RESUMEN
Diacylglycerol lipase-beta (DAGLß) serves as a principal 2-arachidonoylglycerol (2-AG) biosynthetic enzyme regulating endocannabinoid and eicosanoid metabolism in immune cells including macrophages and dendritic cells. Genetic or pharmacological inactivation of DAGLß ameliorates inflammation and hyper-nociception in preclinical models of pathogenic pain. These beneficial effects have been assigned principally to reductions in downstream proinflammatory lipid signaling, leaving alternative mechanisms of regulation largely underexplored. Here, we apply quantitative chemical- and phospho-proteomics to find that disruption of DAGLß in primary macrophages leads to LKB1-AMPK signaling activation, resulting in reprogramming of the phosphoproteome and bioenergetics. Notably, AMPK inhibition reversed the antinociceptive effects of DAGLß blockade, thereby directly supporting DAGLß-AMPK crosstalk in vivo. Our findings uncover signaling between endocannabinoid biosynthetic enzymes and ancient energy-sensing kinases to mediate cell biological and pain responses.
Asunto(s)
Endocannabinoides , Glicéridos , Humanos , Endocannabinoides/metabolismo , Glicéridos/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Lipoproteína Lipasa/metabolismo , Ácidos Araquidónicos/metabolismo , DolorRESUMEN
C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.
Asunto(s)
Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/metabolismo , Ingeniería de Proteínas/métodos , Animales , Dominio Catalítico , Cromatografía Liquida , Diacilglicerol Quinasa/genética , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , Metabolómica/métodos , Sondas Moleculares/química , Ácidos Fosfatidicos/metabolismo , Dominios Proteicos , Proteómica/métodos , Ratas , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.
Asunto(s)
Sondas Moleculares/química , Proteómica/métodos , Azufre/química , Triazoles/química , Tirosina/análisis , Tirosina/química , Células A549 , Sitios de Unión , Flúor/química , Gutatión-S-Transferasa pi/genética , Gutatión-S-Transferasa pi/metabolismo , Células HEK293 , Humanos , Sondas Moleculares/síntesis química , Fosforilación , Fosfotirosina/química , Fosfotirosina/metabolismo , Dominios Proteicos , Procesamiento Proteico-Postraduccional , Ácidos Sulfínicos/química , Tirosina/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.
Asunto(s)
Carcinoma Ductal Pancreático , Proteínas con Homeodominio LIM , Proteínas del Tejido Nervioso , Neoplasias Pancreáticas , Factores de Transcripción , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Epigénesis Genética , Genes Supresores de Tumor , Humanos , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Advancements in chemical proteomics and mass spectrometry lipidomics are providing new opportunities to understand lipid kinase activity, specificity, and regulation on a global cellular scale. Here, we describe recent developments in chemical biology of lipid kinases with a focus on those members that phosphorylate diacylglycerols. We further discuss future implications of how these mass spectrometry-based approaches can be adapted for studies of additional lipid kinase members with the aim of bridging the gap between protein and lipid kinase-focused investigations.
Asunto(s)
Diacilglicerol Quinasa , Proteómica , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/metabolismo , Lípidos , Espectrometría de Masas/métodos , Fosfotransferasas , Proteómica/métodosRESUMEN
Background: Patients with rheumatoid arthritis (RA) experience joint swelling and cartilage destruction resulting in chronic pain, functional disability, and compromised joint function. Current RA treatments, including glucocorticoid receptor agonists, produce adverse side effects and lack prolonged treatment efficacy. Cannabinoids (i.e., cannabis-like signaling molecules) exert anti-inflammatory and analgesic effects with limited side effects compared to traditional immunosuppressants, making them excellent targets for the development of new arthritic therapeutics. Monoacylglycerol lipase (MAGL) inhibition reduces inflammation in mouse models of acute inflammation, through cannabinoid receptor dependent and independent pathways. The current study investigated the efficacy of inhibiting synthetic and catabolic enzymes that regulate the endocannabinoid 2-arachidonoylglycerol (2-AG) in blocking paw inflammation, pain-related behaviors, and functional loss caused by collagen-induced arthritis (CIA). Methods: Male DB1A mice subjected to CIA were administered the glucocorticoid agonist dexamethasone (DEX), MAGL inhibitor JZL184 (8 or 40 mg/kg, s.c.), alone or in combination, or diacylglycerol lipase ß (DAGLß) inhibitor KT109 (40 mg/kg, s.c.). CIA-induced deficits were assayed by arthritic clinical scoring, paw thickness measurements, and behavioral tests of pain and paw function. Results: DEX or dual administration with JZL184 reduced paw thickness and clinical scores, and JZL184 dose-dependently attenuated grip strength and balance beam deficits caused by CIA. Traditional measures of pain-induced behaviors (hyperalgesia and allodynia) were inconsistent. The antiarthritic effects of JZL184 (40 mg/kg) were largely blocked by coadministration of the CB2 antagonist SR144528, and the DAGLß inhibitor KT109 had no effect on CIA, indicating that these effects likely occurred through CB2 activation. Conclusions: MAGL inhibition reduced paw inflammation and pain-depressed behavioral signs of arthritis, likely through an endocannabinoid mechanism requiring CB2. These data support the development of MAGL as a target for therapeutic treatment of inflammatory arthritis.
Asunto(s)
Ácidos Araquidónicos/fisiología , Artritis Experimental/tratamiento farmacológico , Benzodioxoles/farmacología , Endocannabinoides/fisiología , Glicéridos/fisiología , Inflamación/tratamiento farmacológico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Piperidinas/farmacología , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/enzimología , Dexametasona/farmacología , Edema/tratamiento farmacológico , Pie , Hiperalgesia/tratamiento farmacológico , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos DBARESUMEN
Lipid droplets (LDs) are dynamic organelles that undergo dynamic changes in response to changing cellular conditions. During nutrient depletion, LD numbers increase to protect cells against toxic fatty acids generated through autophagy and provide fuel for beta-oxidation. However, the precise mechanisms through which these changes are regulated have remained unclear. Here, we show that the small GTPase RalA acts downstream of autophagy to directly facilitate LD growth during nutrient depletion. Mechanistically, RalA performs this function through phospholipase D1 (PLD1), an enzyme that converts phosphatidylcholine (PC) to phosphatidic acid (PA) and that is recruited to lysosomes during nutrient stress in a RalA-dependent fashion. RalA inhibition prevents recruitment of the LD-associated protein perilipin 3, which is required for LD growth. Our data support a model in which RalA recruits PLD1 to lysosomes during nutrient deprivation to promote the localized production of PA and the recruitment of perilipin 3 to expanding LDs.
Asunto(s)
Gotas Lipídicas/metabolismo , Nutrientes , Fosfolipasa D/metabolismo , Proteínas de Unión al GTP ral/metabolismo , Animales , Autofagia , Fibroblastos/metabolismo , Células HeLa , Humanos , Lisosomas/metabolismo , Ratones Noqueados , Perilipina-3/metabolismo , Ácidos Fosfatidicos/metabolismo , Triglicéridos/metabolismoRESUMEN
Here, we apply quantitative chemical proteomics and untargeted lipidomics to assign a polyunsaturated fatty acid (PUFA)-specific triacylglycerol (TAG) lipase activity for diacylglycerol lipase-beta (DAGLß) in macrophages. We demonstrate that DAGLß but not DAGLα is expressed and active in bone marrow-derived macrophages (BMDMs) as determined by activity-based protein profiling analysis of SILAC BMDMs. Genetic disruption of DAGLß resulted in accumulation of cellular TAGs composed of PUFA but not saturated/low unsaturated fatty acid counterparts, which is recapitulated in wild-type macrophages treated with a DAGLß-selective inhibitor. Biochemical assays with synthetic substrates confirm PUFA-TAGs as authentic DAGLß substrates. In summary, our findings identify DAGLß as a PUFA-specific TAG lipase in primary macrophages.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Macrófagos/metabolismo , Animales , Diferenciación Celular , Cromatografía Liquida , Ácidos Grasos Insaturados/química , Lipasa/química , Lipoproteína Lipasa/química , Espectrometría de Masas , Metabolómica , RatonesRESUMEN
Lipids exert key structural, metabolic, and signaling functions in cells. Lipid diversity found in cells and tissues is regulated principally by metabolic enzymes whose activity is modulated posttranslationally to shape head group and fatty acyl composition of membrane lipids. Methodologies capable of monitoring in vivo changes in the lipidome are needed to assign substrate specificity of metabolic enzymes, which represents a key step toward understanding structure-function of lipids in living systems. The resulting lipid annotations also serve as important biomarkers for understanding mode of action for pharmacological agents targeting metabolic enzymes in cells and animal models. In this chapter, we describe a general metabolomics workflow to complement (chemo)proteomic efforts to modulate lipid pathways for basic science and translational applications.
Asunto(s)
Metabolismo de los Lípidos , Lipidómica/métodos , Espectrometría de Masas en Tándem/métodos , Animales , Biocatálisis , Fraccionamiento Químico/métodos , Cromatografía Liquida/métodos , Humanos , Lípidos/análisis , Lípidos/aislamiento & purificaciónRESUMEN
The metabolic serine hydrolases hydrolyze ester, amide, or thioester bonds found in broad small molecule substrates using a conserved activated serine nucleophile. The mammalian central nervous system (CNS) express a diverse repertoire of serine hydrolases that act as (phospho)lipases or lipid amidases to regulate lipid metabolism and signaling vital for normal neurocognitive function and CNS integrity. Advances in genomic DNA sequencing have provided evidence for the role of these lipid-metabolizing serine hydrolases in neurologic, psychiatric, and neurodegenerative disorders. This review briefly summarizes recent progress in understanding the biochemical and (patho)physiological roles of these lipid-metabolizing serine hydrolases in the mammalian CNS with a focus on serine hydrolases involved in the endocannabinoid system. The development and application of specific inhibitors for an individual serine hydrolase, if available, are also described. This article is part of a Special Issue entitled Novel functions of phospholipase A2 Guest Editors: Makoto Murakami and Gerard Lambeau.