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

Activity-based protein profiling reveals deubiquitinase and aldehyde dehydrogenase targets of a cyanopyrrolidine probe

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1), a deubiquitinating enzyme (DUB), is a potential drug target in various cancers, and liver and lung fibrosis. However, bona fide functions and substrates of UCHL1 remain poorly understood. Herein, we report the characterization of UCHL1 covalent inhibitor MT16-001 based on a thiazole cyanopyrrolidine scaffold. In combination with chemical proteomics, a closely related activity-based probe (MT16-205) was used to generate a comprehensive quantitative profile for on- and off-targets at endogenous cellular abundance. Both compounds are selective for UCHL1 over other DUBs in intact cells but also engage a range of other targets with good selectivity over the wider proteome, including aldehyde dehydrogenases, redox-sensitive Parkinson’s disease related protein PARK7, and glutamine amidotransferase. Taken together, these results underline the importance of robust profiling of activity-based probes as chemical tools and highlight the cyanopyrrolidine warhead as a versatile platform for liganding diverse classes of protein with reactive cysteine residues which can be used for further inhibitor screening, and as a starting point for inhibitor development

Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes

Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets

Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes

Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout. Acyl-cLIP allows sensitive, accurate, real-time measurement of S- or N-palmitoylation, N-myristoylation, S-farnesylation or S-geranylgeranylation. Furthermore, it is applicable to transfer and hydrolysis reactions, and we demonstrate its extension to a high-throughput screening format. We anticipate that Acyl-cLIP will greatly expedite future drug discovery efforts against these challenging targets

Discovery of a potent and selective covalent inhibitor and activity-based probe for the deubiquitylating enzyme UCHL1, with anti-fibrotic activity

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme which is proposed as a potential therapeutic target in neurodegeneration, cancer, and liver and lung fibrosis. Herein we report the discovery of the most potent and selective UCHL1 probe (IMP-1710) to date based on a covalent inhibitor scaffold and apply this probe to identify and quantify target proteins in intact human cells. IMP-1710 stereoselectively labels the catalytic cysteine of UCHL1 at low nanomolar concentration in cells. We further demonstrate that potent and selective UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting the potential of UCHL1 as a potential therapeutic target in fibrotic diseases

Correction to “Discovery of a Potent and Selective Covalent Inhibitor and Activity-Based Probe for the Deubiquitylating Enzyme UCHL1, with Antifibrotic Activity”

Supporting Information, pages S38 and S44. In the PDF Supporting Information, Schemes S1 and S3 contained errors in the synthetic conditions. The conditions for the steps 5 → 6 and 12 → 13 in the respective schemes should be “TFA, DCM, rt” (not “TMSI, K2CO3, CH2Cl2”)

Dual chemical probes enable quantitative system-wide analysis of protein prenylation and prenylation dynamics

Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates the localization and function of over 100 proteins, including the Ras isoforms, and is a therapeutic target in diseases including cancer and infection. Here, we report global and selective profiling of prenylated proteins in living cells enabled by the development of isoprenoid analogues YnF and YnGG in combination with quantitative chemical proteomics. Eighty prenylated proteins were identified in a single human cell line, 64 for the first time at endogenous abundance without metabolic perturbation. We further demonstrate that YnF and YnGG enable direct identification of post-translationally processed prenylated peptides, proteome-wide quantitative analysis of prenylation dynamics and alternative prenylation in response to four different prenyltransferase inhibitors, and quantification of defective Rab prenylation in a model of the retinal degenerative disease choroideremia