Biomedical subjects

B F Cravatt

Publications and source records attributed to B F Cravatt.

At least 19 recordsLinked to original sources

PubMed2005

Desorption/ionization on silicon nanowires.

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.

Chromatography↗

PubMed2004

Segregation of two endocannabinoid-hydrolyzing enzymes into pre- and postsynaptic compartments in the rat hippocampus, cerebellum and amygdala.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) catalyse the hydrolysis of the endocannabinoids anandamide and 2-arachidonoyl glycerol. We investigated their ultrastructural distribution in brain areas where the localization and effects of cannabinoid receptor activation are known. In the hippocampus, FAAH was present in somata and dendrites of principal cells, but not in interneurons. It was located mostly on the membrane surface of intracellular organelles known to store Ca(2+) (e.g. mitochondria, smooth endoplasmic reticulum), less frequently on the somatic or dendritic plasma membrane. MGL immunoreactivity was found in axon terminals of granule cells, CA3 pyramidal cells and some interneurons. In the cerebellum, Purkinje cells and their dendrites are intensively immunoreactive for FAAH, together with a sparse axon plexus at the border of the Purkinje cell/granule cell layers. Immunostaining for MGL was complementary, the axons in the molecular layer were intensively labelled leaving the Purkinje cell dendrites blank. FAAH distribution in the amygdala was similar to that of the CB(1) cannabinoid receptor: evident signal in neuronal somata and proximal dendrites in the basolateral nucleus, and hardly any labelling in the central nucleus. MGL staining was restricted to axons in the neuropil, with similar relative signal intensities seen for FAAH in different nuclei. Thus, FAAH is primarily a postsynaptic enzyme, whereas MGL is presynaptic. FAAH is associated with membranes of cytoplasmic organelles. The differential compartmentalization of the two enzymes suggests that anandamide and 2-AG signalling may subserve functional roles that are spatially segregated at least at the stage of metabolism.

Amidohydrolases↗

PubMed2003

Comparative analysis of fatty acid amide hydrolase and cb(1) cannabinoid receptor expression in the mouse brain: evidence of a widespread role for fatty acid amide hydrolase in regulation of endocannabinoid signaling.

Fatty acid amide hydrolase (FAAH) catalyses hydrolysis of the endocannabinoid arachidonoylethanolamide ("anandamide") in vitro and regulates anandamide levels in the brain. In the cerebellar cortex, hippocampus and neocortex of the rat brain, FAAH is located in the somata and dendrites of neurons that are postsynaptic to axon fibers expressing the CB(1) cannabinoid receptor [Proc R Soc Lond B 265 (1998) 2081]. This complementary pattern of FAAH and CB(1) expression provided the basis for a hypothesis that endocannabinoids may function as retrograde signaling molecules at synapses in the brain [Proc R Soc Lond B 265 (1998) 2081; Phil Trans R Soc Lond 356 (2001) 381] and subsequent experimental studies have confirmed this [Science 296 (2002) 678]. To assess more widely the functions of FAAH in the brain and the potential impact of FAAH activity on the spatiotemporal dynamics of endocannabinoid signaling in different regions of the brain, here we have employed immunocytochemistry to compare the distribution of FAAH and CB(1) throughout the mouse brain, using FAAH(-/-) mice as negative controls to validate the specificity of FAAH-immunoreactivity observed in wild type animals. In many regions of the brain, a complementary pattern of FAAH and CB(1) expression was observed, with FAAH-immunoreactive neuronal somata and dendrites surrounded by CB(1)-immunoreactive fibers. In these regions of the brain, FAAH may regulate postsynaptic formation of anandamide, thereby influencing the spatiotemporal dynamics of retrograde endocannabinoid signaling. However, in some regions of the brain such as the globus pallidus and substantia nigra pars reticulata, CB(1) receptors are abundant but with little or no associated FAAH expression and in these brain regions the spatial impact and/or duration of endocannabinoid signaling may be less restricted than in regions enriched with FAAH. A more complex situation arises in several regions of the brain where both FAAH and CB(1) are expressed but in a non-complementary pattern, with FAAH located in neurons and/or oligodendrocytes that are proximal but not postsynaptic to CB(1)-expressing axon fibers. Here FAAH may nevertheless influence endocannabinoid signaling but more remotely. Finally, there are regions of the brain where FAAH-immunoreactive neurons and/or oligodendrocytes occur in the absence of CB(1)-immunoreactive fibers and here FAAH may be involved in regulation of signaling mediated by other endocannabinoid receptors or by receptors for other fatty acid amide signaling molecules. In conclusion, by comparing the distribution of FAAH and CB(1) in the mouse brain, we have provided a neuroanatomical framework for comparative analysis of the role of FAAH in regulation of the spatiotemporal dynamics of retrograde endocannabinoid signaling in different regions of the brain.

Amidohydrolases↗

PubMed2002

Transcriptional regulation of the mouse fatty acid amide hydrolase gene.

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme that inactivates a family of fatty acid amide molecules which are implicated in physiological processes such as pain and sleep. We cloned a 1.9 kb fragment of the 5'-untranslated region of the mouse FAAH gene into the pGL3 basic luciferase reporter vector and showed that this sequence has promoter activity in vitro. By primer extension analysis, we have determined the transcription start site to be 200 bases upstream of the ATG initiation codon and found that a TATA motif was absent. A number of putative response elements, including those for estrogen and glucocorticoids, were identified in this sequence. We have demonstrated that the estrogen and glucocorticoid receptors down-regulate transcriptional activity independent of their ligand. These data should help in understanding the mechanisms of FAAH gene transcription.

5' Flanking Region↗

PubMed2001

Structural basis for a disfavored elimination reaction in catalytic antibody 1D4.

Murine antibody 1D4 selectively catalyzes a highly disfavored beta-elimination reaction. Crystal structures of unliganded 1D4 and 1D4 in complex with a transition-state analog (TSA) have elucidated a possible general base mode of catalysis. The structures of the unliganded and liganded Fabs were determined to 1.80 and 1.85 A resolution, respectively. The structure of the complex reveals a binding pocket with high shape complementarity to the TSA, which is recruited to coerce the substrate into the sterically demanding, eclipsed conformation that is required for catalysis. A histidine residue and two water molecules are likely involved in the catalysis. The structure supports either a concerted E2 or stepwise E1cB-like mechanism for elimination. Finally, the liganded 1D4 structure shows minor conformational rearrangements in CDR H2, indicative of induced-fit binding of the hapten. 1D4 has pushed the boundaries of antibody-mediated catalysis into the realm of disfavored reactions and, hence, represents an important milestone in the development of this technology.

Animals↗

PubMed2001

Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase.

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB(1)) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB(1) ligand anandamide. Although anandamide binds and activates the CB(1) receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH(-/-)) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB(1)-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH(-/-)-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB(1) antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.

Amidohydrolases↗

PubMed2001

Characterization and manipulation of the acyl chain selectivity of fatty acid amide hydrolase.

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme that catabolizes several neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large group of hydrolytic enzymes termed the amidase signature (AS) family that is defined by a conserved, linear AS sequence of approximately 130 amino acids. Members of the AS family display strikingly different substrate selectivities, yet the primary structural regions responsible for defining substrate recognition in these enzymes remain unknown. In this study, a series of unbranched p-nitroanilide (pNA) substrates ranging from 6 to 20 carbons in length was used to probe the acyl chain binding specificity of FAAH, revealing that this enzyme exhibits a strong preference for acyl chains 9 carbons in length or longer. A fluorophosphonate inhibitor of FAAH containing a photoactivatable benzophenone group was synthesized and used to locate a region of the enzyme implicated in substrate binding. Protease digestion and mass spectrometry analysis of FAAH-inhibitor conjugates identified the major site of cross-linking as residues 487-493. Site-directed mutagenesis revealed that a single residue in this region, I491, strongly influenced substrate specificity of FAAH. For example, an I491A mutant displayed a greatly reduced binding affinity for medium-chain pNA substrates (7-12 carbons) but maintained nearly wild-type binding and catalytic constants for longer chain substrates (14-20 carbons). Mutation of I491 to aromatic or more polar residues generated enzymes with relative hydrolytic efficiencies for medium- versus long-chain pNAs that varied up to 90-fold. Collectively, these studies indicate that I491 participates in hydrophobic binding interactions with medium-chain FAAH substrates. Additionally, the significant changes in substrate selectivity achieved by single amino acid changes suggest that FAAH possesses a rather malleable substrate binding domain and may serve, along with other AS enzymes, as a template for the engineering of amidases with novel and/or tailored specificities.

Alanine↗

PubMed2001

Profiling serine hydrolase activities in complex proteomes.

Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.

Animals↗

PubMed2001

Effect of oleamide on sleep and its relationship to blood pressure, body temperature, and locomotor activity in rats.

Oleamide (cis-9,10-octadecenoamide) is a brain lipid that has recently been isolated from the cerebral fluid of sleep-deprived cats. Intracerebroventricular and intraperitoneal administration of oleamide induces sleep in rats. However, it is unclear whether oleamide's hypnogenic effects are mediated, in part, by its actions on blood pressure and core body temperature. Here we show that systemic administration of oleamide (10 and 20 mg/kg) in rats increased slow-wave sleep 2, without affecting blood pressure and heart rate. In addition, oleamide decreased body temperature and locomotor activity in a dose-dependent manner. These latter effects were not correlated in time with the observed increases in slow-wave sleep. These data suggest that the hypnogenic effects of oleamide are not related to changes in blood pressure, heart rate, or body temperature.

Animals↗

PubMed2001

Proteins regulating the biosynthesis and inactivation of neuromodulatory fatty acid amides.

Fatty acid amides (FAAs) represent a growing family of biologically active lipids implicated in a diverse range of cellular and physiological processes. At present, two general types of fatty acid amides, the N-acylethanolamines (NAEs) and the fatty acid primary amides (FAPAs), have been identified as potential physiological neuromodulators/neurotransmitters in mammals. Representative members of these two subfamilies include the endocannabinoid NAE anandamide and the sleep-inducing FAPA oleamide. In this Chapter, molecular mechanisms proposed for the biosynthesis and inactivation of FAAs are critically evaluated, with an emphasis placed on the biochemical and cell biological properties of proteins thought to mediate these processes.

Amides↗

PubMed2001

Profiling the specific reactivity of the proteome with non-directed activity-based probes.

BACKGROUND: The field of proteomics aims to characterize dynamics in protein function on a global level. However, several classes of proteins, in particular low abundance proteins, remain difficult to characterize using standard proteomics technologies. Recently, chemical strategies have emerged that profile classes of proteins based on activity rather than quantity, thereby greatly facilitating the analysis of low abundance constituents of the proteome. RESULTS: In order to expand the classes of proteins susceptible to analysis by activity-based methods, we have synthesized a library of biotinylated sulfonate esters and applied its members to complex proteomes under conditions that distinguish patterns of specific protein reactivity. Individual sulfonates exhibited unique profiles of proteome reactivity that in extreme cases appeared nearly orthogonal to one another. A robustly labeled protein was identified as a class I aldehyde dehydrogenase and shown to be irreversibly inhibited by members of the sulfonate library. CONCLUSIONS: Through screening the proteome with a non-directed library of chemical probes, diverse patterns of protein reactivity were uncovered. These probes labeled protein targets based on properties other than abundance, circumventing one of the major challenges facing contemporary proteomics research. Considering further that the probes were found to inhibit a target enzyme's catalytic activity, the methods described herein should facilitate the identification of compounds possessing both selective proteome reactivities and novel bioactivities.

Aldehyde Dehydrogenase↗

PubMed2000

Fatty acid amide hydrolase substrate specificity.

Fatty acid amide hydrolase (FAAH), also referred to as oleamide hydrolase and anandamide amidohydrolase, is a serine hydrolase responsible for the degradation of endogenous oleamide and anandamide, fatty acid amides that function as chemical messengers. FAAH hydrolyzes a range of fatty acid amides, and the present study examines the relative rates of hydrolysis of a variety of natural and unnatural fatty acid primary amide substrates using pure recombinant rat FAAH.

Amidohydrolases↗

PubMed2000

Torsin A and its torsion dystonia-associated mutant forms are lumenal glycoproteins that exhibit distinct subcellular localizations.

Early-onset torsion dystonia is an autosomal dominant hyperkinetic movement disorder that has recently been linked to a 3-base pair deletion in the DYT1 gene. The DYT1 gene encodes a 332-amino acid protein, torsin A, that bears low but significant homology to the Hsp100/Clp family of ATPase chaperones. The deletion in DYT1 associated with torsion dystonia results in the loss of one of a pair of glutamic acid residues residing near the C terminus of torsin A (DeltaE-torsin A). At present, little is known about the expression, subcellular distribution, and/or function of either the torsin A or DeltaE-torsin A protein. When transfected into mammalian cells, both torsin A and DeltaE-torsin A were found to behave as lumenally oriented glycoproteins. Immunofluorescence studies revealed that torsin A localized to a diffuse network of intracellular membranes displaying significant co-immunoreactivity for the endoplasmic reticulum resident protein BiP, whereas DeltaE-torsin A resided in large spheroid intracellular structures exclusive of BiP immunoreactivity. These results initially suggested that DeltaE-torsin A might exist as insoluble aggregates. However, both torsin A and DeltaE-torsin A were readily solubilized by nonionic detergents, were similarly accessible to proteases, and displayed equivalent migration patterns on sucrose gradients. Collectively, these data support that both the wild type and torsion dystonia-associated forms of torsin A are properly folded, lumenal proteins of similar oligomeric states. The potential relationship between the altered subcellular distribution of DeltaE-torsin A and the disease-inducing phenotype of the protein is discussed.

Animals↗

PubMed2000

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity.

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability.

Amino Acid Sequence↗

PubMed2000

Clarifying the catalytic roles of conserved residues in the amidase signature family.

Fatty acid amide hydrolase (FAAH) is a mammalian integral membrane enzyme responsible for the hydrolysis of a number of neuromodulatory fatty acid amides, including the endogenous cannabinoid anandamide and the sleep-inducing lipid oleamide. FAAH belongs to a large class of hydrolytic enzymes termed the "amidase signature family," whose members are defined by a conserved stretch of approximately 130 amino acids termed the "amidase signature sequence." Recently, site-directed mutagenesis studies of FAAH have targeted a limited number of conserved residues in the amidase signature sequence of the enzyme, identifying Ser-241 as the catalytic nucleophile and Lys-142 as an acid/base catalyst. The roles of several other conserved residues with potentially important and/or overlapping catalytic functions have not yet been examined. In this study, we have mutated all potentially catalytic residues in FAAH that are conserved among members of the amidase signature family, and have assessed their individual roles in catalysis through chemical labeling and kinetic methods. Several of these residues appear to serve primarily structural roles, as their mutation produced FAAH variants with considerable catalytic activity but reduced expression in prokaryotic and/or eukaryotic systems. In contrast, five mutations, K142A, S217A, S218A, S241A, and R243A, decreased the amidase activity of FAAH greater than 100-fold without detectably impacting the structural integrity of the enzyme. The pH rate profiles, amide/ester selectivities, and fluorophosphonate reactivities of these mutants revealed distinct catalytic roles for each residue. Of particular interest, one mutant, R243A, displayed uncompromised esterase activity but severely reduced amidase activity, indicating that the amidase and esterase efficiencies of FAAH can be functionally uncoupled. Collectively, these studies provide evidence that amidase signature enzymes represent a large class of serine-lysine catalytic dyad hydrolases whose evolutionary distribution rivals that of the catalytic triad superfamily.

Amidohydrolases↗

PubMed2000

Exceptionally potent inhibitors of fatty acid amide hydrolase: the enzyme responsible for degradation of endogenous oleamide and anandamide.

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features-an optimal C12-C8 chain length, pi-unsaturation introduction at the corresponding arachidonoyl Delta(8,9)/Delta(11,12) and oleoyl Delta(9,10) location, and an alpha-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with K(i)s that drop below 200 pM and are 10(2)-10(3) times more potent than the corresponding trifluoromethyl ketones.

Amidohydrolases↗

PubMed2000

Fatty acid amide hydrolase expression in rat choroid plexus: possible role in regulation of the sleep-inducing action of oleamide.

The enzyme fatty acid amide hydrolase (FAAH) catalyses hydrolysis of oleamide, a sleep-inducing lipid whose concentration in the cerebrospinal fluid (CSF) is elevated in sleep-deprived mammals. Previous studies have reported expression of FAAH by distinct populations of neurons in the rat brain. Here we demonstrate using immunocytochemical methods that FAAH is also expressed by non-neuronal epithelial cells of the rat choroid plexus. The choroid plexus is formed by invaginations of the pia mater into the ventricle cavities of the brain and an important function of the choroidal epithelium is to regulate production and composition of CSF. Therefore, the role of FAAH in epithelial cells of the choroid plexus may be to control the concentration of oleamide in the CSF and as such FAAH may exert an important regulatory role in shaping the duration and magnitude of the sleep-inducing effect of endogenously or exogenously derived oleamide.

Amidohydrolases↗

PubMed2000

Chemical strategies for the global analysis of protein function.

As the human genome project nears completion, biological research is entering a new era in which experimental focus will shift from identifying novel genes to determining the function of gene products. Rising to this challenge, several technologies have emerged that aim to characterise genes and/or proteins collectively rather than individually. Of particular interest is a new breed of strategies that employs synthetic chemistry to enrich our understanding of protein function on a global scale.

Animals↗