Comparative Characterization of a Wild Type and Transmembrane Domain-Deleted Fatty Acid Amide Hydrolase:  Identification of the Transmembrane Domain as a Site for Oligomerization

Patricelli, Matthew P.; Lashuel, Hilal A.; Giang, Dan K.; Kelly, Jeffery W.; Cravatt, Benjamin F.

doi:10.1021/bi981733n

Cited by 157 publications

(197 citation statements)

References 52 publications

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“…Human-recombinant FAAH and MAGL were expressed in Escherichia coli as previously described [40,41]. A highthroughput fluorometric screening assay for recombinant FAAH inhibition using the fluorescent substrate arachidonoyl 7-amino-4-methylcoumarin amide was performed as previously reported [40].…”

Section: Faah and Magl Fluorometric Assaysmentioning

confidence: 99%

Equipotent Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase – Dual Targets of the Endocannabinoid System to Protect against Seizure Pathology

et al. 2012

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Advances in the understanding of the endogenous cannabinoid system have led to several therapeutic indications for new classes of compounds that enhance cannabinergic responses. Endocannabinoid levels are elevated during pathogenic conditions, and inhibitors of endocannabinoid inactivation promote such on-demand responses. The endocannabinoids anandamide and 2-arachidonoyl glycerol have been implicated in protective signaling against excitotoxic episodes, including seizures. To better understand modulatory pathways that can exploit such responses, we used the new generation compound AM6701 that blocks both the anandamide-deactivating enzyme fatty acid amide hydrolase (FAAH) and the 2-arachidonoyl glycerol-deactivating enzyme monoacylglycerol lipase (MAGL) with equal potency. Also studied was the structural isomer AM6702 which is 44-fold more potent for inhibiting FAAH versus MAGL. When applied before and during kainic acid (KA) exposure to cultured hippocampal slices, AM6701 protected against the resulting excitotoxic events of calpain-mediated cytoskeletal damage, loss of presynaptic and postsynaptic proteins, and pyknotic changes in neurons. The equipotent inhibitor was more effective than its close relative AM6702 at protecting against the neurodegenerative cascade assessed in the slice model. In vivo, AM6701 was also the more effective compound for reducing the severity of KA-induced seizures and protecting against behavioral deficits linked to seizure damage. Corresponding with the behavioral improvements, cytoskeletal and synaptic protection was elicited by AM6701, as found in the KA-treated hippocampal slice model. It is proposed that the influence of AM6701 on FAAH and MAGL exerts a synergistic action on the endocannabinoid system, thereby promoting the protective nature of cannabinergic signaling to offset excitotoxic brain injury.

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Section: Faah and Magl Fluorometric Assaysmentioning

confidence: 99%

Equipotent Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase – Dual Targets of the Endocannabinoid System to Protect against Seizure Pathology

et al. 2012

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“…Each of these residues was mutated to either aspartate or glutamate, and the resulting FAAH mutants were recombinantly expressed in E. coli as His 6 -tagged fusion proteins and purified as described previously (28). With the exceptions of M191E and T236E, all of the designed FAAH mutants expressed to sufficiently high levels to permit kinetic analysis.…”

Section: Characterization Of Cytoplasmic Access (Ca) Tunnelmentioning

confidence: 99%

Structure-Based Design of a FAAH Variant That Discriminates between the N-Acyl Ethanolamine and Taurine Families of Signaling Lipids

McKinney

Cravatt

2006

Biochemistry

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Fatty acid amide hydrolase (FAAH) inactivates a large and diverse class of endogenous signaling lipids termed fatty acid amides. Representative fatty acid amides include the N-acyl ethanolamines (NAEs) anandamide, which serves as an endogenous ligand for cannabinoid receptors, and N-oleoyl and N-palmitoyl ethanolamine, which produce satiety and anti-inflammatory effects, respectively. Global metabolite profiling studies of FAAH (-/-) mice have recently identified a second class of endogenous FAAH substrates: the N-acyl taurines (NATs). To determine the metabolic and signaling functions performed by NAEs and NATs in vivo, a FAAH variant that discriminates between these two substrate classes would be of value. Here, we report the structure-guided design of a point mutant in the active site of FAAH that selectively disrupts interactions with NATs. This glycine-to-aspartate (G268D) mutant was found to exhibit wildtype kinetic parameters with NAEs, but more than a 100-fold reduction in activity with NATs attributable to combined effects on K m and k cat values. These in vitro properties were also observed in living cells, where WT-FAAH and the G268D mutant displayed equivalent hydrolytic activity with NAEs, but the latter enzyme was severely impaired in its ability to catabolize NATs. The G268D FAAH mutant may thus serve as a valuable research tool to illuminate the unique roles played by the NAE and NAT classes of signaling lipids in vivo.Fatty acid amide hydrolase (FAAH) 1 is a member of the amidase signature (AS) family, a diverse group of metabolic enzymes characterized by a conserved serine-and glycinerich stretch of approximately 130 amino acids (1, 2). The AS family of hydrolases is found in a wide range of organisms, including archaea (3), eubacteria (1, 4-6), fungi (7), nematodes, plants, insects, birds (8), and mammals (9, 10). Unlike most AS enzymes, FAAH is an integral membrane protein (9, 10), which presumably facilitates its role in degrading a large number of amidated lipids in vivo.The endogenous fatty acid amide substrates hydrolyzed by FAAH fall into multiple structural classes that display distinct biological activities. A principal class of endogenous FAAH substrates is the N-acyl ethanolamines (NAEs, Figure 1A), which includes several signaling lipids, such as the endogenous cannabinoid N-arachidonoyl ethanolamine or anandamide (C20:4 NAE) (11), the satiating factor N-oleoyl ethanolamine (C18:1 NAE) (12), and the anti-inflammatory substance N-palmitoyl ethanolamine (C16:0 NAE) (13,14).FAAH also hydrolyzes fatty acid primary amides, including oleamide, which has been isolated from the cerebrospinal fluid of sleep-deprived cats (15) and, upon injection into rats, found to induce physiological sleep (16). More recently, global metabolite profiling has been applied to tissues from FAAH(-/-) mice, revealing a third class of substrates, the N-acyl taurines (NATs, Figure 1B) (17). FAAH regulates complementary sets of NATs in the nervous system and peripheral tissues bearing long chain saturat...

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“…In addition, FAAH is unusual among AS enzymes, because it is integrated into membranes. Deletion of the first 30 amino acids of FAAH, containing a N-terminal transmembrane domain, yields a catalytically active mutant (⌬TM-FAAH) still able to bind to the membranes (14).…”

mentioning

confidence: 99%

Closing the Gate to the Active Site

Mei

Venere

Gasperi

et al. 2007

Journal of Biological Chemistry

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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 (⌬G ≈ 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.Fatty acid amide hydrolase (FAAH) 3 is a dimeric, integral membrane protein (1) that brings about the degradation of important fatty acid neurotransmitters, such as oleamide and anandamide (2). These fatty acid amides (FAAs), collectively termed "endocannabinoids," modulate several aspects of mammalian pathophysiology, both at central and peripheral level (3). It is now widely recognized that the biological activity of FAAs depends on the "metabolic control" of their endogenous tone (3), and in the last few years evidence has accumulated that points to FAAH as the key regulator of FAAs concentration in vivo (4, 5). Therefore, this enzyme is becoming the subject of intense investigation, and the design of ad hoc inhibitors able to tune its catalytic activity is a hot spot in medicinal chemistry (for a recent review see Ref. 6). In fact, FAAH inhibitors might represent next-generation therapeutics for the treatment of drug and alcohol abuse (7), anxiety (8), cancer (9), as well as neurodegenerative (10, 11) and vascular (12) diseases. In addition, interest toward FAAH was boosted by the fact that this enzyme is the first and, to date, the only characterized mammalian member of a large group, referred to as the "amidase signature" (AS) family (13). More than 100 members of this class have been reported in the literature, mostly from bacteria or fungi. Despite its quite remarkable size, struct...

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Comparative Characterization of a Wild Type and Transmembrane Domain-Deleted Fatty Acid Amide Hydrolase: Identification of the Transmembrane Domain as a Site for Oligomerization

Cited by 157 publications

References 52 publications

Equipotent Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase – Dual Targets of the Endocannabinoid System to Protect against Seizure Pathology

Equipotent Inhibition of Fatty Acid Amide Hydrolase and Monoacylglycerol Lipase – Dual Targets of the Endocannabinoid System to Protect against Seizure Pathology

Structure-Based Design of a FAAH Variant That Discriminates between the N-Acyl Ethanolamine and Taurine Families of Signaling Lipids

Closing the Gate to the Active Site

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