The complex pathophysiology of Alzheimer's disease (AD) hampers the development of effective treatments. Attempts to prevent neurodegeneration in AD have failed so far, highlighting the need for further clarification of the underlying cellular and molecular mechanisms. Neuroinflammation seems to play a crucial role in disease progression, although its specific contribution to AD pathogenesis remains elusive. We have previously shown that the modulation of the endocannabinoid system (ECS) renders beneficial effects in a context of amyloidosis, which triggers neuroinflammation. In the 5xFAD model, the genetic inactivation of the enzyme that degrades anandamide (AEA), the fatty acid amide hydrolase (FAAH), was associated with a significant amelioration of the memory deficit.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7-11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845's effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition.

Encapsulation of antigens within protein microcrystals (polyhedra) is a promising approach for the stable delivery of vaccines. In this study, a vaccine was encapsulated into polyhedra against cyprinid herpesvirus II (CyHV-2). CyHV-2 typically infects gibel carp, Carassius auratus gibelio, causing gill hemorrhagic disease. The vaccine was constructed using a codon-optimized sequence, D4ORF, comprising the ORF72 (region 1-186 nt), ORF66 (region 993-1197 nt), ORF81 (region 603-783 nt), and ORF82 (region 85-186 nt) genes of CyHV-2. The H1-D4ORF and D4ORF-VP3 sequences were, respectively, obtained by fusing the H1-helix sequence (region 1-90 nt) ofBombyx mori cypovirus(BmCPV) polyhedrin to the 5' terminal end of D4ORF and by fusing a partial sequence (1-279 nt) of the BmCPV VP3 gene to the 3' terminal end of D4ORF. Furthermore, BmNPV-H1-D4ORF-polh and BmNPV-D4ORF-VP3-polh recombinant B. mori nucleopolyhedroviruses (BmNPVs), belonging to the family Baculoviridae, and co-expressing BmCPV polyhedrin and H1-D4ORF or D4ORF-VP3, were constructed. H1-D4ORF and D4ORF-VP3 fusion proteins were confirmed to be encapsulated into recombinant cytoplasmic polyhedra by Western blotting. Degradation of vaccine proteins was assessed by SDS-PAGE, and the results showed that the encapsulated vaccine proteins in polyhedra could be protected from degradation. Furthermore, when gibel carp were vaccinated with the purified polyhedra from BmNPV-H1-D4ORF-polh and BmNPV-D4ORF-VP3-polh via injection, the antibody titers in the serum of the vaccinated fish reached 1:6400-1:12,800 at 3 weeks post-vaccination. Therelative percentage of survival of immunized gibel carp reached 64.71% and 58.82%, respectively, following challenge with CyHV-2. These results suggest that incorporating vaccine protein into BmCPV polyhedra may be a novel approach for developing aquaculture microencapsulated vaccines.

Ligand-dependent protein degradation has emerged as a compelling strategy to pharmacologically control the protein content of cells. So far, however, only a limited number of E3 ligases have been found to support this process. Here, we use a chemical proteomic strategy that leverages broadly reactive, cysteine-directed electrophilic fragments coupled to selective ligands for intracellular proteins (for example, SLF for FKBP12, JQ1 for BRD4) to screen for heterobifunctional degrader compounds (or proteolysis targeting chimeras, PROTACs) that operate by covalent adduction of E3 ligases. This approach identified DCAF16-a poorly characterized substrate recognition component of CUL4-DDB1 E3 ubiquitin ligases-as a target of electrophilic PROTACs that promote the nuclear-restricted degradation of proteins. We find that only a modest fraction (~10-40%) of DCAF16 needs to be modified to support protein degradation, pointing to the potential for electrophilic PROTACs to induce neosubstrate degradation without substantially perturbing the function of the participating E3 ligase.

Hepatopancreas necrosis disease (HPND) of the Chinese mitten crab Eriocheir sinensis causes huge economic loss in China. However, the pathogenic factors and pathogenesis are still a matter of dissension. To search for potential pathogens, the hepatopancreatic flora of diseased crabs with mild symptoms, diseased crabs with severe symptoms, and crabs without visible symptoms were investigated using metatranscriptomics sequencing. The prevalence of Absidia glauca and Candidatus Synechococcus spongiarum decreased, whereas the prevalence of Spiroplasma eriocheiris increased in the hepatopancreatic flora of crabs with HPND. Homologous sequences of 34 viral species and 4 Microsporidian species were found in the crab hepatopancreas without any significant differences between crabs with and without HPND. Moreover, DEGs in the hepatopancreatic flora between crabs with severe symptoms and without visible symptoms were enriched in the ribosome, retinol metabolism, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450, biosynthesis of unsaturated fatty acids, and other glycan degradation. Moreover, the relative abundance of functions of DEDs in the hepatopancreatic flora changed with the pathogenesis process. These results suggested that imbalance of hepatopancreatic flora was associated with crab HPND. The identified DEGs were perhaps involved in the pathological mechanism of HPND; nonetheless, HPND did not occur due to virus or microsporidia infection.

As a serine hydrolase, monoacylglycerol lipase (MAGL) is principally responsible for the metabolism of 2-arachidonoylglycerol (2-AG) in the central nervous system (CNS), leading to the formation of arachidonic acid (AA). Dysfunction of MAGL has been associated with multiple CNS disorders and symptoms, including neuroinflammation, cognitive impairment, epileptogenesis, nociception and neurodegenerative diseases. Inhibition of MAGL provides a promising therapeutic direction for the treatment of these conditions, and a MAGL positron emission tomography (PET) probe would greatly facilitate preclinical and clinical development of MAGL inhibitors. Herein, we design and synthesize a small library of fluoropyridyl-containing MAGL inhibitor candidates. Pharmacological evaluation of these candidates by activity-based protein profiling identified 14 as a lead compound, which was then radiolabeled with fluorine-18 via a facile SNAr reaction to form 2-[18F]fluoropyridine scaffold. Good blood-brain barrier permeability and high in vivo specific binding was demonstrated for radioligand [18F]14 (also named as [18F]MAGL-1902). This work may serve as a roadmap for clinical translation and further design of potent 18F-labeled MAGL PET tracers.

The endocannabinoid (eCB) system plays an important homeostatic role in the regulation of stress circuits and has emerged as a therapeutic target to treat stress disorders and alcohol use disorder (AUD). Extensive research has elucidated a role for the eCB anandamide (AEA), but less is known about 2-arachidonoylglycerol (2-AG) mediated signalling.

Angiogenesis refers to the process of growing new blood vessels from pre-existing capillaries or post-capillary veins. This process plays a critical role in promoting tumorigenesis and metastasis. As a result, developing antiangiogenic agents has become an attractive strategy for tumor treatment. Sirtuin6 (SIRT6), a member of nicotinamide adenine (NAD+)-dependent histone deacetylases, regulates various biological processes, including metabolism, oxidative stress, angiogenesis, and DNA damage and repair. Some SIRT6 inhibitors have been identified, but the effects of SIRT6 inhibitors on anti-angiogenesis have not been reported. We have identified a pyrrole-pyridinimidazole derivative 8a as a highly effective inhibitor of SIRT6 and clarified its anti-pancreatic-cancer roles. This study investigated the antiangiogenic roles of 8a. We found that 8a was able to inhibit the migration and tube formation of HUVECs and downregulate the expression of angiogenesis-related proteins, including VEGF, HIF-1α, p-VEGFR2, and N-cadherin, and suppress the activation of AKT and ERK pathways. Additionally, 8a significantly blocked angiogenesis in intersegmental vessels in zebrafish embryos. Notably, in a pancreatic cancer xenograft mouse model, 8a down-regulated the expression of CD31, a marker protein of angiogenesis. These findings suggest that 8a could be a promising antiangiogenic and cancer therapeutic agent.

Chronic pain is often a symptom of knee osteoarthritis (OA) for which current analgesics are either inadequate or are associated with serious side effects. The endocannabinoid system may offer alternative targets for pain relief. We evaluated the effects of a potent and selective monoacylglycerol (MAG) lipase inhibitor (MJN110) on OA pain behaviour, spinal mechanisms of action and joint histopathology in the rat.

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

Local environmental cues are indispensable for axonal growth and guidance during brain circuit formation. Here, we combine genetic and pharmacological tools, as well as systems neuroanatomy in human fetuses and mouse models, to study the role of endocannabinoid and Slit/Robo signalling in axonal growth. We show that excess 2-arachidonoylglycerol, an endocannabinoid affecting directional axonal growth, triggers corpus callosum enlargement due to the errant CB1 cannabinoid receptor-containing corticofugal axon spreading. This phenotype mechanistically relies on the premature differentiation and end-feet proliferation of CB2R-expressing oligodendrocytes. We further show the dependence of both axonal Robo1 positioning and oligodendroglial Slit2 production on cell-type-specific cannabinoid receptor activation. Accordingly, Robo1 and/or Slit2 manipulation limits endocannabinoid modulation of axon guidance. We conclude that endocannabinoids can configure focal Slit2/Robo1 signalling to modulate directional axonal growth, which may provide a basis for understanding impaired brain wiring associated with metabolic deficits and prenatal drug exposure.

Obesity develops when energy intake chronically exceeds energy expenditure. Because brown adipose tissue (BAT) dissipates energy in the form of heat, increasing energy expenditure by augmenting BAT-mediated thermogenesis may represent an approach to counter obesity and its complications. The ability of BAT to dissipate energy is dependent on expression of mitochondrial uncoupling protein 1 (UCP1). To facilitate the identification of pharmacological modulators of BAT UCP1 levels, which may have potential as antiobesity medications, we developed a transgenic model in which luciferase activity faithfully mimics endogenous UCP1 expression and its response to physiologic stimuli. Phenotypic screening of a library using cells derived from this model yielded a small molecule that increases UCP1 expression in brown fat cells and mice. Upon adrenergic stimulation, compound-treated mice showed increased energy expenditure. These tools offer an opportunity to identify pharmacologic modulators of UCP1 expression and uncover regulatory pathways that impact BAT-mediated thermogenesis.

Inhibition of endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL) reduces somatic morphine withdrawal signs, but its effects on aversive aspects of withdrawal are unknown. The present study investigated whether Δ(9)-tetrahydrocannabinol (THC), the MAGL inhibitor JZL184, the FAAH inhibitor PF-3845, or the dual FAAH/MAGL inhibitor SA-57 would reduce acquisition of morphine withdrawal-induced conditioned place avoidance (CPA) and jumping.

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.

During Drosophila embryogenesis, establishment of ventral and lateral cell fates requires spatial regulation of an extracellular serine protease cascade composed of Nudel, Gastrulation Defective (GD), Snake, and Easter. Pipe, a sulfotransferase expressed ventrally during oogenesis, sulfates secreted targets that somehow confer positive spatial input to this cascade. Nudel and GD activation are pipe-independent, while Easter activation requires pipe. The effect of pipe on Snake activation has been unknown. Here we show that Snake activation is cascade-dependent but pipe-independent. These findings support a conclusion that Snake's activation of Easter is the first spatially regulated step in the dorsoventral protease cascade.

Fatty acid amides and fatty acid ethanolamides are novel signalling molecules exemplified by the sleep-inducing lipid oleamide and the endocannabinoid anandamide, respectively. These substances are inactivated by fatty acid amide hydrolase (FAAH), an enzyme that is expressed by neurons and non-neuronal cells in the brain. In the rat, FAAH-immunoreactivity has been detected in epithelial cells of the choroid plexus and, in accordance with this finding, here we report FAAH mRNA expression in rat choroid plexus epithelium using in situ hybridisation methods. Surprisingly, a comparative analysis of mouse brain did not reveal FAAH mRNA expression or FAAH-immunoreactivity in the choroid plexus of this species. FAAH-immunoreactivity was, however, detected in non-choroidal ventricular ependymal cells in the mouse brain and the specificity of this immunostaining was confirmed by analysis of FAAH-knockout mice. FAAH-immunoreactivity was detected in ependymal cells throughout the ventricles of the mouse brain but with regional variation in the intensity of immunostaining. Intriguingly, in rat brain, although FAAH expression is observed in choroid plexus epithelial cells, little or no FAAH-immunoreactivity is present in the ventricular ependyma. Thus, there are mutually exclusive patterns of FAAH expression in the ventricular epithelium of rat and mouse brain. Our observations provide the basis for an experimental analysis that exploits differences in FAAH expression in rat and mouse to investigate FAAH function in ventricular epithelial cells and, in particular, the role of FAAH in regulating the sleep-inducing agent oleamide in cerebrospinal fluid.

N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells.

Patients with non-small cell lung cancers that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR whereby the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may affect drug activity and safety.