Biomedical subjects

Benjamin F Cravatt

Publications and source records attributed to Benjamin F Cravatt.

At least 73 records · Page 4Linked to original sources

PubMed2004

Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo.

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.

Amino Acid Sequence↗

PubMed2004

Characterization of the sleep-wake patterns in mice lacking fatty acid amide hydrolase.

STUDY OBJECTIVES: Oleamide and anandamide are fatty acid amides implicated in the regulatory mechanisms of sleep processes. However, due to their prompt catabolism by fatty acid amide hydrolase (FAAH), their pharmacologic and behavioral effects, in vivo, disappear rapidly. To determine if, in the absence of FAAH, the hypnogenic fatty acid amides induce an increase of sleep, we characterized the sleep-wake patters in FAAH-knockout mice [FAAH (-/-)] before and after sleep deprivation. DESIGN: FAAH (-/-), FAAH (+/-), and FAAH (+/+) mice were implanted chronically for sleep, body temperature (Tb), and locomotor activity (LMA) recordings. Sleep-wake states were recorded during a 24-hour baseline session followed by 8 hours of sleep deprivation. Recovery recordings were done during the 16 hours following sleep deprivation. Total amount of wake, slow-wave sleep, and rapid eye movement sleep were calculated and compared between genotypes. The electroencephalographic spectral analysis was performed by fast Fourier transform analysis. Telemetry recordings of Tb and LMA were carried out continuously during 4 days under baseline conditions. SETTING: N/A. PATIENTS OR PARTICIPANTS: FAAH (-/-) mice and their heterozygote (+/-) and control (+/+) littermates were used. INTERVENTIONS: Sleep deprivation. MEASUREMENTS AND RESULTS: FAAH (-/-) mice possess higher values of slow-wave sleep and more intense episodes of slow-wave sleep than do control littermates under baseline conditions that are not related to differences in Tb and LMA. A rebound of slow-wave sleep and rapid eye movement sleep as well an increase in the levels of slow-wave activity were observed after sleep deprivation in all genotypes. CONCLUSION: These findings support the role of fatty acid amides as possible modulators of sleep and indicate that the homeostatic mechanisms of sleep in FAAH (-/-) mice are not disrupted.

Amidohydrolases↗

PubMed2004

Reduced cellular expression and activity of the P129T mutant of human fatty acid amide hydrolase: evidence for a link between defects in the endocannabinoid system and problem drug use.

Fatty acid amide hydrolase (FAAH) inactivates the endogenous cannabinoid (endocannabinoid) anandamide and related lipid transmitters in vivo. A single nucleotide polymorphism (SNP) in the human FAAH gene (385C to A) has recently been described that, in homozygous form, is over-represented in subjects with problem drug use. This SNP, which converts a conserved proline residue in FAAH to threonine (P129T), suggests a potential role for the FAAH-endocannabinoid system in regulating addictive behavior. Nonetheless, the impact of the 385A mutation on the biochemical and cellular function of FAAH remains unknown. Here, we report that T-lymphocytes isolated from patients homozygous for the P129T-FAAH variant express less than half of the FAAH protein and activity observed in wild-type (WT) lymphocytes. Transfected COS-7 cells also expressed significantly lower levels of P129T-FAAH compared with WT-FAAH, indicating that the aberrant expression of the former protein is not a cell type-specific phenomenon. A comparison of the transcription/translation efficiencies and cellular stabilities of WT- and P129T-FAAH proteins revealed that the reduced expression of the mutant enzyme is due to a post-translational mechanism that precedes productive folding. These findings indicate that the natural 385A SNP in the human FAAH gene produces a mutant enzyme with reduced cellular stability, thus fortifying a potential link between functional abnormalities in the endocannabinoid system and drug abuse and dependence.

Amidohydrolases↗

PubMed2004

Functional disassociation of the central and peripheral fatty acid amide signaling systems.

Fatty acid amides (FAAs) constitute a large class of endogenous signaling lipids that modulate several physiological processes, including pain, feeding, blood pressure, sleep, and inflammation. Although FAAs have been proposed to evoke their behavioral effects through both central and peripheral mechanisms, these distinct signaling pathways have remained experimentally challenging to separate. Here, we report a transgenic mouse model in which the central and peripheral FAA systems have been functionally uncoupled. Mice were generated that express the principle FAA-degrading enzyme FAA hydrolase (FAAH) specifically in the nervous system (FAAH-NS mice) by crossing FAAH(-/-) mice with transgenic mice that express FAAH under the neural specific enolase promoter. FAAH-NS mice were found to possess wild-type levels of FAAs in the brain and spinal cord, but significantly elevated concentrations of these lipid transmitters in peripheral tissues. This anatomically restricted biochemical phenotype correlated with a reversion of the reduced pain sensitivity of FAAH(-/-) mice, consistent with the FAA anandamide producing this effect by acting on cannabinoid receptors in the nervous system. Interestingly, however, FAAH-NS mice still exhibited an antiinflammatory phenotype similar in magnitude to FAAH(-/-) mice, indicating that this activity, which was not blocked by cannabinoid receptor antagonists, was mediated by peripherally elevated FAAs. These data suggest that the central and peripheral FAA signaling systems regulate discrete behavioral processes and may be targeted for distinct therapeutic gain.

Amides↗

PubMed2004

Reversible inhibitors of fatty acid amide hydrolase that promote analgesia: evidence for an unprecedented combination of potency and selectivity.

Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of alpha-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.

Amidohydrolases↗

PubMed2004

Comparison of anandamide transport in FAAH wild-type and knockout neurons: evidence for contributions by both FAAH and the CB1 receptor to anandamide uptake.

The cellular inactivation of the endogenous cannabinoid (endocannabinoid) anandamide (AEA) represents a controversial and intensely investigated subject. This process has been proposed to involve two proteins, a transporter that promotes the cellular uptake of AEA and fatty acid amide hydrolase (FAAH), which hydrolyzes AEA to arachidonic acid. However, whereas the role of FAAH in AEA metabolism is well-characterized, the identity of the putative AEA transporter remains enigmatic. Indeed, the indirect pharmacological evidence used to support the existence of an AEA transporter has been suggested also to be compatible with a model in which AEA uptake is driven by simple diffusion coupled to FAAH metabolism. Here, we have directly addressed the contribution of FAAH to AEA uptake by examining this process in neuronal preparations from FAAH(-/-) mice and in the presence of the uptake inhibitor UCM707. The results of these studies reveal that (i) care should be taken to avoid the presence of artifacts when studying the cellular uptake of lipophilic molecules like AEA, (ii) FAAH significantly contributes to AEA uptake, especially with longer incubation times, and (iii) a UCM707-sensitive protein(s) distinct from FAAH also participates in AEA uptake. Interestingly, the FAAH-independent component of AEA transport was significantly reduced by pretreatment of neurons with the cannabinoid receptor 1 (CB1) antagonist SR141716A. Collectively, these results indicate that the protein-dependent uptake of AEA is largely mediated by known constituents of the endocannabinoid system (FAAH and the CB1 receptor), although a partial contribution of an additional UCM707-sensitive protein is also suggested.

Amidohydrolases↗

PubMed2004

Activity-based probes for the proteomic profiling of metalloproteases.

Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activity-based profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

Dipeptides↗

PubMed2004

Structural commonalities among integral membrane enzymes.

The X-ray crystal structures of five distinct enzymes (prostaglandin H(2) synthase, squalene cyclase, fatty acid amide hydrolase, microsomal cytochrome P450, and estrone sulfatase) challenge contemporary descriptions of integral membrane proteins. This structurally divergent group represents an important component of the integral membrane proteome that lies at the bilayer's aqueous interface. We summarize here what is collectively understood about the membrane insertion of these proteins, what roles they may play in lipid biology, and their relationship to soluble structural homologs.

Animals↗

PubMed2004

Mapping enzyme active sites in complex proteomes.

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.

Aldehyde Dehydrogenase↗

PubMed2004

Formation of prostamides from anandamide in FAAH knockout mice analyzed by HPLC with tandem mass spectrometry.

We investigated the formation of PGF(2alpha) 1-ethanolamide, PGE(2) 1-ethanolamide, and PGD(2) 1-ethanolamide (prostamides F(2alpha), E(2), and D(2), respectively) in liver, lung, kidney, and small intestine after a single intravenous bolus administration of 50 mg/kg of anandamide to normal and fatty acid amide hydrolase knockout (FAAH -/-) male mice. One group of three normal mice was not dosed (naïve) while another group of three normal mice received a bolus intravenous injection of 50 mg/kg of anandamide. Three FAAH -/- mice also received an intravenous injection of 50 mg/kg of anandamide. After 30 min, the lung, liver, kidney, and small intestine were harvested and processed by liquid-liquid extraction. The concentrations of prostamide F(2alpha), prostamide E(2), prostamide D(2), and anandamide were determined by HPLC-tandem mass spectrometry. Prostamide F(2alpha) was detected in tissues in FAAH -/- mice after administration of anandamide. Concentrations of anandamide, prostamide E(2), and prostamide D(2) in liver, kidney, lung, and small intestine were much higher in the anandamide-treated FAAH -/- mice than those of the anandamide-treated control mice. This report demonstrates that prostamides, including prostamide F(2alpha), were formed in vivo from anandamide, potentially by the cyclooxygenase-2 pathway when the competing FAAH pathway is lacking.

Amidohydrolases↗

PubMed2004

Chemical strategies for activity-based proteomics.

The assignment of molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes presents a major challenge for the field of proteomics. To address this problem, chemical approaches have been introduced that utilize small-molecule probes to profile dynamics in enzyme activity in complex proteomes. These strategies for activity-based protein profiling enable both the discovery and functional analysis of enzymes associated with human disease.

Animals↗

PubMed2004

The endogenous cannabinoid system and its role in nociceptive behavior.

The analgesic properties of exogenous cannabinoids have been recognized for many years and suggest a regulatory role for the endogenous cannabinoid ("endocannabinoid") system in mammalian nociceptive pathways. The endocannabinoid system includes: (1) at least two families of lipid signaling molecules, the N-acyl ethanolamines (e.g., anandamide) and the monoacylglycerols (e.g., 2-arachidonoyl glycerol); (2) multiple enzymes involved in the biosynthesis and degradation of these lipids, including the integral membrane enzyme fatty acid amide hydrolase; and (3) two G-protein coupled receptors, CB1 and CB2, which are primarily localized to the nervous system and immune system, respectively. Here, we review recent genetic, behavioral, and pharmacological studies that have tested the function of the endocannabinoid system in pain sensation. Collectively, these investigations support a role for endocannabinoids in modulating behavioral responses to acute, inflammatory, and neuropathic pain stimuli.

Animals↗

PubMed2004

The development and application of methods for activity-based protein profiling.

Recent advances in genomic and proteomic technologies have begun to address the challenge of assigning molecular and cellular functions to the numerous protein products encoded by prokaryotic and eukaryotic genomes. In particular, chemical strategies for proteome analysis have emerged that enable profiling of protein activity on a global scale. Herein, we highlight these chemical proteomic methods and their application to the discovery and characterization of disease-related enzyme activities.

Animals↗

PubMed2004

Profiling enzyme activities in vivo using click chemistry methods.

Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. Recently, we introduced a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") to analyze the functional state of enzymes in living cells and organisms. Here, we report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.

Alkynes↗

PubMed2004

Discovering disease-associated enzymes by proteome reactivity profiling.

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes. Quantitative screening of probe-proteome reactions coupled with bioinformatic analysis enabled the selection of a suite of probes that exhibit complementary protein reactivity profiles. This optimal probe set was applied to discover several enzyme activities differentially expressed in lean and obese (ob/ob) mice. Interestingly, one of these enzymes, hydroxypyruvate reductase, which was 6-fold upregulated in ob/ob livers, participates in the conversion of serine to glucose, suggesting that this unusual metabolic pathway may contribute to gluconeogenesis selectively in states of obesity.

Acetamides↗

PubMed2004

Fatty acid amide hydrolase in brain ventricular epithelium: mutually exclusive patterns of expression in mouse and rat.

Fatty acid amides and fatty acid ethanolamides are novel signalling molecules exemplified by the sleep-inducing lipid oleamide and the endocannabinoid anandamide, respectively. These substances are inactivated by fatty acid amide hydrolase (FAAH), an enzyme that is expressed by neurons and non-neuronal cells in the brain. In the rat, FAAH-immunoreactivity has been detected in epithelial cells of the choroid plexus and, in accordance with this finding, here we report FAAH mRNA expression in rat choroid plexus epithelium using in situ hybridisation methods. Surprisingly, a comparative analysis of mouse brain did not reveal FAAH mRNA expression or FAAH-immunoreactivity in the choroid plexus of this species. FAAH-immunoreactivity was, however, detected in non-choroidal ventricular ependymal cells in the mouse brain and the specificity of this immunostaining was confirmed by analysis of FAAH-knockout mice. FAAH-immunoreactivity was detected in ependymal cells throughout the ventricles of the mouse brain but with regional variation in the intensity of immunostaining. Intriguingly, in rat brain, although FAAH expression is observed in choroid plexus epithelial cells, little or no FAAH-immunoreactivity is present in the ventricular ependyma. Thus, there are mutually exclusive patterns of FAAH expression in the ventricular epithelium of rat and mouse brain. Our observations provide the basis for an experimental analysis that exploits differences in FAAH expression in rat and mouse to investigate FAAH function in ventricular epithelial cells and, in particular, the role of FAAH in regulating the sleep-inducing agent oleamide in cerebrospinal fluid.

Amidohydrolases↗

PubMed2004

Mice lacking fatty acid amide hydrolase exhibit a cannabinoid receptor-mediated phenotypic hypoalgesia.

Although the N-arachidonoyl ethanolamine (anandamide) binds to cannabinoid receptors and has been implicated in the suppression of pain, its rapid catabolism in vivo by fatty acid amide hydrolase (FAAH) has presented a challenge in investigating the physiological functions of this endogenous cannabinoid. In order to test whether anandamide and other non-cannabinoid fatty amides modulate nociception, we compared FAAH (+/+) and (-/-) mice in the tail immersion, hot plate, and formalin tests, as well as for thermal hyperalgesia in the carrageenan and the chronic constriction injury (CCI) models. FAAH (-/-) mice exhibited a CB1 receptor-mediated phenotypic hypoalgesia in thermal nociceptive tests. These mice also exhibited CB1 receptor-mediated hypoalgesia in both phases of the formalin test accompanied with a phenotypic anti-edema effect, which was not blocked by either CB1 or CB2 antagonists. Additionally, FAAH (-/-) mice displayed thermal anti-hyperalgesic and anti-inflammatory effects in the carrageenan model that were mediated, in part, by CB2, but not CB1 receptors. In contrast, no genotype differences in pain behavior were evident following CCI, which was instead found to obliterate the phenotypic hypoalgesia displayed by FAAH (-/-) mice in the tail immersion and hot plate tests, suggesting that nerve injury may promote adaptive changes in these animals. Collectively, these findings demonstrate a cannabinoid receptor-mediated analgesic phenotype in FAAH (-/-) mice. In more general terms, these findings suggest that selective inhibitors of FAAH might represent a viable pharmacological approach for the clinical treatment of pain disorders.

Adaptation, Physiological↗

PubMed2004

The endogenous cannabinoid system protects against colonic inflammation.

Excessive inflammatory responses can emerge as a potential danger for organisms' health. Physiological balance between pro- and anti-inflammatory processes constitutes an important feature of responses against harmful events. Here, we show that cannabinoid receptors type 1 (CB1) mediate intrinsic protective signals that counteract proinflammatory responses. Both intrarectal infusion of 2,4-dinitrobenzene sulfonic acid (DNBS) and oral administration of dextrane sulfate sodium induced stronger inflammation in CB1-deficient mice (CB1(-/-)) than in wild-type littermates (CB1(+/+)). Treatment of wild-type mice with the specific CB1 antagonist N-(piperidino-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-pyrazole-3-carboxamide (SR141716A) mimicked the phenotype of CB1(-/-) mice, showing an acute requirement of CB1 receptors for protection from inflammation. Consistently, treatment with the cannabinoid receptor agonist R(-)-7-hydroxy-Delta(6)-tetra-hydrocannabinol-dimethylheptyl (HU210) or genetic ablation of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) resulted in protection against DNBS-induced colitis. Electrophysiological recordings from circular smooth muscle cells, performed 8 hours after DNBS treatment, revealed spontaneous oscillatory action potentials in CB1(-/-) but not in CB1(+/+) colons, indicating an early CB1-mediated control of inflammation-induced irritation of smooth muscle cells. DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. Our results indicate that the endogenous cannabinoid system represents a promising therapeutic target for the treatment of intestinal disease conditions characterized by excessive inflammatory responses.

Amidohydrolases↗