A New L-Proline Amide Hydrolase with Potential Application within the Amidase Process

L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.

Potential of Fatty Acid Amide Hydrolase (FAAH), Monoacylglycerol Lipase (MAGL), and Diacylglycerol Lipase (DAGL) Enzymes as Targets for Obesity Treatment: A Narrative Review

The endocannabinoid system (ECS) plays an integral role in maintaining metabolic homeostasis and may affect hunger, caloric intake, and nutrient absorption. Obesity has been associated with higher levels of the endogenous cannabinoid transmitters (endocannabinoids). Therefore, the ECS is an important target in obesity treatment. Modulating the enzymes that synthesize and degrade endocannabinoids, namely fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL), and diacylglycerol lipase (DAGL), may be a promising strategy to treat obesity. This review aims to synthesize all studies investigating pharmacological or genetic manipulation of FAAH, MAGL, or DAGL enzymes in association with obesity-related measures. Pharmacological inhibition or genetic deletion of FAAH tended to promote an obesogenic state in animal models, though the relationships between human FAAH polymorphisms and obesity-related outcomes were heterogeneous, which could be due to FAAH having both pro-appetitive and anti-appetitive substrates. Genetic deletion of Mgll and Dagla as well as pharmacological inhibition of DAGL tended to reduce body weight and improve metabolic state in animal studies, though the effects of Mgll manipulation were tissue-dependent. Monitoring changes in body weight in ongoing clinical trials of FAAH inhibitors may clarify whether FAAH inhibition is a potential therapeutic strategy for treatment obesity. More preclinical work is needed to characterize the role of MAGL and DAGL modulation in obesity-related outcomes.

FAAH polymorphism (rs324420) modulates extinction recall in healthy humans: an fMRI study

Gold standard treatments for anxiety- and trauma-related disorders focus on exposure therapy promoting extinction learning and extinction retention. However, its efficacy is limited. Preclinical and particularly animal research has been able to demonstrate that homozygosity for the fatty acid amide hydrolase (FAAH) C385A allele, similar to FAAH inhibition, is associated with elevated concentrations of anandamide (AEA) and facilitates extinction learning and extinction recall. However, in humans, the underlying neurobiological processes are less well understood, and further knowledge might enhance the development of more effective therapies. In this functional magnetic resonance imaging (fMRI) study, a fear conditioning, fear extinction and extinction recall paradigm was conducted with 55 healthy male adults. They were genotyped for the FAAH single-nucleotide polymorphism (SNP) rs324420 to investigate differences related to extinction recall in neural activation and State–Trait Anxiety Inventory (STAI) ratings between AC heterozygotes and CC homozygotes (FAAH C385A SNP). Differential brain activation upon an unextinguished relative to an extinguished stimulus, was greater in AC heterozygotes as compared to CC homozygotes in core neural structures previously related to extinction recall, such as the medial superior frontal gyrus, the dorsal anterior cingulate and the anterior and middle insular cortex. Furthermore, AC heterozygotes displayed higher AEA levels and lower STAI-state ratings. Our data can be interpreted in line with previous suggestions of more successful extinction recall in A-allele carriers with elevated AEA levels. Data corroborate the hypothesis that the endocannabinoid system, particularly AEA, plays a modulatory role in the extinction of aversive memory.

Genetic Variants of Fatty Acid Amide Hydrolase Modulate Acute Inflammatory Responses to Colitis in Adult Male Mice

Cannabinoids, including cannabis derived phytocannabinoids and endogenous cannabinoids (endocannabinoids), are typically considered anti-inflammatory. One such endocannabinoid is N-arachidonoylethanolamine (anandamide, AEA), which is metabolized by fatty acid amide hydrolase (FAAH). In humans, there is a loss of function single nucleotide polymorphism (SNP) in the FAAH gene (C385A, rs324420), that leads to increases in the levels of AEA. Using a mouse model with this SNP, we investigated how this SNP affects inflammation in a model of inflammatory bowel disease. We administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) intracolonically, to adult male FAAH SNP mice and examined colonic macroscopic tissue damage and myeloperoxidase activity, as well as levels of plasma and amygdalar cytokines and chemokines 3 days after administration, at the peak of colitis. We found that mice possessing the loss of function alleles (AC and AA), displayed no differences in colonic damage or myeloperoxidase activity compared to mice with wild type alleles (CC). In contrast, in plasma, colitis-induced increases in interleukin (IL)-2, leukemia inhibitory factor (LIF), monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF) were reduced in animals with an A allele. A similar pattern was observed in the amygdala for granulocyte colony stimulating factor (G-CSF) and MCP-1. In the amygdala, the mutant A allele led to lower levels of IL-1α, IL-9, macrophage inflammatory protein (MIP)-1β, and MIP-2 independent of colitis—providing additional understanding of how FAAH may serve as a regulator of inflammatory responses in the brain. Together, these data provide insights into how FAAH regulates inflammatory processes in disease.

Interplay Between Endocannabinoid System and Neurodegeneration: Focus on Polypharmacology

: Pharmacological treatment of complex pathologies such as neurodegenerative diseases still represents a major challenge, due to the networked pathways involved in their onset and progression that may require equally complex therapeutic approaches. Polypharmacology, based on the simultaneous modulation of multiple targets involved in the disease, may offer the potential to increase effectiveness and reduce the drawbacks related to the use of drug combinations. Clearly, this approach requires both the knowledge of the systems responsible for disease development and the discovery of new attractive targets to be exploited to design a multitarget drug. Over the last years, an ever increasing interest has focused on the endocannabinoid system, implicated in the modulation of several physiological functions, among which neuroinflammation, a crucial process for most neurodegenerative diseases. In this respect, the cannabinoid receptor subtype 2 represents a promising therapeutic target, being overexpressed in microglia cells and thus involved in neuroinflammation. The indirect modulation of this system through the inhibition of the main enzymes responsible for endocannabinoids metabolism, namely fatty acid amide hydrolase and monoacylglycerol lipase, may also significantly affect neurodegenerative processes. The aim of this review is to give an overview of the opportunities posed by the endocannabinoid system for neurodegenerative diseases management, mainly focusing on the potential for a multitarget strategy.

Intrasexual cuticular hydrocarbon dimorphism in a wasp sheds light on hydrocarbon biosynthesis genes in Hymenoptera

Cuticular hydrocarbons (CHCs) cover the cuticle of insects and serve as desiccation barrier and for chemical communication. While the main enzymatic steps of CHC biosynthesis are well understood, few of the underlying genes have been identified. Here we show how exploitation of intrasexual CHC dimorphism in a mason wasp, Odynerus spinipes, in combination with whole-genome sequencing and comparative transcriptomics facilitated identification of such genes. RNAi-mediated knockdown of twelve candidate gene orthologs in honey bees, Apis mellifera, confirmed nine genes impacting CHC profile composition. Most of them have predicted functions consistent with current knowledge of CHC metabolism. However, we found first-time evidence for a fatty acid amide hydrolase also influencing CHC profile composition. In situ hybridization experiments furthermore suggest trophocytes participating in CHC biosynthesis. Our results set the base for experimental CHC profile manipulation in Hymenoptera and imply that the evolutionary origin of CHC biosynthesis predates the arthropods' colonization of land.