Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualisation, identification and quantification of N-myristoylation, N- and S-acylation, Ocholesterylation, S-farnesylation and S-geranylgeranylation

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry–based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional ‘capture reagents’ by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease

Myristoylation profiling in human cells and zebrafish.

Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour

New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish

Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in the progression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requires attachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidated Shh has been challenging due to the limited array of tools available, and the roles of these posttranslational modifications are poorly understood. Herein, we describe the development and validation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable its visualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown to be an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shh from signalling cells, formation of multimeric transport complexes and signalling. We have used this probe to determine the size of transport complexes of lipidated Shh in culture medium and expression levels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitative chemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy and detection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probes provide a set of novel and well-validated tools that can be used to investigate the role of lipidation on activity of Shh, and potentially other members of the Hedgehog protein family

Localization of Hhat in PANC1 cells.

<p>A. Localization of Hhat in PANC1 cells was assessed using Hhat-EGFP transfection and confocal microscopy combined with immunofluorescence localization of ER (PDI) and Golgi (GM130). B. HEK293a Hhat-V5 stable cells were co-stained for the V5 epitope with ER (Calnexin) or Golgi (GM130) and nuclei (DAPI). The data show that both Hhat-EGFP and Hhat-V5 localize primarily in ER with little if any in Golgi apparatus. Scale bar = 10 µm.</p

Hhat RNAi KD in PANC1 and HEK293a-Hhat-V5 cells.

<p>A. Quantitative RT-PCR was performed using Hhat-specific primers to confirm target gene knockdown in PANC1 cells following Hhat-#1 and Hhat-#2 siRNA transfection. Hhat expression is normalized with GAPDH and compared to Non-targeting siRNA control. Error bar represents the standard error of at least three independent experiments performed in duplicate (**, <i>P</i><0.01; ***, <i>P</i><0.001). B. To confirm Hhat KD at the protein level, HEK293a cells stably expressing a pcDNA-DEST40-Hhat-V5 construct at moderate levels were transfected with the Hhat-#1, Hhat-#2 and Non-targeting siRNAs. 72 h post transfection cells were lysed and Hhat expression examined by SDS-PAGE followed by western blotting with an anti-V5 antibody. Blots were probed for tubulin as a loading control. C. Densitometry was performed on the blots in panel B; values were then normalized to the tubulin loading control and compared to the Non-targeting siRNA control.</p

Hhat KD inhibits Shh palmitoylation and multimeric complex formation.

<p>A. 48 h after Hhat-#1 siRNA transfection PANC1 cells were labeled with YnC15, then medium and cell lysates were collected for 5E1 immunoprecipitation, treated by click chemistry and analyzed by Shh Western blot with H-160 Ab. In both medium and cell lysates, YnC15-labeled Shh was reduced in Hhat-#1 KD cells compared to Mutated Hhat-#1 and control cells. In contrast, YnC15-unlabeled Shh was more abundant in the medium of Hhat-#1 KD cells, but less abundant in cell lysates, demonstrating increased release when Hhat is knocked down. B. To examine the role of Hhat in Shh oligomerization, 72 h after siRNA transfection of PANC1 cells the media were subjected to gel filtration chromatography (Superdex200 10/300 GL column). After TCA precipitation, the fractions were probed by dot blot with anti-Shh H-160 antibody. Untreated and control Mutated Hhat-#1-treated cells showed an abundance of large complexes migrating near the void volume (Vo), whereas Hhat-#1 siRNA-treated KD cells had a much higher proportion of monomer (Vt represents the total volume of the column). Experiments were repeated three times with similar results.</p

Hhat KD inhibits PANC1 proliferation and matrix invasion.

<p>A. PANC1 cells were labeled with CFSE at the start of the experiment and treated with Shh neutralizing antibody 5E1 on Day 1 (5E1 D1) to test whether PANC1 proliferation is Shh dependent. 5E1 treatment on Day 4 (5E1 D4) was used to mimic the kinetics of siRNA KD. Cells were allowed to grow for 8 days during which CFSE dilution gave a measure of cell division. 8 days after transfection with Hhat-#1 siRNA the cells were analyzed by flow cytometry. The Y-axis shows the CFSE mean fluorescence intensity (MFI) observed from 30,000 cells in each condition. The experiments were repeated three times in triplicate, and statistical significance was measured by using the two-tailed t test (***, <i>P</i><0.001). B, C. 72 h after transfection with Hhat-1 siRNA or control Mutated Hhat siRNA PANC1 cells were plated onto Matrigel invasion chambers for 24 h. Cells that had migrated from the upper to the lower side of the filter were photographed (B) and counted with a light microscope (40 fields/filter, C). Non-treated (NT) and 5E1-treated cells are shown for comparison in B. The experiments were repeated four times, and statistical significance was measured by using the two-tailed t test (***, <i>P</i><0.001).</p