ABSTRACT
Flowers are the most vulnerable plant organ to infection by the necrotrophic fungus Botrytis cinerea. Here we show that pre-treatment of chrysanthemum (Chrysanthemum morifolium) flowers with phenylalanine (Phe) significantly reduces their susceptibility to B. cinerea. To comprehend how Phe treatment induces resistance, we monitored the dynamics of metabolites (by GC/LC-MS) and transcriptomes (by RNAseq) in flowers after Phe treatment and B. cinerea infection. Phe treatment resulted in accumulation of 3-phenyllactate and benzaldehyde, and in particular induced the expression of genes related to Ca2+ signaling and receptor kinases, implicating an induction of the defense response. Interestingly, the main effects of Phe treatment were observed in flowers exposed to B. cinerea infection, stabilizing the global fluctuations in the levels of metabolites and transcripts while reducing susceptibility to the fungus. We suggest that Phe-induced resistance is associated to cell priming, enabling rapid and targeted reprogramming of cellular defense responses to resist disease development. After Phe pre-treatment, the levels of the anti-fungal volatiles phenylacetaldehyde and eugenol were maintained and the level of coniferin, a plausible monolignol precursor in cell wall lignification, was strongly increased. In addition, Phe pre-treatment reduced ROS generation, prevented ethylene emission, and caused changes in the expression of a minor number of genes related to cell wall biogenesis, encoding the RLK THESEUS1, or involved in Ca2+ and hormonal signaling processes. Our findings point to Phe pre-treatment as a potential orchestrator of a broad-spectrum defense response which may not only provide an ecologically friendly pest control strategy but also offers a promising way of priming plants to induce defense responses against B. cinerea.
Subject(s)
Botrytis , Chrysanthemum/physiology , Flowers/physiology , Phenylalanine/physiology , Plant Diseases/immunology , Chrysanthemum/immunology , Chrysanthemum/microbiology , Ethylenes/metabolism , Flowers/immunology , Phenylalanine/metabolism , Plant Diseases/microbiology , Plant Growth Regulators/metabolism , Reactive Oxygen SpeciesABSTRACT
The role of Phe213 in the allosteric mechanism of human cytochrome P450 CYP3A4 was studied using a combination of progesterone (PGS) and carbamazepine (CBZ) as probe substrates. We expressed, purified, and incorporated into POPC Nanodiscs three mutants, F213A, F213S, and F213Y, and compared them with wild-type (WT) CYP3A4 by monitoring spectral titration, the rate of NADPH oxidation, and steady-state product turnover rates with pure substrates and substrate mixtures. All mutants demonstrated higher activity with CBZ, lower activity with PGS, and a reduced level of activation of CBZ epoxidation by PGS, which was most pronounced in the F213A mutant. Using all-atom molecular dynamics simulations, we compared the dynamics of WT CYP3A4 and the F213A mutant incorporated into the lipid bilayer and the effect of the presence of the PGS molecule at the allosteric peripheral site and evaluated the critical role of Phe213 in mediating the heterotropic allosteric interactions in CYP3A4.
Subject(s)
Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Phenylalanine/metabolism , Allosteric Site , Carbamazepine/chemistry , Cytochrome P-450 CYP3A/physiology , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/physiology , Humans , Hydroxylation , Kinetics , Molecular Dynamics Simulation , Oxidation-Reduction , Phenylalanine/physiology , Progesterone/chemistryABSTRACT
Glycine receptors (GlyRs) are Cys-loop receptors that mediate fast synaptic inhibition in the brain stem and spinal cord. They are involved in the generation of motor rhythm, reflex circuit coordination, and sensory signal processing and therefore represent targets for therapeutic interventions. The extracellular domains (ECDs) of Cys-loop receptors typically contain many aromatic amino acids, but only those in the receptor binding pocket have been extensively studied. Here, we show that many Phe residues in the ECD that are not located in the binding pocket are also involved in GlyR function. We examined these Phe residues by creating several GlyR variants, characterizing these variants with the two-electrode voltage clamp technique in Xenopus oocytes, and interpreting changes in receptor parameters by using currently available structural information on the open and closed states of the GlyR. Substitution of six of the eight Phe residues in the ECD with Ala resulted in loss of function or significantly increased the EC50 and also altered the maximal response to the partial GlyR agonist taurine compared with glycine in those receptor variants that were functional. Substitutions with other amino acids, combined with examination of nearby residues that could potentially interact with these Phe residues, suggested interactions that could be important for GlyR function, and possibly similar interactions could contribute to the function of other members of the Cys-loop receptor family. Overall, our results suggest that many ECD regions are important for GlyR function and that these regions could inform the design of therapeutic agents targeting GlyR activity.
Subject(s)
Phenylalanine/genetics , Receptors, Glycine/genetics , Amino Acid Substitution , Animals , Humans , Loss of Function Mutation , Phenylalanine/physiology , Protein Binding/genetics , Protein Domains/genetics , Protein Engineering/methods , Receptors, Glycine/physiology , Taurine/metabolismABSTRACT
The role of dopaminergic system in decision-making is well documented, and evidence suggests that it could play a significant role in response selection processes. The N-40 is a fronto-central event-related potential, generated by the supplementary motor areas (SMAs) and a physiological index of response selection processes. The aim of the present study was to determine whether infraclinical effects of dopamine depletion on response selection processes could be evidenced via alterations of the N-40. We obtained a dopamine depletion in healthy volunteers with the acute phenylalanine and tyrosine depletion (APTD) method which consists in decreasing the availability of dopamine precursors. Subjects realized a Simon task in the APTD condition and in the control condition. When the stimulus was presented on the same side as the required response, the stimulus-response association was congruent and when the stimulus was presented on the opposite side of the required response, the stimulus-response association was incongruent. The N-40 was smaller for congruent associations than for incongruent associations. Moreover, the N-40 was sensitive to the level of dopaminergic activity with a decrease in APTD condition compared to control condition. This modulation of the N-40 by dopaminergic level could not be explained by a global decrease of cerebral electrogenesis, since negativities and positivities indexing the recruitment of the primary motor cortex (anatomically adjacent to the SMA) were unaffected by APTD. The specific sensitivity of N-40 to ATPD supports the model of Keeler et al. (Neuroscience 282:156-175, 2014) according to which the dopaminergic system is involved in response selection.
Subject(s)
Dopamine/physiology , Motor Cortex/physiopathology , Phenylalanine/physiology , Reaction Time/physiology , Tyrosine/physiology , Adult , Analysis of Variance , Decision Making , Electroencephalography , Evoked Potentials/physiology , Female , Humans , Inhibition, Psychological , Male , Phenylalanine/blood , Tyrosine/blood , Young AdultABSTRACT
GPR139 is an orphan G-protein-coupled receptor expressed in the central nervous system. To identify its physiologic ligand, we measured GPR139 receptor activity from recombinant cells after treatment with amino acids, orphan ligands, serum, and tissue extracts. GPR139 activity was measured using guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding, calcium mobilization, and extracellular signal-regulated kinases phosphorylation assays. Amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) activated GPR139, with EC50 values in the 30- to 300-µM range, consistent with the physiologic concentrations of L-Trp and L-Phe in tissues. Chromatography of rat brain, rat serum, and human serum extracts revealed two peaks of GPR139 activity, which corresponded to the elution peaks of L-Trp and L-Phe. With the purpose of identifying novel tools to study GPR139 function, a high-throughput screening campaign led to the identification of a selective small-molecule agonist [JNJ-63533054, (S)-3-chloro-N-(2-oxo-2-((1-phenylethyl)amino)ethyl) benzamide]. The tritium-labeled JNJ-63533054 bound to cell membranes expressing GPR139 and could be specifically displaced by L-Trp and L-Phe. Sequence alignment revealed that GPR139 is highly conserved across species, and RNA sequencing studies of rat and human tissues indicated its exclusive expression in the brain and pituitary gland. Immunohistochemical analysis showed specific expression of the receptor in circumventricular regions of the habenula and septum in mice. Together, these findings suggest that L-Trp and L-Phe are candidate physiologic ligands for GPR139, and we hypothesize that this receptor may act as a sensor to detect dynamic changes of L-Trp and L-Phe in the brain.
Subject(s)
Habenula/chemistry , Nerve Tissue Proteins/physiology , Phenylalanine/physiology , Receptors, G-Protein-Coupled/physiology , Septum of Brain/chemistry , Tryptophan/physiology , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , HEK293 Cells , Humans , Male , Mice , Molecular Sequence Data , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/drug effects , Phenylalanine/blood , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/analysis , Receptors, G-Protein-Coupled/drug effects , Tryptophan/bloodABSTRACT
Phenylketonuria (PKU), if not detected and treated in newborns, causes severe neurological dysfunction and cognitive and behavioral deficiencies. Despite the biochemical characterization of PKU, the molecular mechanisms underlying PKU-associated brain dysfunction remain poorly understood. The aim of this study was to gain insights into the pathogenesis of this neurological damage by analyzing protein expression profiles in brain tissue of Black and Tan BRachyury-PahEnu2 mice (a mouse model of PKU). We compared the cerebral protein expression of homozygous PKU mice with that of their heterozygous counterparts using two-dimensional difference gel electrophoresis analysis, and identified 21 differentially expressed proteins, four of which were over-expressed and 17 under-expressed. An in silico bioinformatic approach indicated that protein under-expression was related to neuronal differentiation and dendritic growth, and to such neurological disorders as progressive motor neuropathy and movement disorders. Moreover, functional annotation analyses showed that some identified proteins were involved in oxidative metabolism. To further investigate the proteins involved in the neurological damage, we validated two of the proteins that were most strikingly under-expressed, namely, Syn2 and Dpysl2, which are involved in synaptic function and neurotransmission. We found that Glu2/3 and NR1 receptor subunits were over-expressed in PKU mouse brain. Our results indicate that differential expression of these proteins may be associated with the processes underlying PKU brain dysfunction, namely, decreased synaptic plasticity and impaired neurotransmission. We identified a set of proteins whose expression is affected by hyperphenylalaninemia. We think that phenylketonuria (PKU) brain dysfunction also depends on reduced Syn2 and Dpysl2 levels, increased Glu2/3 and NR1 levels, and decreased Pkm, Ckb, Pgam1 and Eno1 levels. These findings finally confirm that alteration in synaptic function, in transmission and in energy metabolism underlie brain damage provoked by hyperphenylalaninemias.
Subject(s)
Brain Chemistry/physiology , Nervous System Diseases/genetics , Nervous System Diseases/pathology , Phenylketonurias/genetics , Phenylketonurias/pathology , Protein Biosynthesis/physiology , Animals , Biomarkers , Blotting, Western , Chromatography, High Pressure Liquid , Computational Biology , Computer Simulation , Disease Models, Animal , Electrophoresis, Gel, Two-Dimensional , Gene Regulatory Networks , Mice , Phenylalanine/metabolism , Phenylalanine/physiology , Tandem Mass SpectrometryABSTRACT
Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.
Subject(s)
Apoptosis , Fas Ligand Protein/physiology , Neurons/physiology , Phenylalanine/physiology , fas Receptor/metabolism , Animals , Cells, Cultured , Cerebral Cortex/pathology , Endoplasmic Reticulum Stress , Phenylketonurias/metabolism , Phenylketonurias/pathology , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Signal TransductionABSTRACT
Despite interest in dopamine's role in emotion-based decision-making, few reports of the effects of dopamine manipulations are available in this area in humans. This study investigates dopamine's role in emotion-based decision-making through a common measure of this construct, the Iowa Gambling Task (IGT), using Acute Tyrosine Phenylalanine Depletion (ATPD). In a between-subjects design, 40 healthy adults were randomized to receive either an ATPD beverage or a balanced amino acid beverage (a control) prior to completing the IGT, as well as pre- and post-manipulation blood draws for the neurohormone prolactin. Together with conventional IGT performance metrics, choice selections and response latencies were examined separately for good and bad choices before and after several key punishment events. Changes in response latencies were also used to predict total task performance. Prolactin levels increased significantly in the ATPD group but not in the control group. However, no significant group differences in performance metrics were detected, nor were there sex differences in outcome measures. However, the balanced group's bad deck latencies speeded up across the task, while the ATPD group's latencies remained adaptively hesitant. Additionally, modulation of latencies to the bad decks predicted total score for the ATPD group only. One interpretation is that ATPD subtly attenuated reward salience and altered the approach by which individuals achieved successful performance, without resulting in frank group differences in task performance.
Subject(s)
Decision Making , Emotions , Phenylalanine/physiology , Tyrosine/physiology , Adult , Female , Humans , Male , Phenylalanine/deficiency , Reference Values , Tyrosine/deficiencyABSTRACT
Voltage-gated sodium, potassium, and calcium channels are made of a pore domain (PD) controlled by four voltage-sensing domains (VSDs). The PD contains the ion permeation pathway and the activation gate located on the intracellular side of the membrane. A large number of small molecules are known to inhibit the PD by acting as open channel blockers. The voltage-gated proton channel Hv1 is made of two VSDs and lacks the PD. The location of the activation gate in the VSD is unknown and open channel blockers for VSDs have not yet been identified. Here, we describe a class of small molecules which act as open channel blockers on the Hv1 VSD and find that a highly conserved phenylalanine in the charge transfer center of the VSD plays a key role in blocker binding. We then use one of the blockers to show that Hv1 contains two intracellular and allosterically coupled gates.
Subject(s)
Ion Channel Gating/physiology , Ion Channels/chemistry , Ion Channels/physiology , Porins/chemistry , Porins/physiology , Animals , Conserved Sequence/genetics , Female , Humans , Ion Channels/antagonists & inhibitors , Phenylalanine/chemistry , Phenylalanine/physiology , Porins/antagonists & inhibitors , Protein Binding/physiology , Protein Structure, Tertiary , XenopusABSTRACT
RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.
Subject(s)
Ubiquitin/metabolism , Ubiquitination , X-Linked Inhibitor of Apoptosis Protein/physiology , Amino Acid Sequence , Humans , Models, Biological , Models, Molecular , Phenylalanine/genetics , Phenylalanine/physiology , Protein Binding/genetics , Protein Binding/physiology , Protein Interaction Domains and Motifs/genetics , Protein Interaction Domains and Motifs/physiology , Protein Multimerization/genetics , Protein Multimerization/physiology , Substrate Specificity , Ubiquitin/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/physiology , Ubiquitination/genetics , Ubiquitination/physiology , X-Linked Inhibitor of Apoptosis Protein/chemistry , X-Linked Inhibitor of Apoptosis Protein/genetics , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O(2) and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O(2)/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.
Subject(s)
Bacterial Proteins/physiology , Mycobacterium tuberculosis/metabolism , Nitric Oxide/metabolism , Truncated Hemoglobins/physiology , Bacterial Proteins/chemistry , Binding Sites , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Computer Simulation , Crystallography, X-Ray , Ligands , Mutagenesis, Site-Directed , Nitric Oxide/chemistry , Oxygen/chemistry , Oxygen/metabolism , Phenylalanine/chemistry , Phenylalanine/metabolism , Phenylalanine/physiology , Protein Structure, Tertiary , Truncated Hemoglobins/chemistryABSTRACT
Multidrug resistance-associated protein 4 (MRP4) is a membrane transporter that mediates the cellular efflux of a wide range of anionic drugs and endogenous molecules. MRP4 transport can influence the pharmacokinetics of drugs and their metabolites, therefore more knowledge about the molecular determinants important for its transport function would be of relevance. Here, we substituted amino acids Phe(368), Trp(995), and Arg(998) with conservative or non-conservative residues, and determined the effect on transport of the model substrates estradiol 17-ß-d-glucuronide (E(2)17ßG), cyclic guanosine monophosphate (cGMP), methotrexate (MTX), and folic acid into membrane vesicles isolated from baculovirus transduced HEK293 cells overexpressing the mutant MRP4 proteins. This revealed that all Arg(998) mutations appeared to be deleterious, whereas the effect of a Phe(368) or Trp(995) replacement was dependent on the amino acid introduced and the substrate studied. Substitution of Phe(368) with Trp (F368W) induced a gain-of-function of E(2)17ßG transport and a loss-of-function of MTX transport, which could not be attributed to an altered substrate binding. Moreover, we did not observe any modification in ATP or ADP handling for F368W. These results, in combination with docking of substrates in a homology model of MRP4 in the inward- and outward-facing conformation, suggest that Phe(368) and Trp(995) do not play an important role in the initial binding of substrates. They, however, might interact with the substrates during rearrangement of helixes for substrate translocation, funneling the substrates to the exit site in the outward-facing conformation.
Subject(s)
Multidrug Resistance-Associated Proteins/chemistry , Multidrug Resistance-Associated Proteins/physiology , Phenylalanine/physiology , Amino Acid Sequence , Amino Acid Substitution/genetics , Down-Regulation/genetics , HEK293 Cells , Humans , Molecular Sequence Data , Multidrug Resistance-Associated Proteins/genetics , Phenylalanine/chemistry , Phenylalanine/genetics , Protein Structure, Secondary/genetics , Protein Transport/genetics , Substrate Specificity/geneticsABSTRACT
Inhibitors of insulin-regulated aminopeptidase (IRAP) improve memory and are being developed as a novel treatment for memory loss. In this study, the binding of a class of these inhibitors to human IRAP was investigated using molecular docking and site-directed mutagenesis. Four benzopyran-based IRAP inhibitors with different affinities were docked into a homology model of the catalytic site of IRAP. Two 4-pyridinyl derivatives orient with the benzopyran oxygen interacting with the Zn(2+) ion and a direct parallel ring-stack interaction between the benzopyran rings and Phe544. In contrast, the two 4-quinolinyl derivatives orient in a different manner, interacting with the Zn(2+) ion via the quinoline nitrogen, and Phe544 contributes an edge-face hydrophobic stacking point with the benzopyran moiety. Mutagenic replacement of Phe544 with alanine, isoleucine, or valine resulted in either complete loss of catalytic activity or altered hydrolysis velocity that was substrate-dependent. Phe544 is also important for inhibitor binding, because these mutations altered the K(i) in some cases, and docking of the inhibitors into the corresponding Phe544 mutant models revealed how the interaction might be disturbed. These findings demonstrate a key role of Phe544 in the binding of the benzopyran IRAP inhibitors and for optimal positioning of enzyme substrates during catalysis.
Subject(s)
Benzopyrans/metabolism , Cystinyl Aminopeptidase/antagonists & inhibitors , Cystinyl Aminopeptidase/metabolism , Phenylalanine/physiology , Benzopyrans/chemistry , Benzopyrans/pharmacology , Binding Sites/drug effects , Binding Sites/physiology , Catalytic Domain/drug effects , Catalytic Domain/physiology , Cell Line , Humans , Hydrophobic and Hydrophilic Interactions , Phenylalanine/chemistry , Substrate Specificity/physiologyABSTRACT
The deletion of Phe-508 (F508del) constitutes the most prevalent cystic fibrosis-causing mutation. This mutation leads to cystic fibrosis transmembrane conductance regulator (CFTR) misfolding and retention in the endoplasmic reticulum and altered channel activity in mammalian cells. This folding defect can however be partially overcome by growing cells expressing this mutant protein at low (27 degrees C) temperature. Chemical "correctors" have been identified that are also effective in rescuing the biosynthetic defect in F508del-CFTR, thereby permitting its functional expression at the cell surface. The mechanism of action of chemical correctors remains unclear, but it has been suggested that certain correctors [including 4-cyclohexyloxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-325)] may act to promote trafficking by interacting directly with the mutant protein. To test this hypothesis, we assessed the effect of VRT-325 addition on the channel activity of F508del-CFTR after its surface expression had been "rescued" by low temperature. It is noteworthy that short-term pretreatment with VRT-325 [but not with an inactive analog, 4-hydroxy-2-(1-[4-(4-methoxy-benzenesulfonyl)-piperazin-1-yl]-ethyl)-quinazoline (VRT-186)], caused a modest but significant inhibition of cAMP-mediated halide flux. Furthermore, VRT-325 decreased the apparent ATP affinity of purified and reconstituted F508del-CFTR in our ATPase activity assay, an effect that may account for the decrease in channel activity by temperature-rescued F508del-CFTR. These findings suggest that biosynthetic rescue mediated by VRT-325 may be conferred (at least in part) by direct modification of the structure of the mutant protein, leading to a decrease in its ATP-dependent conformational dynamics. Therefore, the challenge for therapy discovery will be the design of small molecules that bind to promote biosynthetic maturation of the major mutant without compromising its activity in vivo.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Animals , Cricetinae , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/physiology , Mutation , Phenylalanine/genetics , Phenylalanine/metabolism , Phenylalanine/physiology , Piperazines , Protein Transport/genetics , Protein Transport/physiology , Quinazolines/metabolism , Sequence DeletionABSTRACT
Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two noncatalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the alpha,beta-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low K(M) that MsJHE shows for JH. This low K(M), however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance-stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appears to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism.
Subject(s)
Carboxylic Ester Hydrolases/metabolism , Manduca/enzymology , Phenylalanine/physiology , Sesquiterpenes/metabolism , Threonine/physiology , Animals , Binding Sites , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/genetics , Chromatography, Thin Layer , Hydrolysis , Kinetics , Larva , Models, Molecular , Mutation/genetics , Substrate SpecificityABSTRACT
A new phenylboric acid derivative entrapped lipiodol (PBAD-lipiodol) was developed as a boron carrier for the boron neutron capture therapy (BNCT) of hepatoma in Taiwan. The biodistribution of both PBAD-lipiodol and BPA-fructose was assayed in GP7TB hepatoma-bearing rat model. The highest uptake of PBAD-lipiodol was found at 2h post injection. The application of BNCT for the hepatoma treatment in tumor-bearing rats is suggested to be 2-4h post PBAD-lipiodol injection.
Subject(s)
Boron Neutron Capture Therapy/methods , Boronic Acids/pharmacokinetics , Fluorine Radioisotopes/pharmacokinetics , Iodized Oil/pharmacokinetics , Liver Neoplasms, Experimental/metabolism , Phenylalanine/analogs & derivatives , Animals , Boronic Acids/chemical synthesis , Boronic Acids/pharmacology , Fluorine Radioisotopes/pharmacology , Iodized Oil/chemical synthesis , Iodized Oil/pharmacology , Liver Neoplasms, Experimental/radiotherapy , Magnetic Resonance Imaging , Male , Phenylalanine/chemical synthesis , Phenylalanine/pharmacokinetics , Phenylalanine/physiology , Rats , Rats, Inbred F344 , Tissue DistributionABSTRACT
The human family of MAPK (mitogen-activated protein kinase) signal-integrating kinases (Mnks) comprises four related proteins derived from two genes by alternative splicing. The MNK1 gene gives rise to two proteins, Mnk1a and Mnk1b, which possess distinct C-termini and properties. Despite lacking the C-terminal MAPK-binding site, Mnk1b shows higher basal activity than Mnk1a. In contrast, the activity of Mnk1a is tightly regulated by signalling through ERK (extracellular-signal-regulated kinase) and p38 MAPK. We show that the short C-terminus of Mnk1b confers on it a 'default' behaviour of substantial, but unregulated, activity. In contrast, the longer C-terminus of Mnk1a represses the basal activity and T (activation)-loop phosphorylation of this isoenzyme while allowing both properties to be stimulated by upstream MAPK signalling. Two features of the C-terminus of Mnk1a appear to account for this behaviour: the known MAPK-binding site and a region (predicted to be alpha-helical) which occludes access to the catalytic domain and the T-loop. The activation of Mnk1a results in a marked conformational change leading to a more 'open' structure. We also identified a conserved phenylalanine residue in an Mnk-specific insert as playing a key role in governing the ease with which Mnk1a can be phosphorylated. These studies help to identify the features that give rise to the diverse properties of human Mnk isoforms.
Subject(s)
Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Catalytic Domain/physiology , Cells, Cultured , Enzyme Activation/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Intracellular Signaling Peptides and Proteins/physiology , Isoenzymes/chemistry , Isoenzymes/metabolism , Isoenzymes/physiology , Models, Biological , Molecular Sequence Data , Phenylalanine/metabolism , Phenylalanine/physiology , Phosphorylation , Protein Conformation , Protein Serine-Threonine Kinases/physiology , Protein Structure, Tertiary/physiology , Sequence Homology, Amino Acid , Structure-Activity Relationship , Threonine/chemistry , Threonine/metabolism , Threonine/physiologyABSTRACT
BACKGROUND: A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2beta2-2beta3 loops of the heptameric precursor to generate a 14-strand transmembrane beta barrel. METHODOLOGY/PRINCIPAL FINDINGS: We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the beta barrel, and a third one, F324, that lies part way up the barrel. CONCLUSIONS/SIGNIFICANCE: Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus.
Subject(s)
Antigens, Bacterial/chemistry , Bacterial Toxins/chemistry , Phenylalanine/physiology , Protein ConformationABSTRACT
Recent studies show that the extrahepatic human UDP-glucuronosyltransferase (UGT)1A10 is capable of phase II glucuronidation of several major cytochrome P450 metabolites of warfarin (i.e., 6-, 7-, and 8-hydroxywarfarin). This study expands on this finding by testing the hypothesis that the UGT1A10 F(90)-M(91)-V(92)-F(93) amino acid motif is important for proper recognition and conjugation of hydroxywarfarin derivatives. Site-directed mutagenesis studies demonstrate that F(90) is critical for 6- and 7-hydroxywarfarin glucuronidation based on the complete loss of enzymatic activity toward these substrates. In contrast, V92A and F93A mutants lead to higher rates of substrate turnover, have minimum changes in K(m) values, and demonstrate substrate inhibition kinetics. A completely different activity profile is observed in the presence of 8-hydroxywarfarin. No change in either activity or affinity is observed with F90A when compared with wild type, whereas F93A and V92A mutants show increases in V(max) (3- and 10-fold, respectively) and minimum changes in K(m). Liquid chromatographytandem mass spectrometry studies show that enzymatic products produced by mutants are identical to wild-type products produced in the presence of 6-, 7-, and 8-hydroxywarfarin. Because F(90) is not critical for the glucuronidation of 8-hydroxywarfarin, there is likely another, different amino acid responsible for binding this compound. In addition, an inhibitory binding site may be formed in the presence of 6- and 7-hydroxywarfarin. This new knowledge and continued characterization of the hydroxywarfarin binding site(s) for UGT1A10 will help elucidate the molecular mechanism of hydroxywarfarin glucuronidation and potentially result in more effective anticoagulant therapies.
Subject(s)
Glucuronides/metabolism , Glucuronosyltransferase/metabolism , Phenylalanine/metabolism , Warfarin/analogs & derivatives , Binding Sites/physiology , Binding, Competitive/genetics , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/physiology , Humans , Phenylalanine/chemistry , Phenylalanine/physiology , Warfarin/chemistry , Warfarin/metabolismABSTRACT
LC3 belongs to a novel ubiquitin-like protein family that is involved in different intracellular trafficking processes, including autophagy. All members of this family share a unique three-dimensional structure composed of a C-terminal ubiquitin core and two N-terminal alpha-helices. Here, we focus on the specific contribution of these regions to autophagy induced by amino acid deprivation. We show that the ubiquitin core by itself is sufficient for LC3 processing through the conjugation machinery and for its consequent targeting to the autophagosomal membrane. The N-terminal region was found to be important for interaction between LC3 and p62/SQSTM1 (hereafter termed p62). This interaction is dependent on the first 10 amino acids of LC3 and on specific residues located within the ubiquitin core. Knockdown of LC3 isoforms and overexpression of LC3 mutants that fail to interact with p62 blocked the incorporation of p62 into autophagosomes. The accumulation of p62 was accompanied by elevated levels of polyubiquitylated detergent-insoluble structures. p62, however, is not required for LC3 lipidation, autophagosome formation and targeting to lysosomes. Our results support the proposal that LC3 is responsible for recruiting p62 into autophagosomes, a process mediated by phenylalanine 52, located within the ubiquitin core, and the N-terminal region of the protein.