Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases.

The initial adoption of penicillin as an antibiotic marked the start of exploring other compounds essential for pharmaceuticals, yet resistance to penicillins and their side effects has compromised their efficacy. The N-terminal nucleophile (Ntn) amide-hydrolases S45 family plays a key role in catalyzing amide bond hydrolysis in various compounds, including antibiotics like penicillin and cephalosporin. This study comprehensively analyzes the structural and functional traits of the bacterial N-terminal nucleophile (Ntn) amide-hydrolases S45 family, covering penicillin G acylases, cephalosporin acylases, and D-succinylase. Utilizing structural bioinformatics tools and sequence analysis, the investigation delineates structurally conserved regions (SCRs) and substrate binding site variations among these enzymes. Notably, sixteen SCRs crucial for substrate interaction are identified solely through sequence analysis, emphasizing the significance of sequence data in characterizing functionally relevant regions. These findings introduce a novel approach for identifying targets to enhance the biocatalytic properties of N-terminal nucleophile (Ntn) amide-hydrolases, while facilitating the development of more accurate three-dimensional models, particularly for enzymes lacking structural data. Overall, this research advances our understanding of structure-function relationships in bacterial N-terminal nucleophile (Ntn) amide-hydrolases, providing insights into strategies for optimizing their enzymatic capabilities.

Endocannabinoid Hydrolase Inhibitors: Potential Novel Anxiolytic Drugs.

Over the past decade, the idea of targeting the endocannabinoid system to treat anxiety disorders has received increasing attention. Previous studies focused more on developing cannabinoid receptor agonists or supplementing exogenous cannabinoids, which are prone to various adverse effects due to their strong pharmacological activity and poor receptor selectivity, limiting their application in clinical research. Endocannabinoid hydrolase inhibitors are considered to be the most promising development strategies for the treatment of anxiety disorders. More recent efforts have emphasized that inhibition of two major endogenous cannabinoid hydrolases, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH), indirectly activates cannabinoid receptors by increasing endogenous cannabinoid levels in the synaptic gap, circumventing receptor desensitization resulting from direct enhancement of endogenous cannabinoid signaling. In this review, we comprehensively summarize the anxiolytic effects of MAGL and FAAH inhibitors and their potential pharmacological mechanisms, highlight reported novel inhibitors or natural products, and provide an outlook on future directions in this field.

Evaluating the Impact of Probiotic Therapy on the Endocannabinoid System, Pain, Sleep and Fatigue: A Randomized, Double-Blind, Placebo-Controlled Trial in Dancers.

Emerging research links the endocannabinoid system to gut microbiota, influencing nociception, mood, and immunity, yet the molecular interactions remain unclear. This study focused on the effects of probiotics on ECS markers-cannabinoid receptor type 2 (CB2) and fatty acid amide hydrolase (FAAH)-in dancers, a group selected due to their high exposure to physical and psychological stress. In a double-blind, placebo-controlled trial (ClinicalTrials.gov NCT05567653), 15 dancers were assigned to receive either a 12-week regimen of Lactobacillus helveticus Rosell-52 and Bifidobacterium longum Rosell-17 or a placebo (PLA: n = 10, PRO: n = 5). There were no significant changes in CB2 (probiotic: 0.55 to 0.29 ng/mL; placebo: 0.86 to 0.72 ng/mL) or FAAH levels (probiotic: 5.93 to 6.02 ng/mL; placebo: 6.46 to 6.94 ng/mL; p > 0.05). A trend toward improved sleep quality was observed in the probiotic group, while the placebo group showed a decline (PRO: from 1.4 to 1.0; PLA: from 0.8 to 1.2; p = 0.07841). No other differences were noted in assessed outcomes (pain and fatigue). Probiotic supplementation showed no significant impact on CB2 or FAAH levels, pain, or fatigue but suggested potential benefits for sleep quality, suggesting an area for further research.

Cytosolic N-terminal formyl-methionine deformylation derives cancer stem cell features and tumor progression.

Eukaryotic cells can synthesize formyl-methionine (fMet)-containing proteins not only in mitochondria but also in the cytosol to some extent. Our previous study revealed substantial upregulation of N-terminal (Nt)-fMet-containing proteins in the cytosol of SW480 colorectal cancer cells. However, the functional and pathophysiological implications remain unclear. Here, we demonstrated that removal of the Nt-formyl moiety of Nt-fMet-containing proteins (via expressing Escherichia coli PDF peptide deformylase) resulted in a dramatic increase in the proliferation of SW480 colorectal cancer cells. This proliferation coincided with the acquisition of cancer stem cell features, including reduced cell size, enhanced self-renewal capacity, and elevated levels of the cancer stem cell surface marker CD24 and pluripotent transcription factor SOX2. Furthermore, deformylation of Nt-fMet-containing proteins promoted the tumorigenicity of SW480 colorectal cancer cells in an in vivo xenograft mouse model. Taken together, these findings suggest that cytosolic deformylation has a tumor-enhancing effect, highlighting its therapeutic potential for cancer treatment.

Structural study for substrate recognition of human N-terminal glutamine amidohydrolase 1 in the arginine N-degron pathway.

The N-degron pathway determines the half-life of proteins by selectively destabilizing the proteins bearing N-degrons. N-terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N-degron (Arg/N-degron) pathway as an initializing enzyme via the deamidation of the N-terminal (Nt) glutamine (Gln). However, the Nt-serine-bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate-binding conformations of hNTAQ1 depending on the subsequent residue of Nt-Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt-Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate-recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt-Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N-degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.

Connection between protein-tyrosine kinase inhibition and coping with oxidative stress in Bacillus subtilis.

In bacteria, attenuation of protein-tyrosine phosphorylation occurs during oxidative stress. The main described mechanism behind this effect is the H2O2-triggered conversion of bacterial phospho-tyrosines to protein-bound 3,4-dihydroxyphenylalanine. This disrupts the bacterial tyrosine phosphorylation-based signaling network, which alters the bacterial polysaccharide biosynthesis. Herein, we report an alternative mechanism, in which oxidative stress leads to a direct inhibition of bacterial protein-tyrosine kinases (BY-kinases). We show that DefA, a minor peptide deformylase, inhibits the activity of BY-kinase PtkA when Bacillus subtilis is exposed to oxidative stress. High levels of PtkA activity are known to destabilize B. subtilis pellicle formation, which leads to higher sensitivity to oxidative stress. Interaction with DefA inhibits both PtkA autophosphorylation and phosphorylation of its substrate Ugd, which is involved in exopolysaccharide formation. Inactivation of defA drastically reduces the capacity of B. subtilis to cope with oxidative stress, but it does not affect the major oxidative stress regulons PerR, OhrR, and Spx, indicating that PtkA inhibition is the main pathway for DefA involvement in this stress response. Structural analysis identified DefA residues Asn95, Tyr150, and Glu152 as essential for interaction with PtkA. Inhibition of PtkA depends also on the presence of a C-terminal α-helix of DefA, which resembles PtkA-interacting motifs from known PtkA activators, TkmA, SalA, and MinD. Loss of either the key interacting residues or the inhibitory helix of DefA abolishes inhibition of PtkA in vitro and impairs postoxidative stress recovery in vivo, confirming the involvement of these structural features in the proposed mechanism.

An Extremely Sensitive Ultra-High Throughput Growth Selection Assay for the Identification of Amidase Activity.

In the last decades, biocatalysis has offered new perspectives for the synthesis of (chiral) amines, which are essential building blocks for pharmaceuticals, fine and bulk chemicals. In this regard, amidases have been employed due to their broad substrate scope and their independence from expensive cofactors. To expand the repertoire of amidases, tools for their rapid identification and characterization are greatly demanded. In this work an ultra-high throughput growth selection assay based on the production of the folate precursor p-aminobenzoic acid (PABA) is introduced to identify amidase activity. PABA-derived amides structurally mimic the broad class of commonly used chromogenic substrates derived from p-nitroaniline. This suggests that the assay should be broadly applicable for the identification of amidases. Unlike conventional growth selection assays that rely on substrates as nitrogen or carbon source, our approach requires PABA in sub-nanomolar concentrations, making it exceptionally sensitive and ideal for engineering campaigns that aim at enhancing amidase activities from minimally active starting points, for example. The presented assay offers flexibility in the adjustment of sensitivity to suit project-specific needs using different expression systems and fine-tuning with the antimetabolite sulfathiazole. Application of this PABA-based assay facilitates the screening of millions of enzyme variants on a single agar plate within two days, without the need for laborious sample preparation or expensive instruments, with transformation efficiency being the only limiting factor. KEY POINTS: • Ultra-high throughput assay (tens of millions on one agar plate) for amidase screening • High sensitivity by coupling selection to folate instead of carbon or nitrogen source • Highly adjustable in terms of sensitivity and expression of the engineering target.

DDAH-1 maintains endoplasmic reticulum-mitochondria contacts and protects dopaminergic neurons in Parkinson's disease.

The loss of dopaminergic neurons in the substantia nigra is a hallmark of pathology in Parkinson's disease (PD). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the critical enzyme responsible for the degradation of asymmetric dimethylarginine (ADMA) which inhibits nitric oxide (NO) synthase and has been implicated in neurodegeneration. Mitochondrial dysfunction, particularly in the mitochondria-associated endoplasmic reticulum membrane (MAM), plays a critical role in this process, although the specific molecular target has not yet been determined. This study aims to examine the involvement of DDAH-1 in the nigrostriatal dopaminergic pathway and PD pathogenesis. The distribution of DDAH-1 in the brain and its colocalization with dopaminergic neurons were observed. The loss of dopaminergic neurons and aggravated locomotor disability after rotenone (ROT) injection were showed in the DDAH-1 knockout rat. L-arginine (ARG) and NO donors were employed to elucidate the role of NO respectively. In vitro, we investigated the effects of DDAH-1 knockdown or overexpression on cell viability and mitochondrial functions, as well as modulation of ADMA/NO levels using ADMA or ARG. MAM formation was assessed by the Mitofusin2 oligomerization and the mitochondrial ubiquitin ligase (MITOL) phosphorylation. We found that DDAH-1 downregulation resulted in enhanced cell death and mitochondrial dysfunctions, accompanied by elevated ADMA and reduced NO levels. However, the recovered NO level after the ARG supplement failed to exhibit a protective effect on mitochondrial functions and partially restored cell viability. DDAH-1 overexpression prevented ROT toxicity, while ADMA treatment attenuated these protective effects. The declines of MAM formation in ROT-treated cells were exacerbated by DDAH-1 downregulation via reduced MITOL phosphorylation, which was reversed by DDAH-1 overexpression. Together, the abundant expression of DDAH-1 in nigral dopaminergic neurons may exert neuroprotective effects by maintaining MAM formation and mitochondrial function probably via ADMA, indicating the therapeutic potential of targeting DDAH-1 for PD.

Monofluorophore-based Two-Photon Ratiometric Fluorescent Probe for the Quantitative Imaging of Fatty Acid Amide Hydrolase in Live Neurons and Mouse Brain Tissues.

Fatty acid amide hydrolase (FAAH) plays a crucial role in the metabolism of the endocannabinoid system by hydrolyzing a series of bioactive amides, whose abnormal levels are associated with neuronal disorders including Alzheimer's disease (AD). However, due to the lack of suitable quantitative sensing tools, real-time and accurate monitoring of the activity of FAAH in living systems remains unresolved. Herein, a novel enzyme-activated near-infrared two-photon ratiometric fluorescent probe (CANP) based on a naphthylvinylpyridine monofluorophore is successfully developed, in which the electron-withdrawing amide moiety is prone to be hydrolyzed to an electron-donating amine group under the catalysis of FAAH, leading to the activation of the intramolecular charge transfer process and the emergence of a new 80 nm red-shifted emission, thereby achieving a ratiometric luminescence response. Benefiting from the high selectivity, high sensitivity, and ratiometric response to FAAH, the probe CANP is successfully used to quantitatively monitor and image the FAAH levels in living neurons, by which an amyloid ß (Aß)-induced upregulation of endogenous FAAH activity is observed. Similar increases in FAAH activity are found in various brain regions of AD model mice, indicating a potential fatty acid amide metabolite-involved pathway for the pathological deterioration of AD. Moreover, our quantitative FAAH inhibition experiments further demonstrate the great value of CANP as an efficient visual probe for in situ and precise assessment of FAAH inhibitors in complex living systems, assisting the discovery of FAAH-related therapeutic agents.

Probing the enzymatic activity and maturation process of the EcAIII Ntn-amidohydrolase using local random mutagenesis.

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.

Impact of miR-222-3p-mediated downregulation of arylacetamide deacetylase on drug hydrolysis and lipid accumulation.

Arylacetamide deacetylase (AADAC) is involved in drug hydrolysis and lipid metabolism. In 23 human liver samples, no significant correlation was observed between AADAC mRNA (19.7-fold variation) and protein levels (137.6-fold variation), suggesting a significant contribution of post-transcriptional regulation to AADAC expression. The present study investigated whether AADAC is regulated by microRNA in the human liver and elucidate its biological significance. Computational analysis predicted two potential miR-222-3p recognition elements in the 3'-untranslated region (UTR) of AADAC. Luciferase assay revealed that the miR-222-3p recognition element was functional in downregulating AADAC expression. In HEK293 cells transfected with an AADAC expression plasmid containing 3'-UTR, miR-222-3p overexpression decreased AADAC protein level and activity, whereas miR-222-3p inhibition increased them. Similar results were observed in human hepatoma-derived Huh-1 cells endogenously expressing AADAC and HepaSH cells that are hepatocytes from chimeric mice with humanized livers. In individual human liver samples, AADAC protein levels inversely correlated with miR-222-3p levels. Overexpression of miR-222-3p resulted in increased lipid accumulation in Huh-1 cells, which was reversed by AADAC overexpression. In contrast, miR-222-3p inhibition decreased lipid accumulation, which was reversed by AADAC knockdown. In conclusion, we found that hepatic AADAC was downregulated by miR-222-3p, resulting in decreased drug hydrolysis and increased lipid accumulation.

Two different types of hydrolases co-degrade ochratoxin A in a highly efficient degradation strain Lysobacter sp. CW239.

Ochratoxin A (OTA) is a toxic secondary metabolite that widely contaminates agro-products and poses a significant dietary risk to human health. Previously, a carboxypeptidase CP4 was characterized for OTA degradation in Lysobacter sp. CW239, but the degradation activity was much lower than its host strain CW239. In this study, an amidohydrolase ADH2 was screened for OTA hydrolysis in this strain. The result showed that 50 µg/L OTA was completely degraded by 1.0 µg/mL rADH2 within 5 min, indicating ultra-efficient activity. Meanwhile, the two hydrolases (i.e., CP4 and ADH2) in the strain CW239 showed the same degradation manner, which transformed the OTA to ochratoxin α (OTα) and l-ß-phenylalanine. Gene mutants (Δcp4, Δadh2 and Δcp4-adh2) testing result showed that OTA was co-degraded by carboxypeptidase CP4 and amidohydrolase ADH2, and the two hydrolases are sole agents in strain CW239 for OTA degradation. Hereinto, the ADH2 was the overwhelming efficient hydrolase, and the two types of hydrolases co-degraded OTA in CW239 by synergistic effect. The results of this study are highly significant to ochratoxin A contamination control during agro-products production and postharvest.

The three-dimensional structure of DapE from Enterococcus faecium reveals new insights into DapE/ArgE subfamily ligand specificity.

DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.

Combined in vivo effect of N-acylethanolamine-hydrolyzing acid amidase and glycogen synthase kinase-3ß inhibition to treat multiple sclerosis.

The current pharmacological approaches to multiple sclerosis (MS) target its inflammatory and autoimmune components, but effective treatments to foster remyelination and axonal repair are still lacking. We therefore selected two targets known to be involved in MS pathogenesis: N-acylethanolamine-hydrolyzing acid amidase (NAAA) and glycogen synthase kinase-3ß (GSK-3ß). We tested whether inhibiting these targets exerted a therapeutic effect against experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The combined inhibition of NAAA and GSK-3ß by two selected small-molecule compounds, ARN16186 (an NAAA inhibitor) and AF3581 (a GSK-3ß inhibitor), effectively mitigated disease progression, rescuing the animals from paralysis and preventing a worsening of the pathology. The complementary activity of the two inhibitors reduced the infiltration of immune cells into the spinal cord and led to the formation of thin myelin sheaths around the axons post-demyelination. Specifically, the inhibition of NAAA and GSK-3ß modulated the over-activation of NF-kB and STAT3 transcription factors in the EAE-affected mice and induced the nuclear translocation of ß-catenin, reducing the inflammatory insult and promoting the remyelination process. Overall, this work demonstrates that the dual-targeting of key aspects responsible for MS progression could be an innovative pharmacological approach to tackle the pathology.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

A Hadal Streptomyces-Derived Echinocandin Acylase Discovered through the Prioritization of Protein Families.

Naturally occurring echinocandin B and FR901379 are potent antifungal lipopeptides featuring a cyclic hexapeptide nucleus and a fatty acid side chain. They are the parent compounds of echinocandin drugs for the treatment of severe fungal infections caused by the Candida and Aspergilla species. To minimize hemolytic toxicity, the native fatty acid side chains in these drug molecules are replaced with designer acyl side chains. The deacylation of the N-acyl side chain is, therefore, a crucial step for the development and manufacturing of echinocandin-type antibiotics. Echinocandin E (ECE) is a novel echinocandin congener with enhanced stability generated via the engineering of the biosynthetic machinery of echinocandin B (ECB). In the present study, we report the discovery of the first echinocandin E acylase (ECEA) using the enzyme similarity tool (EST) for enzymatic function mining across protein families. ECEA is derived from Streptomyces sp. SY1965 isolated from a sediment collected from the Mariana Trench. It was cloned and heterologously expressed in S. lividans TK24. The resultant TKecea66 strain showed efficient cleavage activity of the acyl side chain of ECE, showing promising applications in the development of novel echinocandin-type therapeutics. Our results also provide a showcase for harnessing the essentially untapped biodiversity from the hadal ecosystems for the discovery of functional molecules.

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

Transcriptomic signature, bioactivity and safety of a non-hepatotoxic analgesic generating AM404 in the midbrain PAG region.

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids' addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund's adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001's safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.