Problem drug use and dependence are neurobehavioral disorders of complex origin. Although environmental factors contribute to drug abuse and addiction, genetic factors also play a significant role estimated at 40 -60% of the total risk. Nonetheless, the precise identities of human genes that confer vulnerability to problem drug use remain mostly unknown. Here, we describe a natural single nucleotide polymorphism in the human gene that encodes the principal endocannabinoid-inactivating enzyme, fatty acid amide hydrolase (FAAH), that in homozygous form is strongly associated with both street drug use and problem drug͞alcohol use. This single nucleotide polymorphism results in a missense mutation (385C3 A) that converts a conserved proline residue to threonine (Pro1293 Thr), producing a FAAH variant that displays normal catalytic properties but an enhanced sensitivity to proteolytic degradation. Collectively, these results suggest that genetic mutations in FAAH may constitute important risk factors for problem drug use and support a potential link between functional abnormalities in the endogenous cannabinoid system and drug abuse and dependence. D rug abuse and dependence are neurobehavioral disorders of complex origin in which both environmental and genetic factors are perceived to contribute to vulnerability (1). Genetic factors have been estimated to account for 40-60% of the risk in susceptible individuals (2). Although the primary molecular sites of action for many drugs of abuse are well characterized, efforts to identify genetic alterations in these neural signaling systems that are associated with problem drug use and addiction have, to date, been mostly unsuccessful (2). One neural signaling pathway generally implicated in drug abuse and addiction is the endogenous cannabinoid system (3). This system includes a G protein-coupled receptor CB1 that binds the principal psychoactive component of marijuana, ⌬-9-tetrahydrocannabinol, and the putative endogenous CB1 ligands, anandamide and 2-arachidonoylglycerol (4). Recent evidence suggests that the endogenous cannabinoid system may contribute not only to the development of dependence on marijuana (5) but also other drugs of abuse (6-8). Mice with a targeted disruption in the CB1 receptor exhibit reduced withdrawal responses to morphine (9), suggesting that significant crosstalk exists between endogenous opioid and cannabinoid systems in neural pathways that mediate addiction. Consistent with this notion, withdrawal from cannabinoids is significantly reduced in mice lacking preproenkephalin (10) or the -opioid receptor (11).Recently, a third central component of the endogenous cannabinoid system, the integral membrane enzyme fatty acid amide hydrolase (FAAH), has been identified (12-14). Several lines of evidence suggest that FAAH serves as a primary catabolic regulator of anandamide and related fatty acid amide-signaling molecules in vivo. Specifically, mice with a targeted disruption in the FAAH gene (FAAH Ϫ/Ϫ ) are severely impaired in their ability to degra...
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.
Hundreds, if not thousands, of uncharacterized enzymes currently populate the human proteome. Assembly of these proteins into the metabolic and signaling pathways that govern cell physiology and pathology constitutes a grand experimental challenge. Here, we address this problem by using a multidimensional profiling strategy that combines activity-based proteomics and metabolomics. This approach determined that KIAA1363, an uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and lysophosphatidic acid. Biochemical studies confirmed that KIAA1363 regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of KIAA1363 disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo. The integrated molecular profiling method described herein should facilitate the functional annotation of metabolic enzymes in any living system.
Benzoxazoles pevent misfolding: Benzoxazole‐based inhibitors of transthyretin (TTR) amyloid fibril formation are among the most effective found to date. They stabilize TTR against both acid‐mediated misfolding and urea denaturation by raising the activation barrier to tetramer dissociation, the rate‐limiting step for amyloid formation. The figure depicts the cocrystal structure of one of the better benzoxazole inhibitors bound to TTR.
Post-translational modification of histones plays an integral role in regulation of genomic expression through modulation of chromatin structure and function. Chemical preparations of histones bearing these modifications allows for comprehensive in vitro mechanistic investigation into their action to deconvolute observations from genome-wide studies in vivo. Previously, we reported the semisynthesis of ubiquitylated histone H2B (uH2B) using two orthogonal expressed protein ligation (EPL) reactions. Semisynthetic uH2B, when incorporated into nucleosomes, directly stimulates methylation of histone H3 lysine 79 (K79) by the methyltransferase, disruptor of telomeric silencinglike (Dot1L). Although recruitment of Dot1L to the nucleosomal surface by uH2B could be excluded, comprehensive mechanistic analysis was precluded by systematic limitations in the ability to generate uH2B in large scale. Here we report a highly optimized synthesis of ubiquitylated H2B bearing a Gly76Ala point mutation (uH2BG76A), yielding tens of milligrams of ubiquitylated protein. uH2BG76A is indistinguishable from the native uH2B by Dot1L, allowing for detailed studies of the resultant trans-histone crosstalk. Kinetic and structure activity relationship analyses using uH2BG76A suggest a non-canonical role for ubiquitin in the enhancement of the chemical step of H3K79 methylation. Furthermore, titration of the level of uH2B within the nucleosome revealed a 1:1 stoichiometry of Dot1L activation.Histones harbor an extraordinary density of post-translational modifications, including acetylation, methylation, phosphorylation, and ubiquitylation (1,2). Acting directly through enhancement or abbrogation of internucleosomal contacts (3) or indirectly through the recruitment of position-specific binding modules (4), these modifications direct the complex expression of the genome. Monoubiquitylation of H2B on K120 (5) is implicated in diverse nuclear processes, ranging from DNA damage repair and cell-cycle checkpoint regulation, to transcriptional elongation and stimulation of histone lysine methyltransferase activity (6-8). However, the mechanistic role of ubiquitin in these contexts remains elusive. In organisms ranging from yeast to humans, ubiquitylation of H2B is a prerequisite for efficient methylation
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