducted on Merck silica gel plates with fluorescent indicator on glass (5–20 μm, 60 Å)

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