Post-translational farnesylation or geranylgeranylation at a C-terminal cysteine residue regulates 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 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.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitylating enzyme that 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.

A highly accurate and versatile fluorescence polarisation assay for any enzyme adding or removing lipid posttranslational modifications, with the potential to accelerate drug discovery against these targets.

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure, and subcellular localisation. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets due to 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 labelling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as bio-orthogonal labelling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional 'capture reagents' using click chemistry, allowing in-gel fluorescence visualisation 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, Ocholesterylation, 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 labelling to data processing within three 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.

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...

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, and we show that a previously claimed UCHL1 inhibitor (LDN-57444) fails to engage UCHL1 in cells. We further demonstrate that potent UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting a potential therapeutic role for UCHL1 inhibition and providing a basis for future therapeutic development of selective UCHL1 inhibitors.

“Immobilisation of pyrrolidinyl peptide nucleic acids on paper resulted in a new DNA sensor with great specificity”.

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, and we show that a previously claimed UCHL1 inhibitor (LDN-57444) fails to engage UCHL1 in cells. We further demonstrate that potent UCHL1 inhibitors block pro-fibrotic responses in a cellular model of idiopathic pulmonary fibrosis, supporting a potential therapeutic role for UCHL1 inhibition and providing a basis for future therapeutic development of selective UCHL1 inhibitors.