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The JAK family of non-receptor tyrosine kinases includes four subtypes (JAK1, JAK2, JAK3, and TYK2) and is responsible for signal transduction downstream of diverse cytokine receptors. JAK inhibitors have emerged as important therapies for immuno(onc)ological disorders, but their use is limited by undesirable side effects presumed to arise from poor subtype 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 transphosphorylation 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 subtype selectivity.

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.

Background: Parents play a key role in infants’ development through their interactions and the type of environment they provide for their child 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 parents’ perceptions of their infants’ active play by parental working status. The purpose of this study was to explore parents’ perceptions 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 the proportion of working and stay at home parents who responded within each theme were used to compare for differences between 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 listen to a healthcare provider.Conclusions: Working status of parents seems to have implications on certain aspects and perceptions of active play which in turn may influence infants’ physical 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.

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.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here, we report a series of in vivo-active 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed excellent selectivity for DAGLβ over other serine hydrolases, including DAGLα (~60-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 DAGLβ 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 (PLA2G4A). We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

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.

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.

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.

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 ≈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 ≈11 kcal/mol that accounted for ≈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 (ΔG ≈3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈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.

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.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol synthesis. The peroxisomal targeting signals for phosphomevalonate kinase and isopentenyl diphosphate isomerase have been identified. In the current study we identify the peroxisomal targeting signals required for four other enzymes of the cholesterol biosynthetic pathway: acetoacetyl-CoA (AA-CoA) thiolase, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, mevalonate diphosphate decarboxylase (MPPD), and farnesyl diphosphate (FPP) synthase. Data are presented that demonstrate that mitochondrial AA-CoA thiolase contains both a mitochondrial targeting signal at the amino terminus and a peroxisomal targeting signal (PTS-1) at the carboxy terminus. We also analyze a new variation of PTS-2 sequences required to target HMG-CoA synthase and MPPD to peroxisomes. In addition, we show that FPP synthase import into peroxisomes is dependent on the PTS-2 receptor and identify at the amino terminus of the protein a 20-amino acid region that is required for the peroxisomal localization of the enzyme. These data provide further support for the conclusion that peroxisomes play a critical role in cholesterol biosynthesis.—Olivier, L. M., W. Kovacs, K. Masuda, G-A. Keller, and S. K. Krisans. Identification of peroxisomal targeting signals in cholesterol biosynthetic enzymes: AA-CoA thiolase, HMG-CoA synthase, MPPD, and FPP synthase. J. Lipid Res. 2000. 41: 1921–1935.

In the liver 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is present not only in the endoplasmic reticulum but also in the peroxisomes. However, to date no information is available regarding the function of the peroxisomal HMG-CoA reductase in cholesterol/isoprenoid metabolism, and the structure of the peroxisomal HMG-CoA reductase has yet to be determined. We have identified a mammalian cell line that expresses only one HMG-CoA reductase protein and that is localized exclusively to peroxisomes. This cell line was obtained by growing UT2 cells (which lack the endoplasmic reticulum HMG-CoA reductase) in the absence of mevalonate. The cells exhibited a marked increase in a 90-kDa HMG-CoA reductase that was localized exclusively to peroxisomes. The wild type Chinese hamster ovary cells contain two HMG-CoA reductase proteins, the well characterized 97-kDa protein, localized in the endoplasmic reticulum, and a 90-kDa protein localized in peroxisomes. The UT2 cells grown in the absence of mevalonate containing the up-regulated peroxisomal HMG-CoA reductase are designated UT2*. A detailed characterization and analysis of this cell line is presented in this study.

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.

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.

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.