ABSTRACT
Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.
Subject(s)
Carboxypeptidases , Glutamates , Microtubules , Tubulin , Animals , Humans , Binding Sites , Brain/metabolism , Carboxypeptidases/chemistry , Carboxypeptidases/metabolism , Carboxypeptidases/ultrastructure , Cryoelectron Microscopy , Crystallography, X-Ray , Glutamates/metabolism , Glutamates/chemistry , Homeostasis , Magnetic Resonance Spectroscopy , Microtubules/chemistry , Microtubules/metabolism , Microtubules/ultrastructure , Models, Molecular , Protein Binding , Sf9 Cells , Substrate Specificity , Tubulin/metabolism , Tubulin/chemistry , Tubulin/ultrastructureABSTRACT
L-theanine (N-ethyl-γ-glutamine) is the main amino acid in tea leaves. It not only contributes to tea flavor but also possesses several health benefits. Compared with its sedative and calming activities, the immunomodulatory effects of L-theanine have received less attention. Clinical and epidemiological studies have shown that L-theanine reduces immunosuppression caused by strenuous exercise and prevents colds and influenza by improving immunity. Numerous cell and animal studies have proven that theanine plays an immunoregulatory role in inflammation, nerve damage, the intestinal tract, and tumors by regulating γδT lymphocyte function, glutathione (GSH) synthesis, and the secretion of cytokines and neurotransmitters. In addition, theanine can be used as an immunomodulator in animal production. This article reviews the research progress of L-theanine on immunoregulation and related mechanisms, as well as its application in poultry and animal husbandry. It is hoped that this work will be beneficial to future related research.
Subject(s)
Cytokines , Glutamates , Animals , Glutamates/chemistry , Immunity , Tea/chemistryABSTRACT
EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.
Subject(s)
Antiporters , Catalytic Domain , Escherichia coli Proteins , Escherichia coli , Glutamates , Static Electricity , Antiporters/chemistry , Antiporters/metabolism , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Glutamates/chemistry , Glutamates/metabolism , Protons , Substrate Specificity , Protein Binding , Nuclear Magnetic Resonance, Biomolecular , Hydrogen-Ion Concentration , Drug Resistance, Multiple, Bacterial , Ion TransportABSTRACT
Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.
Subject(s)
Cell Membrane , Electron Transport Complex I , Escherichia coli Proteins , NADH Dehydrogenase , Amino Acid Substitution , Cell Membrane/chemistry , Conserved Sequence , Electron Transport Complex I/chemistry , Electron Transport Complex I/genetics , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Glutamates/chemistry , Glutamates/genetics , Lysine/chemistry , Lysine/genetics , NADH Dehydrogenase/chemistry , NADH Dehydrogenase/genetics , Protons , Quinones/chemistryABSTRACT
Prostate-specific membrane antigen (PSMA) is highly overexpressed in most prostate cancers and is clinically visualized using PSMA-specific probes incorporating glutamate-ureido-lysine (GUL). PSMA is effectively absent from certain high-mortality, treatment-resistant subsets of prostate cancers, such as neuroendocrine prostate cancer (NEPC); however, GUL-based PSMA tracers are still reported to have the potential to identify NEPC metastatic tumors. These probes may bind unknown proteins associated with PSMA-suppressed cancers. We have identified the up-regulation of PSMA-like aminopeptidase NAALADaseL and the metabotropic glutamate receptors (mGluRs) in PSMA-suppressed prostate cancers and find that their expression levels inversely correlate with PSMA expression and are associated with GUL-based radiotracer uptake. Furthermore, we identify that NAALADaseL and mGluR expression correlates with a unique cell cycle signature. This provides an opportunity for the future study of the biology of NEPC and potential therapeutic directions. Computationally predicting that GUL-based probes bind well to these targets, we designed and synthesized a fluorescent PSMA tracer to investigate these proteins in vitro, where it shows excellent affinity for PSMA, NAALADaseL, and specific mGluRs associated with poor prognosis.
Subject(s)
Antigens, Surface/metabolism , Glutamate Carboxypeptidase II/metabolism , Glutamates , Lysine , Molecular Probes , Prostatic Neoplasms/diagnosis , Prostatic Neoplasms/metabolism , Urea , Animals , Antigens, Surface/chemistry , Binding Sites , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Disease Models, Animal , Disease Progression , Fluorescent Antibody Technique , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Gene Expression , Glutamate Carboxypeptidase II/chemistry , Glutamates/chemistry , Humans , Immunohistochemistry , Lysine/chemistry , Male , Mice , Models, Molecular , Molecular Conformation , Molecular Imaging/methods , Molecular Probes/chemistry , Prostatic Neoplasms/genetics , Protein Binding , Receptors, Kainic Acid/genetics , Receptors, Kainic Acid/metabolism , Structure-Activity Relationship , Urea/analogs & derivatives , Urea/chemistryABSTRACT
The pathogenesis of Alzheimer's disease (AD) is still unclear, and presently there is no cure for the disease that can be used for its treatment or to stop its progression. Here, we investigated the therapeutic potential of ramalin (isolated from the Antarctic lichen, Ramalina terebrata), which exhibits various physiological activities, in AD. Specifically, derivatives were synthesized based on the structure of ramalin, which has a strong antioxidant effect, BACE-1 inhibition activity, and anti-inflammatory effects. Therefore, ramalin and its derivatives exhibit activity against multiple targets associated with AD and can serve as potential therapeutic agents for the disease.
Subject(s)
Alzheimer Disease/drug therapy , Anti-Inflammatory Agents/therapeutic use , Antioxidants/therapeutic use , Glutamates/therapeutic use , Alzheimer Disease/metabolism , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Anti-Inflammatory Agents/chemical synthesis , Anti-Inflammatory Agents/chemistry , Antioxidants/chemical synthesis , Antioxidants/chemistry , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Biphenyl Compounds/antagonists & inhibitors , Glutamates/chemical synthesis , Glutamates/chemistry , Humans , Molecular Structure , Picrates/antagonists & inhibitorsABSTRACT
A water-soluble perylene imide derivative (PDI-Glu) was synthesized and their supramolecular aggregates composed of PDI-Glu and Al3+ were prepared as a "turn on" fluorometric probe to monitor F- in a purely aqueous system. Based on an "indicator displacement assay" (IDA) approach, the sensing performance and mechanism of PDI-Glu/Al3+ complex toward F- were investigated by absorption and emission spectra. It was suggested that disassembly of PDI-Glu/Al3+ aggregates was promoted by addition of F- through the competitive binding between Al3+ and F-. The detection limit is 240 nmol/L. This method featured simple preparation, excellent water solubility, adjustable self-assembly performance, ease of observation and operation, and high selectivity and sensitivity. It was used for monitoring F- in toothpaste and tap water samples with excellent accuracy and recovery. To the best of our knowledge, this is the first water-soluble perylene diimide-based probe for F- detection in 100% aqueous media. We believe this work could not only extend the sensing scope of water-soluble perylene diimide, but also bring some useful information for the rapid detection of anionic analytes in aqueous media. The disassembly of supramolecular aggregates of PDI-Glu/Al3+ along with significant fluorescence recovery enable a rapid and visual detection of F- based on an "indicator displacement assay" strategy.
Subject(s)
Fluorescent Dyes/chemistry , Fluorides/analysis , Imides/chemistry , Perylene/analogs & derivatives , Aluminum/chemistry , Dentifrices/analysis , Drinking Water/analysis , Glutamates/chemistry , Limit of Detection , Perylene/chemistry , Spectrometry, Fluorescence/methods , Water Pollutants, Chemical/analysisABSTRACT
Surfactant aggregates have long been considered as a tool to improve drug delivery and have been widely used in medical products. The pH-responsive aggregation behavior in anionic gemini surfactant 1,3-bis(N-dodecyl-N-propanesulfonate sodium)-propane (C12C3C12(SO3)2) and its mixture with a cationic monomeric surfactant cetyltrimethylammonium bromide (CTAB) have been investigated. The spherical-to-wormlike micelle transition was successfully realized in C12C3C12(SO3)2 through decreasing the pH, while the rheological properties were perfectly enhanced for the formation of wormlike micelles. Especially at 140 mM and pH 6.7, the mixture showed high viscoelasticity, and the maximum of the zero-shear viscosity reached 1530 Pa·s. Acting as a sulfobetaine zwitterionic gemini surfactant, the electrostatic attraction, the hydrogen bond and the short spacer of C12C3C12(SO3)2 molecules were all responsible for the significant micellar growth. Upon adding CTAB, the similar transition could also be realized at a low pH, and the further transformation to branched micelles occurred by adjusting the total concentration. Although the mixtures did not approach the viscosity maximum appearing in the C12C3C12(SO3)2 solution, CTAB addition is more favorable for viscosity enhancement in the wormlike-micelle region. The weakened charges of the headgroups in a catanionic mixed system minimizes the micellar spontaneous curvature and enhances the intermolecular hydrogen-bonding interaction between C12C3C12(SO3)2, facilitating the formation of a viscous solution, which would greatly induce entanglement and even the fusion of wormlike micelles, thus resulting in branched microstructures and a decline of viscosity.
Subject(s)
Rheology , Surface-Active Agents/chemistry , Cetrimonium/chemistry , Glutamates/chemistry , Hydrogen-Ion Concentration , Micelles , ViscosityABSTRACT
Tea (Camellia sinensis) is one of the most important cash crops in the world. Theanine, as an important amino acid component in tea, is a key quality index for excellent tea quality and high economic value. People increase theanine accumulation in tea mainly through the application of nitrogen fertilizer, shading and pruning. However, these methods are not effective. In this study, we treated tea buds with a 100 µM solution of GA3 containing 1 tween-20, investigated the effects of GA3 on theanine accumulation, bud yield, chlorophyll fluorescence parameters and expression level of theanine biosynthesis pathway genes in tea plant by qPCR, LC-MS/MS etc. Results showed that change trends of theanine and GA3 was extremely positively correlated with each other. Exogenous GA3 upregulated the expression level of theanine biosynthesis pathway genes, caused an increase of theanine content (mg·g-1) by 27% in tea leaves compared with Mock, and accelerated the germination of buds and elongation of shoots, which lead to a significant increase of tea yield by 56% (w/w). Moreover, the decrease of chlorophyll contents, photochemical quenching coefficient (qP) and relative electron transport rate (rETR) under GA3 treatment suggested that GA3 reduced photosynthesis in the tender tea leaves, indicating that the decline of carbon assimilation in tea plants was conducive to the nitrogen metabolism, and it was beneficial to the accumulation of theanine. This study provided a new technical and theoretical support for the precise control of tea quality components and phenophase.
Subject(s)
Camellia sinensis/growth & development , Camellia sinensis/metabolism , Gibberellins/pharmacology , Plant Leaves/metabolism , Tea/metabolism , Amino Acids/chemistry , Chlorophyll/chemistry , Chromatography, Liquid , Gibberellins/chemistry , Glutamates/chemistry , Nitrogen/metabolism , Photosynthesis , Plant Proteins/genetics , Plant Shoots , Polymerase Chain Reaction , Tandem Mass SpectrometryABSTRACT
l-Theanine is a characteristic amino acid in tea with various effects including antioxidant and anti-inflammatory effects. Previously, most studies had reported that l-theanine regulates the immune function in vivo by inhibiting the expression of the inflammatory factors, but how l-theanine regulates the inflammatory factors' pathway is not known. In this study, we innovatively found the binding target of l-theanine in vivo-cannabinoid receptor 1, and demonstrated that l-theanine regulated the immune function and glutamine metabolism by competitively binding cannabinoid receptor 1. Mechanistically, l-theanine competitively binds cannabinoid receptor 1, leading to inhibition of cannabinoid receptor 1 activity, and regulates glutamine metabolism and immune function in normal and E44813-stressed rats. In normal rats, l-theanine inhibits ERK1/2 phosphorylation through Gßy by antagonizing cannabinoid receptor 1, thus affecting GS expression. From the point of view of immune signaling, after LTA antagonizes the activity of cannabinoid receptor 1, it relieves the inhibition of cannabinoid receptor 1 on COX-2 expression, downregulates Pdcd4 expression and NFκB, and ultimately enhances the expression of the anti-inflammatory factor IL-10. In E44813-stressed rats, l-theanine promotes the nuclear translocation of p-ERK1/2 by inhibiting the activity of cannabinoid receptor 1, and finally acts on GS. At the same time, it decreases the expression of the pro-inflammatory factor TNF-α and increases the expression of the anti-inflammatory factor IL-10 in stressed rats through the COX2-Pdcd4-NFκB-IL10 and TNFα pathways. In summary, these results demonstrate that l-theanine regulates glutamine metabolism and immune function by competitively binding to cannabinoid receptor 1.
Subject(s)
Glutamates/pharmacokinetics , Glutamine/metabolism , Immunity/drug effects , Receptors, Cannabinoid/metabolism , Animals , Anti-Inflammatory Agents/pharmacology , Antioxidants/pharmacology , Apoptosis Regulatory Proteins/metabolism , Down-Regulation , Glutamates/chemistry , Glutamine/chemistry , MAP Kinase Signaling System , Male , Molecular Docking Simulation , NF-kappa B , Phosphorylation , Rats , Signal Transduction/drug effectsABSTRACT
Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of L-Glu or L-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous L-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport L-Gln.
Subject(s)
Brain/metabolism , Glutamates/administration & dosage , Glutamine/administration & dosage , Synaptosomes/metabolism , Animals , Behavior, Animal/drug effects , Chromatography, Liquid , Dose-Response Relationship, Drug , Female , Glutamates/chemistry , Glutamates/pharmacokinetics , Glutamine/chemistry , Glutamine/pharmacokinetics , Male , Mice , Primary Cell Culture , Stereoisomerism , Tandem Mass Spectrometry , gamma-Aminobutyric Acid/metabolismABSTRACT
One of the most prevalent cancers in men is prostate cancer and could be managed with immunotoxins or antibody treatment. Because of the substantial rise of the Prostate-Specific Antigen and the Prostate-Specific Membrane Antigen (PSMA), cancer vaccination should be rendered with these antigens. Through pharmacodynamic experiments in a library of natural compounds from ZINC database, the current research sought to identify compounds that could suppress PSMA protein. To test the most productive compounds for further research, the Library has been scanned with Pharmacophore and ADMET analysis followed by molecular docking methods in the first phase. After selecting 15 ligands with the best pose related to docking results, to evaluate the stability of the ligand-protein bounds of the compounds, a molecular dynamics simulation considering the effect of the presence of zinc ions on the protein structure was performed. The measurement of ligand binding modes and free energy has shown that four compounds, including Z10, Z06, Z01, and Z03, have formed critical interactions with the active site's residues. Besides, multiple approaches were employed to determine their inhibition rating and describe the variables that facilitate the attachment of ligands to the protein active site. The results are obtained from the MMPBSA/GBSA analysis of four selected small molecules (Z10, Z06, Z01, and Z03), which are very close to the IC50 value of reference ligand (DCIBzl); they are -13.85 kcal/mol, -12.58 kcal/mol, -10.71 kcal/mol and -9.39 kcal/mol respectively. Finally, we evaluate the results obtained from selected ligands using hydrogen bond and decomposition analyzes. We have examined the effective interactions between ligands and S1/S1'pockets in protein. Our computational results illustrate the design of more efficient inhibitors of PSMA.
Subject(s)
Antigens, Surface/metabolism , Enzyme Inhibitors/metabolism , Glutamate Carboxypeptidase II/metabolism , Glutamates/metabolism , Urea/analogs & derivatives , Antigens, Surface/chemistry , Catalytic Domain , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Glutamate Carboxypeptidase II/chemistry , Glutamates/chemistry , Glutamates/pharmacokinetics , Humans , Ligands , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Thermodynamics , Urea/chemistry , Urea/metabolism , Urea/pharmacokineticsABSTRACT
Autism spectrum disorder (ASD) is a neurodevelopmental disease, and the number of patients has increased rapidly in recent years. The causes of ASD involve both genetic and environmental factors, but the details of causation have not yet been fully elucidated. Many reports have investigated genetic factors related to synapse formation, and alcohol and tobacco have been reported as environmental factors. This review focuses on endoplasmic reticulum stress and amino acid cycle abnormalities (particularly glutamine and glutamate) induced by many environmental factors. In the ASD model, since endoplasmic reticulum stress is high in the brain from before birth, it is clear that endoplasmic reticulum stress is involved in the development of ASD. On the other hand, one report states that excessive excitation of neurons is caused by the onset of ASD. The glutamine- glutamate cycle is performed between neurons and glial cells and controls the concentration of glutamate and GABA in the brain. These neurotransmitters are also known to control synapse formation and are important in constructing neural circuits. Theanine is a derivative of glutamine and a natural component of green tea. Theanine inhibits glutamine uptake in the glutamine-glutamate cycle via slc38a1 without affecting glutamate; therefore, we believe that theanine may prevent the onset of ASD by changing the balance of glutamine and glutamate in the brain.
Subject(s)
Autism Spectrum Disorder/drug therapy , Autism Spectrum Disorder/etiology , Glutamates/chemistry , Glutamic Acid/metabolism , Glutamine/metabolism , Plant Extracts/chemistry , Tea/chemistry , Brain/metabolism , Endoplasmic Reticulum Stress , Glutamates/metabolism , Glutamates/pharmacology , Humans , Neurogenesis , Neuroglia , Neurons , Plant Extracts/metabolism , Plant Extracts/pharmacology , gamma-Aminobutyric Acid/metabolismABSTRACT
N-(2-18F-fluoropropionyl)-l-glutamate (18F-FPGLU), a new N-substituted 18F-labeling l-glutamate, is a potential amino acid tracer for oncology PET imaging with good tumor-to-background contrast in several tumor-bearing mice. Herein, we evaluated the potential value of 18F-FPGLU for PET imaging of glioma in orthotopic glioma-bearing SD rats. A series of competitive inhibition experiments with various types of inhibitors were conducted with C6 cells to investigate the transport mechanism of 18F-FPGLU in glioma. Establishment of orthotopic rat C6 glioma-bearing SD rats models was confirmed by MRI. Then PET imaging of 18F-FPGLU was performed on the orthotopic C6 glioma-bearing SD rats and compared with that of 18F-FDG. After the rats sacrificed, the whole brain was collected and immunofluorescence staining of glial fibrillary acidic protein (GFAP) and matrix metalloproteinase 2 (MMP2) were processed. Na+-dependent system XAG- and Na+-independent system XC- are the mainly transporters of 18F-FPGLU in C6 cells. N-methyl-d-aspartate (NMDA) receptor, which is associated with the invasiveness and proliferation of glioma cells, is also involved in the uptake of 18F-FPGLU. High uptake and retention of 18F-FPGLU was observerd in orthotopic glioma with good visualization and the tumor/background ratio reached 2.35 at 60 min post-injection, which was significantly higher than that of 18F-FDG (1.72) in small-animal PET images. High expression of MMP-2 and GFAP was observed in the immunofluorescence staining of glioma xerography slices. 18F-FPGLU seems to be a better potential PET tracer than 18F-FDG for brain glioma imaging with good visualization and ability to assess the tumor activity.
Subject(s)
Brain/diagnostic imaging , Fluorine Radioisotopes/chemistry , Glioma/diagnostic imaging , Glutamates/chemistry , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemistry , Animals , Cell Line, Tumor , Fluorescent Antibody Technique , Glutamates/pharmacokinetics , Heterografts , Magnetic Resonance Imaging/methods , Rats , Rats, Sprague-DawleyABSTRACT
(4S)-4-(3-[18F]Fluoropropyl)-L-glutamic acid ([18F]FSPG) is a positron emission tomography (PET) imaging agent for measuring the system xC- transporter activity. It has been used for the detection of various cancers and metastasis in clinical trials. [18F]FSPG is also a promising diagnostic tool for evaluation of multiple sclerosis, drug resistance in chemotherapy, inflammatory brain diseases, and infectious lesions. Due to the very short half-life (110 min) of 18F nuclide, [18F]FSPG needs to be produced on a daily basis; therefore, fast and efficient synthesis and analytical methods for quality control must be established to assure the quality and safety of [18F]FSPG for clinical use. To manufacture cGMP-compliant [18F]FSPG, all four nonradioactive stereoisomers of FSPG were prepared as reference standards for analysis. (2S,4S)-1 and (2R,4R)-1 were synthesized starting from protected L- and D-glutamate derivatives in three steps, whereas (2S,4R)-1 and (2R,4S)-1 were prepared in three steps from protected (S)- and (R)-pyroglutamates. A chiral HPLC method for simultaneous determination of four FSPG stereoisomers was developed by using a 3-cm Chirex 3126 column and a MeCN/CuSO4(aq) mobile phase. In this method, (2R,4S)-1, (2S,4S)-1, (2R,4R)-1, and (2S,4R)-1 were eluted in sequence with sufficient resolution in less than 25 min without derivatization. Scale-up synthesis of intermediates for the production of [18F]FSPG in high optical purity was achieved via stereo-selective synthesis or resolution by recrystallization. The enantiomeric excess of intermediates was determined by HPLC using a Chiralcel OD column and monitored at 220 nm. The nonradioactive precursor with >98% ee can be readily distributed to other facilities for the production of [18F]FSPG. Based on the above accomplishments, cGMP-compliant [18F]FSPG met the acceptance criteria in specifications and was successfully manufactured for human use. It has been routinely prepared and used in several pancreatic ductal adenocarcinoma metastasis-related clinical trials.
Subject(s)
Glutamates/chemistry , Glutamates/chemical synthesis , Chromatography, High Pressure Liquid , Crystallization , Humans , Injections , StereoisomerismABSTRACT
Glutamate delta (GluD) receptors belong to the ionotropic glutamate receptor family, yet they don't bind glutamate and are considered orphan. Progress in defining the ion channel function of GluDs in neurons has been hindered by a lack of pharmacological tools. Here, we used a chemo-genetic approach to engineer specific and photo-reversible pharmacology in GluD2 receptor. We incorporated a cysteine mutation in the cavity located above the putative ion channel pore, for site-specific conjugation with a photoswitchable pore blocker. In the constitutively open GluD2 Lurcher mutant, current could be rapidly and reversibly decreased with light. We then transposed the cysteine mutation to the native receptor, to demonstrate with high pharmacological specificity that metabotropic glutamate receptor signaling triggers opening of GluD2. Our results assess the functional relevance of GluD2 ion channel and introduce an optogenetic tool that will provide a novel and powerful means for probing GluD2 ionotropic contribution to neuronal physiology.
Neurotransmitters are chemicals released by the body that trigger activity in neurons. Receptors on the surface of neurons detect these neurotransmitters, providing a link between the inside and the outside of the cell. Glutamate is one of the major neurotransmitters and is involved in virtually all brain functions. Glutamate binds to two different types of receptors in neurons. Ionotropic receptors have pores known as ion channels, which open when glutamate binds. This is a fast-acting response that allows sodium ions to flow into the neuron, triggering an electrical signal. Metabotropic receptors, on the other hand, trigger a series of events inside the cell that lead to a response. Metabotropic receptors take more time than ionotropic receptors to elicit a response in the cell, but their effects last much longer. One type of receptor, known as the GluD family, is very similar to ionotropic glutamate receptors but does not directly respond to glutamate. Instead, the ion channel of GluD receptors opens after being activated by glutamate metabotropic receptors. GluD receptors are produced throughout the brain and play roles in synapse formation and activity, but the way they work remains unclear. An obstacle to understanding how GluD receptors work is the lack of molecules that can specifically block these receptors' ion channel activity. Lemoine et al. have developed a tool that enables control of the ion channel in GluD receptors using light. Human cells grown in the lab were genetically modified to produce a version of GluD2 (a member of the GluD family) with a light-sensitive molecule attached. In darkness or under green light, the light-sensitive molecule blocks the channel and prevents ions from passing through. Under violet light, the molecule twists, and ions can flow through the channel. With this control over the GluD2 ion channel activity, Lemoine et al. were able to validate previous research showing that the activation of metabotropic glutamate receptors can trigger GluD2 to open. The next step will be to test this approach in neurons. This will help researchers to understand what role GluD ion channels play in neuron to neuron communication.
Subject(s)
Glutamate Dehydrogenase/genetics , Glutamate Dehydrogenase/metabolism , Binding Sites , Genetic Engineering , Glutamate Dehydrogenase/chemistry , Glutamates/chemistry , Glutamates/metabolism , HEK293 Cells , Humans , Light , MutationABSTRACT
α-Ketoglutaramic acid (KGM, α-ketoglutaramate), also known as 2-oxoglutaramic acid (OGM, 2-oxoglutaramate), is a substrate of ω-amidase, also known as Nitrilase 2 (NIT2), and is essential for studying the canonical role of ω-amidase, as well as its role in multiple diseases. Until now, KGM used for biological studies has been prepared most often by the enzymatic oxidation of l-glutamine using snake venom l-amino acid oxidase, which provides KGM as an aqueous solution, containing by-products including 5-oxoproline and α-ketoglutarate. The enzymatic method for KGM preparation, therefore, cannot provide pure product or an accurate percent yield evaluation. Here, we report a synthetic method for the preparation of this important substrate, KGM, in 3 steps, from l-2-hydroxyglutaramic acid, in pure form, in 53% overall yield.
Subject(s)
Ketoglutaric Acids/chemical synthesis , Ketoglutaric Acids/metabolism , Amidohydrolases/metabolism , Aminohydrolases/metabolism , Animals , Catalysis , Glutamates/chemistry , Glutamine/chemistry , L-Amino Acid Oxidase/metabolism , Pyrrolidonecarboxylic Acid/chemistry , Snake Venoms/chemistryABSTRACT
PURPOSE: The present study describes the analysis of amino acid transporters ASCT1, ASCT2, LAT1, and xc- in breast cancer under normoxic and hypoxic conditions. [18F]FDOPA-PET and [18F]FSPG-PET were used as imaging biomarkers to probe L-type amino acid transporter (LAT1) and cystine-glutamate antiporter (xc-) in breast cancer models. PROCEDURES: LAT1 and xc- transporters were studied under normoxic and hypoxic conditions with radiotracers [18F]FDOPA and [18F]FSPG in estrogen receptor-positive (ER+) MCF7 and triple-negative MDA-MB231 cells and in human mammary epithelial MCF10A control cells. Protein expression was analyzed using Western blot and immunohistochemistry. RESULTS: ASCT1 protein expression levels were comparable in all three cell lines, while noticeable ASCT2 expression levels were only found in MCF10A control cells. Higher LAT1 protein expression was detected in ER+ MCF7 cells. High xc- protein expression levels were detected in MDA-MB231 cells. Uptake of [18F]FDOPA through LAT1 was significantly higher in MCF7 versus MDA-MB231 cells, while the uptake of [18F]FSPG through xc- resulted in the opposite confirming expression and functional differences for both amino acid transporters in different breast cancer models. Hypoxia significantly increased [18F]FDOPA uptake in MCF7 cells and [18F]FSPG uptake in MDA-MB231 cells. In vivo PET imaging revealed substantially higher tumor uptake of [18F]FDOPA in MCF7 tumors as well as [18F]FSPG uptake in MDA-MB231 tumors confirming differences detected in vitro. CONCLUSIONS: ER+ breast cancer cells express higher levels of amino acid transporter LAT1, whereas triple-negative breast cancer cells express more xc-. Cellular uptake and PET imaging experiments with [18F]FDOPA and [18F]FSPG confirmed functional LAT1 and xc- expression profiles. There was initial evidence that hypoxia regulates the function of both amino acid transporters in breast cancer. The results further indicated that [18F]FDOPA and [18F]FSPG are suitable radiotracer to distinguish between ER+ and triple-negative breast cancer types.
Subject(s)
Amino Acid Transport System y+/metabolism , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/metabolism , Dihydroxyphenylalanine/analogs & derivatives , Glutamates/chemistry , Large Neutral Amino Acid-Transporter 1/metabolism , Positron-Emission Tomography , Amino Acid Transport System y+/genetics , Animals , Breast Neoplasms/genetics , Cell Hypoxia/genetics , Cell Line, Tumor , Dihydroxyphenylalanine/chemistry , Female , Gene Expression Regulation, Neoplastic , Humans , Large Neutral Amino Acid-Transporter 1/genetics , Mice, Nude , RNA, Messenger/genetics , RNA, Messenger/metabolism , Radiopharmaceuticals/chemistryABSTRACT
Lascivol was identified as the bitter compound in two Tricholoma species, T. aestuans and T. virgatum, and was previously isolated from the European mushroom T. lascivum. The structure of lascivol was previously solved by X-ray crystallographical analysis but its stereochemistry at C3 remained ambiguous. We thus re-examined the absolute configuration of C3 bearing a hydroxy group using the modified Mosher's method.
Subject(s)
Agaricales/chemistry , Glutamates/isolation & purification , Crystallography, X-Ray , Glutamates/chemistry , TasteABSTRACT
Functional foods have created an open environment for the development of new solutions to health-related issues. In celiac disease, there is still no therapeutic alternative other than the observance of a gluten-free diet. In this context, we developed a wheat flour enriched in l-theanine aimed to be a potential alternative to the gluten-free diet. Through microbial transglutaminase-catalysed transamidation of gluten proteins using ethylamine as amine nucleophile, substantial amounts of glutamine residues were converted in theanine residues. Furthermore, using T-cell lines generated from intestinal biopsy specimens of celiac disease patients, this treatment showed the potential to strongly reduce the ability of gluten proteins to stimulate a T-cell-mediated immune response. From a rheological point of view, the functionality of gluten was retained. Considering L-theanine's evidence-based health benefits, a novel functional food is presented here and for celiac disease can be a path towards the development of an alternative to the gluten-free diet.