RESUMO
The Janus tyrosine kinase (JAK) family of non-receptor tyrosine kinases includes four isoforms (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immun(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor isoform selectivity, a common challenge for inhibitors targeting the ATP-binding pocket of kinases. Here we describe the chemical proteomic discovery of a druggable allosteric cysteine present in the non-catalytic pseudokinase domain of JAK1 (C817) and TYK2 (C838), but absent from JAK2 or JAK3. Electrophilic compounds selectively engaging this site block JAK1-dependent trans-phosphorylation and cytokine signaling, while appearing to act largely as 'silent' ligands for TYK2. Importantly, the allosteric JAK1 inhibitors do not impair JAK2-dependent cytokine signaling and are inactive in cells expressing a C817A JAK1 mutant. Our findings thus reveal an allosteric approach for inhibiting JAK1 with unprecedented isoform selectivity.
Assuntos
Cisteína , Proteômica , Transdução de Sinais , Citocinas , Isoformas de ProteínasRESUMO
Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.
Assuntos
Eicosanoides/metabolismo , Endocanabinoides/metabolismo , Imunidade Inata/imunologia , Macrófagos/imunologia , Fosfolipase C gama/metabolismo , Animais , Antígenos CD/biossíntese , Antígenos de Diferenciação Mielomonocítica/biossíntese , Células COS , Linhagem Celular , Chlorocebus aethiops , Citocinas/imunologia , Diglicerídeos/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Células HEK293 , Humanos , Inflamação/imunologia , Lipopolissacarídeos/imunologia , Lipase Lipoproteica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Monoacilglicerol Lipases/metabolismo , Fosfolipase C gama/genética , Prostaglandinas/biossíntese , Receptores Fc/imunologia , Transdução de Sinais/imunologiaRESUMO
BACKGROUND: Parents play a key role in infant's development through their interactions and the type of environment they provide to promote active play. The amount of time parents are able to spend with their infant is dependent on their working status, yet few studies have explored parent perception of their infant's active play by working status. The purpose of this study was to explore parent perception of active play and compare responses between working and stay-at-home parents. METHODS: Twenty-nine parents participated in this qualitative study by completing a one-time, in-person semi-structured interview based on the Theory of Planned Behavior. Themes were developed and compared based on parental working status using a directed content analysis approach. RESULTS: All parents believed active play could have a positive effect on their child's development through physical, social and emotional, cognitive, and/or language and communication development. However, stay-at-home parents reported a broader impact of active play across these domains; whereas working parents most often referenced active play as impacting infant's physical development. Social and emotional interactions were the highest reported form of active play among all parents. Additionally, all parents described similar barriers to increasing the time for active play. The most commonly reported barrier for all parents was time or schedule followed by care needs of the infant, environmental concerns, and need for restrictive devices (e.g., car seats). More stay-at-home parents than working parents reported the care needs of the infant as being a barrier. Recommendations for active play were not widely known amongst all parents, with a higher percentage of working parents reporting they would desire advice from a healthcare provider. CONCLUSIONS: Working status of parents appears to have implications on perceptions of active play which in turn may influence infants' development. Future studies should objectively assess the impact of parents' working status on infant development and explore how gender of the parent may serve as a confounding variable.
Assuntos
Desenvolvimento Infantil , Pais , Criança , Comunicação , Humanos , Lactente , Pesquisa QualitativaRESUMO
PHARC (polyneuropathy, hearing loss, cerebellar ataxia, retinitis pigmentosa, and cataract) is a human neurological disorder caused by deleterious mutations in the ABHD12 gene, which encodes an integral membrane lyso-phosphatidylserine (lyso-PS) lipase. Pharmacological or genetic disruption of ABHD12 leads to higher levels of lyso-PS lipids in human cells and the central nervous system (CNS) of mice. ABHD12 loss also causes rapid rewiring of PS content, resulting in selective increases in the level of arachidonoyl (C20:4) PS and decreases in the levels of other PS species. The biochemical basis for ABHD12-dependent PS remodeling and its pathophysiological significance remain unknown. Here, we show that genetic deletion of the lysophospholipid acyltransferase LPCAT3 blocks accumulation of brain C20:4 PS in mice lacking ABHD12 and concurrently produces hyper-increases in the level of lyso-PS in these animals. These lipid changes correlate with exacerbated auditory dysfunction and brain microgliosis in mice lacking both ABHD12 and LPCAT3. Taken together, our findings reveal that ABHD12 and LPCAT3 coordinately regulate lyso-PS and C20:4 PS content in the CNS and point to lyso-PS lipids as the likely bioactive metabolites contributing to PHARC-related neuropathologies.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Monoacilglicerol Lipases/metabolismo , Doenças do Sistema Nervoso/metabolismo , Fosfatidilserinas/metabolismo , 1-Acilglicerofosfocolina O-Aciltransferase/deficiência , 1-Acilglicerofosfocolina O-Aciltransferase/genética , Animais , Camundongos , Camundongos Knockout , Estrutura Molecular , Monoacilglicerol Lipases/deficiência , Monoacilglicerol Lipases/genéticaRESUMO
Complex hereditary spastic paraplegia (HSP) is a genetic disorder that causes lower limb spasticity and weakness and intellectual disability. Deleterious mutations in the poorly characterized serine hydrolase DDHD2 are a causative basis for recessive complex HSP. DDHD2 exhibits phospholipase activity in vitro, but its endogenous substrates and biochemical functions remain unknown. Here, we report the development of DDHD2(-/-) mice and a selective, in vivo-active DDHD2 inhibitor and their use in combination with mass spectrometry-based lipidomics to discover that DDHD2 regulates brain triglycerides (triacylglycerols, or TAGs). DDHD2(-/-) mice show age-dependent TAG elevations in the central nervous system, but not in several peripheral tissues. Large lipid droplets accumulated in DDHD2(-/-) brains and were localized primarily to the intracellular compartments of neurons. These metabolic changes were accompanied by impairments in motor and cognitive function. Recombinant DDHD2 displays TAG hydrolase activity, and TAGs accumulated in the brains of wild-type mice treated subchronically with a selective DDHD2 inhibitor. These findings, taken together, indicate that the central nervous system possesses a specialized pathway for metabolizing TAGs, disruption of which leads to massive lipid accumulation in neurons and complex HSP syndrome.
Assuntos
Lipase/metabolismo , Fosfolipases A1/metabolismo , Paraplegia Espástica Hereditária/enzimologia , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Cognição , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Marcação de Genes , Células HEK293 , Humanos , Lipase/antagonistas & inibidores , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/ultraestrutura , Locomoção , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fosfolipases , Fosfolipases A1/antagonistas & inibidores , Fosfolipases A1/deficiência , Reprodutibilidade dos Testes , Paraplegia Espástica Hereditária/genética , Triglicerídeos/metabolismoRESUMO
The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLß. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLß in living systems. Optimized inhibitors showed high selectivity for DAGLß over other serine hydrolases, including DAGLα (â¼60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLß inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLß is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.
Assuntos
Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipase Lipoproteica/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Animais , Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/biossíntese , Linhagem Celular , Citocinas/metabolismo , Descoberta de Drogas , Endocanabinoides/biossíntese , Glicerídeos/biossíntese , Lipase Lipoproteica/genética , Lipase Lipoproteica/fisiologia , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Prostaglandinas/metabolismo , Isoformas de Proteínas , Proteoma/efeitos dos fármacos , Relação Quantitativa Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacos , Triazóis/síntese química , Triazóis/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
We recently described a fluorescence polarization platform for competitive activity-based protein profiling (fluopol-ABPP) that enables high-throughput inhibitor screening for enzymes with poorly characterized biochemical activity. Here, we report the discovery of a class of oxime ester inhibitors for the unannotated serine hydrolase RBBP9 from a full-deck (200,000+ compound) fluopol-ABPP screen conducted in collaboration with the Molecular Libraries Screening Center Network (MLSCN). We show that these compounds covalently inhibit RBBP9 by modifying enzyme's active site serine nucleophile and, based on competitive ABPP in cell and tissue proteomes, are selective for RBBP9 relative to other mammalian serine hydrolases.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Oximas/química , Oximas/farmacologia , Animais , Encéfalo/metabolismo , Linhagem Celular , Ésteres/química , Ésteres/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Proteoma/metabolismoRESUMO
BACKGROUND: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. RESULTS: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly. CONCLUSION: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.
Assuntos
Ácidos Araquidônicos/biossíntese , Glicina/análogos & derivados , Redes e Vias Metabólicas , Alcamidas Poli-Insaturadas/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/deficiência , Animais , Ácidos Araquidônicos/química , Ácidos Araquidônicos/metabolismo , Benzamidas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Química Encefálica , Carbamatos/farmacologia , Linhagem Celular Tumoral , Endocanabinoides , Glicina/biossíntese , Glicina/química , Glicina/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Knockout , Oxirredução , Alcamidas Poli-Insaturadas/química , Ratos , Proteínas Recombinantes/metabolismoRESUMO
N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated N-acyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nano-high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGly-induced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F96365), and La3+. Furthermore, PalGly contributed to the production of NO through calcium-sensitive nitric-oxide synthase enzymes present in F-11 cells and was inhibited by the nitric-oxide synthase inhibitor 7-nitroindazole.
Assuntos
Cálcio/metabolismo , Glicina/análogos & derivados , Glicina/farmacologia , Neurônios Aferentes/metabolismo , Óxido Nítrico/biossíntese , Ácidos Palmíticos/farmacologia , Receptores de Canabinoides/metabolismo , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Anticorpos , Benzamidas/farmacologia , Química Encefálica , Carbamatos/farmacologia , Linhagem Celular , Cruzamentos Genéticos , Relação Dose-Resposta a Droga , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/química , Gânglios Espinais/citologia , Glicina/análise , Glicina/química , Glicina/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptores/efeitos dos fármacos , Ácidos Palmíticos/química , Toxina Pertussis/farmacologia , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Regulação para CimaRESUMO
Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1-1 mM was the most active, causing 8.5-9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice.
Assuntos
Amidoidrolases/metabolismo , Fosfolipídeos/fisiologia , Amidoidrolases/biossíntese , Amidoidrolases/deficiência , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfolipídeos/farmacologia , RatosRESUMO
The potent regulatory properties of NKT cells render this subset of lipid-specific T cells a promising target for immunotherapeutic interventions. The marine sponge glycolipid alpha-galactosylceramide (alphaGalCer) is the proto-typic NKT cell agonist, which elicits this function when bound to CD1d. However, our understanding of the in vivo properties of NKT cell agonists and the host factors that control their bioactivity remains very limited. In this report, we isolated the enzyme fatty acid amide hydrolase (FAAH) from mouse serum as an alphaGalCer-binding protein that modulates the induction of key effector functions of NKT cells in vivo. FAAH bound alphaGalCer in vivo and in vitro and was required for the efficient targeting of lipid antigens for CD1d presentation. Immunization of Faah-deficient mice with alphaGalCer resulted in a reduced systemic cytokine production, but enhanced expansion of splenic NKT cells. This distinct NKT response conferred a drastically increased adjuvant effect and strongly promoted protective CTL responses. Thus, our findings identify not only the presence of FAAH in normal mouse serum, but also its critical role in the tuning of immune responses to lipid antigens by orchestrating their transport and targeting for NKT cell activation. Our results suggest that the serum transport of lipid antigens directly shapes the quality of NKT cell responses, which could potentially be modulated in support of novel vaccination strategies.
Assuntos
Amidoidrolases/imunologia , Antígenos/imunologia , Galactosilceramidas/imunologia , Glicolipídeos/imunologia , Células T Matadoras Naturais/imunologia , Adjuvantes Imunológicos/metabolismo , Adjuvantes Imunológicos/farmacologia , Amidoidrolases/metabolismo , Animais , Antígenos/metabolismo , Galactosilceramidas/metabolismo , Galactosilceramidas/farmacologia , Glicolipídeos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
N-arachidonoyl dopamine (NADA) is an endogenous ligand that activates the cannabinoid type 1 receptor and the transient receptor potential vanilloid type 1 channel. Two potential biosynthetic pathways for NADA have been proposed, though no conclusive evidence exists for either. The first is the direct conjugation of arachidonic acid with dopamine and the other is via metabolism of a putative N-arachidonoyl tyrosine (NA-tyrosine). In the present study we investigated these biosynthetic mechanisms and report that NADA synthesis requires TH in dopaminergic terminals; however, NA-tyrosine, which we identify here as an endogenous lipid, is not an intermediate. We show that NADA biosynthesis primarily occurs through an enzyme-mediated conjugation of arachidonic acid with dopamine. While this conjugation likely involves a complex of enzymes, our data suggest a direct involvement of fatty acid amide hydrolase in NADA biosynthesis either as a rate-limiting enzyme that liberates arachidonic acid from AEA, or as a conjugation enzyme, or both.
Assuntos
Ácido Araquidônico/metabolismo , Ácidos Araquidônicos/biossíntese , Moduladores de Receptores de Canabinoides/biossíntese , Dopamina/análogos & derivados , Dopamina/metabolismo , Endocanabinoides , Amidoidrolases/metabolismo , Animais , Ácido Araquidônico/química , Ácidos Araquidônicos/química , Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides/química , Dopamina/biossíntese , Dopamina/química , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of approximately 30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of approximately 11 kcal/mol that accounted for approximately 80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (DeltaG approximately 3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by approximately 2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Ácidos Araquidônicos/química , Inibidores Enzimáticos/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Organofosfonatos/química , Amidoidrolases/química , Animais , Sítios de Ligação , Estabilidade Enzimática , Lipossomos/metabolismo , Proteínas de Membrana/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , RatosRESUMO
Cellular communication in the nervous system is mediated by chemical messengers that include amino acids, monoamines, peptide hormones, and lipids. An interesting question is how neurons regulate signals that are transmitted by membrane-embedded lipids. Here, we report the 2.8 angstrom crystal structure of the integral membrane protein fatty acid amide hydrolase (FAAH), an enzyme that degrades members of the endocannabinoid class of signaling lipids and terminates their activity. The structure of FAAH complexed with an arachidonyl inhibitor reveals how a set of discrete structural alterations allows this enzyme, in contrast to soluble hydrolases of the same family, to integrate into cell membranes and establish direct access to the bilayer from its active site.
Assuntos
Amidoidrolases/química , Proteínas de Bactérias , Membrana Celular/enzimologia , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Ácidos Araquidônicos/metabolismo , Sítios de Ligação , Moduladores de Receptores de Canabinoides , Catálise , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Endocanabinoides , Sequências Hélice-Volta-Hélice , Bicamadas Lipídicas , Modelos Moleculares , Organofosfonatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , SolubilidadeRESUMO
Cancer research depends on the use of human cell lines for both the in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo, where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a functional proteomic analysis of the human breast cancer line MDA-MB-231 grown in culture and as orthotopic xenograft tumors in the mammary fad pad of immunodeficient mice. Using a suite of activity-based chemical probes, we identified carcinoma (human) enzyme activities that were expressed selectively in culture or in xenograft tumors. Likewise, distinct groups of stromal (mouse) enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific host components to breast cancer development. MDA-MB-231 cells isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated with significantly greater tumor growth rates and metastases for xenograft-derived MDA-MB-231 cells upon reintroduction into mice. Collectively, these data indicate that the in vivo environment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying proteomic signatures that depict such changes in cancer cell biology.