2-Arachidonoylglycerol (2-AG) is a signaling lipid in the central nervous system that is a key regulator of neurotransmitter release. 2-AG is an endocannabinoid that activates the cannabinoid CB1 ...receptor. It is involved in a wide array of (patho)physiological functions, such as emotion, cognition, energy balance, pain sensation and neuroinflammation. In this review, we describe the biosynthetic and metabolic pathways of 2-AG and how chemical and genetic perturbation of these pathways has led to insight in the biological role of this signaling lipid. Finally, we discuss the potential therapeutic benefits of modulating 2-AG levels in the brain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Abstract
The cannabinoid CB
2
receptor (CB
2
R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed ...and widely used to target CB
2
R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB
2
R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB
2
R agonists to study the role of CB
2
R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.
S-Palmitoylation is the covalent attachment of C14:0–C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly abundant in neurons, where it plays ...a role in neuronal development and is implicated in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. The knowledge of S-palmitoylation in neurodevelopment is limited due to technological challenges in analyzing this highly hydrophobic protein modification. Here, we used two orthogonal methods, acyl-biotin exchange (ABE) and lipid metabolic labeling (LML), to identify S-palmitoylated proteins and sites during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. We identified 2002 putative S-palmitoylated proteins in total, of which 650 were found with both methods. Significant changes in the abundance of S-palmitoylated proteins were detected, in particular for several processes and protein classes that are known to be important for neuronal differentiation, which include proto-oncogene tyrosine-protein kinase receptor (RET) signal transduction, SNARE protein-mediated exocytosis, and neural cell adhesion molecules. Overall, S-palmitoylation profiling by employing ABE and LML in parallel during RA-induced differentiation of SH-SY5Y cells revealed a subset of high confidence bona fide S-palmitoylated proteins and suggested an important role for S-palmitoylation in neuronal differentiation.
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IJS, KILJ, NUK, PNG, UL, UM
Tight regulation of protein translation drives the proteome to undergo changes under influence of extracellular or intracellular signals. Despite mass spectrometry–based proteomics being an excellent ...method to study differences in protein abundance in complex proteomes, analyzing minute or rapid changes in protein synthesis and abundance remains challenging. Therefore, several dedicated techniques to directly detect and quantify newly synthesized proteins have been developed, notably puromycin-based, bio-orthogonal noncanonical amino acid tagging–based, and stable isotope labeling by amino acids in cell culture–based methods, combined with mass spectrometry. These techniques have enabled the investigation of perturbations, stress, or stimuli on protein synthesis. Improvements of these methods are still necessary to overcome various remaining limitations. Recent improvements include enhanced enrichment approaches and combinations with various stable isotope labeling techniques, which allow for more accurate analysis and comparison between conditions on shorter timeframes and in more challenging systems. Here, we aim to review the current state in this field.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Diacylglycerol lipases (DAGLα and DAGLβ) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that ...can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLα is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo ...short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC.
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•ZDHHC20 is identified as mediator of PDAC metastasis•ZDHHC20 KO phenotype is abrogated in immunocompromised animals•Substrates of ZDHHC20 are identified using a chemical genetics platform
Tomić et al. uncover regulators of PDAC metastasis and validate the pivotal role of ZDHHC20. Despite no impact on in vitro cell phenotype and primary tumor growth, ZDHHC20 depletion curbs metastasis in mice. This effect is dependent on the immune system, particularly natural killer cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To ...address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography–enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID–ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID–ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID–ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID–ABPP providing the advantage of direct activity-based probe interaction site identification.
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•IMAC-enrichable affinity tags (PhosID) are compatible with ABPP studies.•PhosID in combination with ABPP (PhosID–ABPP) enables ABP binding site identification.•PhosID–ABPP can be used in complex contexts, such as intact cells.•We observe and quantify differences in binding sites in intact cells and cell lysates.•Trypsin and pepsin provide complementary views of the ABP-interactome.
Here, we apply IMAC-enrichable phosphonate affinity tags for activity-based protein profiling, enabling the enrichment of peptides bound to the activity-based probe. Key advantages of PhosID–activity-based protein profiling are its high selectivity, efficiency and ease of use. PhosID–activity-based protein profiling allows the direct identification of the probe binding sites and is compatible with intact cell and cell lysate inhibitor treatment. Clear differences in binding sites were revealed in intact cells and in lysates of the same cell line.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The endocannabinoid 2-arachidonoylglycerol (2-AG) is predominantly biosynthesized by sn-1-diacylglycerol lipase α (DAGL-α) in the CNS. Selective inhibitors of DAGL-α will provide valuable insights in ...the role of 2-AG in endocannabinoid signaling processes and are potential therapeutics for the treatment of obesity and neurodegenerative diseases. Here, we describe the development of a natural substrate-based fluorescence assay for DAGL-α, using a coupled enzyme approach. The continuous setup of our assay allows monitoring of DAGL-α activity in real-time and in a 96-well plate format. This constitutes a major improvement to the currently available radiometric and LC/MS-based methods, which can be executed only in low-throughput formats. In addition, our assay circumvents the use of radioactive material. We demonstrate that our assay can be used to screen inhibitors of DAGL-α activity, using 1-stearoyl-2-arachidonoyl-sn-glycerol as the physiologically relevant natural substrate of DAGL-α. Furthermore, our method can be employed to measure DAGL activity and inhibition in the mouse brain membrane proteome. Consequently, our assay should serve as a valuable tool for rapid hit validation and lead optimization of DAGL-α inhibitors.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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