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Identification of residue-to-residue contact between a peptide ligand and its G protein-coupled receptor using periodate-mediated dihydroxyphenylalanine cross-linking and mass spectrometry.
Umanah GK, Huang L, Ding FX, Arshava B, Farley AR, Link AJ, Naider F, Becker JM
(2010) J Biol Chem 285: 39425-36
MeSH Terms: Binding, Competitive, Cross-Linking Reagents, Dihydroxyphenylalanine, Kinetics, Ligands, Mass Spectrometry, Mitogens, Mutagenesis, Site-Directed, Peptides, Periodic Acid, Protein Binding, Protein Conformation, Receptors, G-Protein-Coupled, Saccharomyces cerevisiae, Signal Transduction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added February 20, 2015
Fundamental knowledge about how G protein-coupled receptors and their ligands interact is important for understanding receptor-ligand binding and the development of new drug discovery strategies. We have used cross-linking and tandem mass spectrometry analyses to investigate the interaction of the N terminus of the Saccharomyces cerevisiae tridecapeptide pheromone, α-factor (WHWLQLKPGQPMY), and Ste2p, its cognate G protein-coupled receptor. The Trp(1) residue of α-factor was replaced by 3,4-dihydroxyphenylalanine (DOPA) for periodate-mediated chemical cross-linking, and biotin was conjugated to Lys(7) for detection purposes to create the peptide [DOPA(1),Lys(7)(BioACA),Nle(12)]α-factor, called Bio-DOPA(1)-α-factor. This ligand analog was a potent agonist and bound to Ste2p with ∼65 nanomolar affinity. Immunoblot analysis of purified Ste2p samples that were treated with Bio-DOPA(1)-α-factor showed that the peptide analog cross-linked efficiently to Ste2p. The cross-linking was inhibited by the presence of either native α-factor or an α-factor antagonist. MALDI-TOF and immunoblot analyses revealed that Bio-DOPA(1)-α-factor cross-linked to a fragment of Ste2p encompassing residues Ser(251)-Met(294). Fragmentation of the cross-linked fragment and Ste2p using tandem mass spectrometry pinpointed the cross-link point of the DOPA(1) of the α-factor analog to the Ste2p Lys(269) side chain near the extracellular surface of the TM6-TM7 bundle. This conclusion was confirmed by a greatly diminished cross-linking of Bio-DOPA(1)-α-factor into a Ste2p(K269A) mutant. Based on these and previously obtained binding contact data, a mechanism of α-factor binding to Ste2p is proposed. The model for bound α-factor shows how ligand binding leads to conformational changes resulting in receptor activation of the signal transduction pathway.
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16 MeSH Terms
Estrogen treatment impairs cognitive performance after psychosocial stress and monoamine depletion in postmenopausal women.
Newhouse PA, Dumas J, Wilkins H, Coderre E, Sites CK, Naylor M, Benkelfat C, Young SN
(2010) Menopause 17: 860-73
MeSH Terms: Affect, Aged, Aged, 80 and over, Anxiety, Cognition Disorders, Double-Blind Method, Estradiol, Estrogens, Female, Follicle Stimulating Hormone, Humans, Hydrocortisone, Mental Recall, Middle Aged, Neuropsychological Tests, Phenylalanine, Postmenopause, Stress, Psychological, Tryptophan, Tyrosine
Show Abstract · Added March 3, 2020
OBJECTIVE - Recent studies have shown that women experience an acceleration of cognitive problems after menopause and that estrogen treatment can improve or at least maintain current levels of cognitive functioning in postmenopausal women. However, we have previously shown that the negative emotional effects of psychosocial stress are magnified in normal postmenopausal women after estrogen treatment. This study examined whether estradiol (E2) administration can modify cognitive performance after exposure to psychological stress and monoamine depletion.
METHODS - Participants consisted of 22 postmenopausal women placed on either oral placebo or 17beta-E2 (1 mg/d for 1 mo, then 2 mg/d for 2 mo). At the end of the 3-month treatment phase, participants underwent three depletion challenges in which they ingested one of three amino acid mixtures: deficient in tryptophan, deficient in phenylalanine/tyrosine, or balanced. Five hours later, participants performed the Trier Social Stress Test (TSST), followed by mood and anxiety ratings and cognitive testing. Cognitive measures included tests of attention, psychomotor function, and verbal episodic memory.
RESULTS - E2-treated compared with placebo-treated participants exhibited significant worsening of cognitive performance on tasks measuring attentional performance and psychomotor speed. Similar trends for impairment were seen in measures of long-term episodic memory compared with placebo-treated postmenopausal women. E2-treated participants also showed a significant increase in negative mood and anxiety compared with placebo-treated women after, but not before, the TSST, although the worsening of both cognitive and behavioral functioning was not correlated. These effects were independent of tryptophan or tyrosine/phenylalanine depletion and were not manifested before the TSST or at baseline.
CONCLUSIONS - These data suggest that the relationship between estrogen administration and cognitive/behavioral performance in postmenopausal women may be more complex than initially appreciated and that the effects of psychosocial stress may influence whether hormone effects are beneficial.
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MeSH Terms
Plasma tryptophan and tyrosine levels are independent risk factors for delirium in critically ill patients.
Pandharipande PP, Morandi A, Adams JR, Girard TD, Thompson JL, Shintani AK, Ely EW
(2009) Intensive Care Med 35: 1886-92
MeSH Terms: Aged, Amino Acid Transport System L, Analysis of Variance, Critical Illness, Delirium, Disease Progression, Female, Humans, Logistic Models, Male, Markov Chains, Middle Aged, Phenylalanine, Pilot Projects, Predictive Value of Tests, Regression Analysis, Risk Assessment, Risk Factors, Severity of Illness Index, Tennessee, Time Factors, Tryptophan, Tyrosine
Show Abstract · Added September 23, 2015
AIM - The pathophysiology of delirium remains elusive though neurotransmitters and their precursor large neutral amino acids (LNAAs) may play a role. This pilot study investigated whether alterations of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) plasma levels were associated with a higher risk of transitioning to delirium in critically ill patients.
METHODS - Plasma LNAA concentrations were determined on days 1 and 3 in mechanically ventilated (MV) patients from the MENDS randomized controlled trial (dexmedetomidine vs. lorazepam sedation). Three independent variables were calculated by dividing plasma concentrations of Trp, Phe, and Tyr by the sum of all other LNAA concentrations. Delirium was assessed daily using the confusion assessment method for the intensive care unit (CAM-ICU). Markov regression models were used to analyze independent associations between plasma LNAA ratios and transition to delirium after adjusting for covariates.
RESULTS - The 97 patients included in the analysis had a high severity of illness (median APACHE II, 28; IQR, 24-32). After adjusting for confounders, only high or very low tryptophan/LNAA ratios (p = 0.0003), and tyrosine/LNAA ratios (p = 0.02) were associated with increased risk of transitioning to delirium, while phenylalanine levels were not (p = 0.27). Older age, higher APACHE II scores and increasing fentanyl exposure were also associated with higher probabilities of transitioning to delirium.
CONCLUSIONS - In this pilot study, plasma tryptophan/LNAA and tyrosine/LNAA ratios were associated with transition to delirium in MV patients, suggesting that alterations of amino acids may be important in the pathogenesis of ICU delirium. Future studies evaluating the role of amino acid precursors of neurotransmitters are warranted in critically ill patients.
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23 MeSH Terms
Endotoxin priming of neutrophils requires NADPH oxidase-generated oxidants and is regulated by the anion transporter ClC-3.
Moreland JG, Davis AP, Matsuda JJ, Hook JS, Bailey G, Nauseef WM, Lamb FS
(2007) J Biol Chem 282: 33958-67
MeSH Terms: Anaerobiosis, Animals, Cell Line, Chloride Channels, Enzyme Activation, Humans, Lipopolysaccharides, Mice, N-Formylmethionine Leucyl-Phenylalanine, NADPH Oxidases, Neisseria meningitidis, Neutrophil Activation, Neutrophils, Oxidation-Reduction, Respiratory Burst, Secretory Vesicles, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added February 22, 2016
Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.
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17 MeSH Terms
A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore.
Krantz BA, Melnyk RA, Zhang S, Juris SJ, Lacy DB, Wu Z, Finkelstein A, Collier RJ
(2005) Science 309: 777-81
MeSH Terms: Amino Acid Sequence, Amino Acid Substitution, Antigens, Bacterial, Bacillus anthracis, Bacterial Toxins, Binding Sites, Cell Membrane, Cytosol, Electron Spin Resonance Spectroscopy, Endosomes, Hydrogen-Ion Concentration, Hydrophobic and Hydrophilic Interactions, Lipid Bilayers, Models, Biological, Models, Molecular, Molecular Sequence Data, Mutagenesis, Onium Compounds, Organophosphorus Compounds, Phenylalanine, Protein Conformation, Protein Folding, Quaternary Ammonium Compounds
Show Abstract · Added May 7, 2013
The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.
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23 MeSH Terms
Identification of determinants of ligand binding affinity and selectivity in the prostaglandin D2 receptor CRTH2.
Hata AN, Lybrand TP, Breyer RM
(2005) J Biol Chem 280: 32442-51
MeSH Terms: Alanine, Animals, Anti-Inflammatory Agents, Non-Steroidal, Binding, Competitive, Cell Line, Cell Movement, Chemotactic Factors, Chemotaxis, Cyclic AMP, Dose-Response Relationship, Drug, Eicosanoids, Enzyme-Linked Immunosorbent Assay, Flow Cytometry, Glutamic Acid, Humans, Hypersensitivity, Indomethacin, Inflammation, Kinetics, Ligands, Mice, Models, Biological, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Phenylalanine, Prostaglandins, Protein Binding, Protein Structure, Tertiary, Receptors, G-Protein-Coupled, Receptors, Immunologic, Receptors, Prostaglandin, Tyrosine
Show Abstract · Added December 21, 2013
The chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) is a G protein-coupled receptor that mediates the pro-inflammatory effects of prostaglandin D(2) (PGD(2)) generated in allergic inflammation. The CRTH2 receptor shares greatest sequence similarity with chemoattractant receptors compared with prostanoid receptors. To investigate the structural determinants of CRTH2 ligand binding, we performed site-directed mutagenesis of putative mCRTH2 ligand-binding residues, and we evaluated mutant receptor ligand binding and functional properties. Substitution of alanine at each of three residues in the transmembrane (TM) helical domains (His-106, TM III; Lys-209, TM V; and Glu-268, TM VI) and one in extracellular loop II (Arg-178) decreased PGD(2) binding affinity, suggesting that these residues play a role in binding PGD(2). In contrast, the H106A and E268A mutants bound indomethacin, a nonsteroidal anti-inflammatory drug, with an affinity similar to the wild-type receptor. HEK293 cells expressing the H106A, K209A, and E268A mutants displayed reduced inhibition of intracellular cAMP and chemotaxis in response to PGD(2), whereas the H106A and E268A mutants had functional responses to indomethacin similar to the wild-type receptor. Binding of PGE(2) by the E268A mutant was enhanced compared with the wild-type receptor, suggesting that Glu-268 plays a role in determining prostanoid ligand selectivity. Replacement of Tyr-261 with phenylalanine did not affect PGD(2) binding but decreased the binding affinity for indomethacin. These results provided the first details of the ligand binding pocket of an eicosanoid-binding chemoattractant receptor.
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33 MeSH Terms
Proteinase activity in human and murine saliva as a biomarker for proteinase inhibitor efficacy.
Fingleton B, Menon R, Carter KJ, Overstreet PD, Hachey DL, Matrisian LM, McIntyre JO
(2004) Clin Cancer Res 10: 7865-74
MeSH Terms: Amidines, Amylases, Animals, Biomarkers, Blood Proteins, Chromatography, Liquid, Endopeptidases, Female, Humans, Imidazoles, Male, Mass Spectrometry, Matrix Metalloproteinase 9, Matrix Metalloproteinase Inhibitors, Metalloendopeptidases, Mice, Organic Chemicals, Phenylalanine, Protease Inhibitors, Saliva, Thiophenes, Urokinase-Type Plasminogen Activator
Show Abstract · Added March 5, 2014
As molecularly targeted agents reach the clinic, there is a need for assays to detect their presence and effectiveness against target molecules in vivo. Proteinase inhibitors are one example of a class of therapeutic agent for which satisfactory methods of identifying successful target modulation in vivo are lacking. This is of particular importance while these drugs are in clinical trials because standard maximum-tolerated dose-finding studies often are not suitable due to lack of toxicity. Saliva represents a readily accessible bodily fluid that can be repeatedly sampled and used for assaying in vivo effects of systemic drugs. Here we show the development of a simple assay that can be used to measure proteinase activity in saliva and proteinase inhibition after systemic treatment with three different proteinase inhibitors. A variety of gelatinolytic activities present in human and murine saliva have been assayed with a fluorescent dye-labeled substrate and assigned to different proteinase categories by inclusion of specific classes of inhibitors. Treatment of mice with either matrix metalloproteinase inhibitors or a urokinase inhibitor for a period as short as 48 hours results in levels of the drugs that can be detected in saliva by mass spectrometry and concomitant decreases in salivary proteinase activity, thus demonstrating that these inhibitors successfully modulate their targets in vivo.
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22 MeSH Terms
A matrix metalloproteinase inhibitor promotes granuloma formation during the early phase of Mycobacterium tuberculosis pulmonary infection.
Izzo AA, Izzo LS, Kasimos J, Majka S
(2004) Tuberculosis (Edinb) 84: 387-96
MeSH Terms: Animals, Colony Count, Microbial, Cytokines, Drug Administration Schedule, Female, Granuloma, Leukocyte Count, Lung, Matrix Metalloproteinase Inhibitors, Mice, Mice, Inbred C57BL, Phenylalanine, Thiophenes, Tuberculosis, Pulmonary
Show Abstract · Added August 4, 2015
OBJECTIVE - The host response to pulmonary Mycobacterium tuberculosis (Mtb) infection results in granuloma formation in an effort to limit infection, but the host immune cells also provide an environment in which Mtb persists. Granuloma formation requires immune cell infiltration and concurrent extensive remodeling of pulmonary tissue which we hypothesize to be the result of increased matrix metalloproteinases (MMP) activity.
DESIGN - C57BL/6 mice infected with virulent Mtb (H37Rv) via intratracheal inoculation were treated with a synthetic inhibitor of MMP activity (BB-94). Mice were assessed for colony forming units, granuloma morphology, leukocyte recruitment and cytokine levels over 90 days of infection.
RESULTS - BB-94 treated mice had significantly decreased numbers of pulmonary and blood-borne Mtb early during disease, increased collagen deposition within early granulomas and significantly decreased pulmonary leukocyte recruitment when compared to vehicle-treated, Mtb-infected mice. Cytokine expression did not differ significantly between groups.
CONCLUSION - Events of early granuloma formation can be modified by inhibiting MMP activity, by decreasing leukocyte recruitment, a major source of MMPs during infection, enhancing the establishment of granulomas and decreasing blood-borne dissemination of Mtb.
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14 MeSH Terms
Water ingestion as prophylaxis against syncope.
Lu CC, Diedrich A, Tung CS, Paranjape SY, Harris PA, Byrne DW, Jordan J, Robertson D
(2003) Circulation 108: 2660-5
MeSH Terms: Adolescent, Adult, Cross-Over Studies, Dihydroxyphenylalanine, Female, Heart Rate, Humans, Hypotension, Male, Posture, Reference Values, Syncope, Tilt-Table Test, Treatment Outcome, Water
Show Abstract · Added December 10, 2013
BACKGROUND - Water ingestion raises blood pressure substantially in patients with perturbed autonomic control and more modestly in older subjects. It is unclear whether prophylactic water drinking improves orthostatic tolerance in normal healthy adults.
METHODS AND RESULTS - Twenty-two healthy subjects, 18 to 42 years of age, with no history of syncope underwent head-up tilt-table testing at 60 degrees for 45 minutes or until presyncope or syncope occurred. In their initial test, participants were randomized to either 16 oz (473 mL) of water drinking 5 minutes before tilt-table testing or tilt-table testing alone, with the alternative in a second test on a different day. During the first 30 minutes of tilt, 8 of 22 subjects without water experienced presyncope but only 1 of 22 who had ingested water (P=0.016). Water drinking attenuated the heart rate increase associated with tilt (P<0.001) while accentuating the increase in total peripheral resistance (P=0.012). The average time study participants tolerated head-up tilt was 26% longer after water (41.1+/-8.1 versus 32.6+/-14.3 minutes, mean+/-SD), with a pairwise mean difference of 8.5+/-14.0 minutes (95% CI, 2.3 to 14.7 minutes; P=0.011).
CONCLUSIONS - Water enhances tolerance of upright posture. The effect of water is mediated by increased peripheral vascular resistance. Water ingestion may constitute a simple and effective prophylaxis against vasovagal reactions in healthy subjects, such as those associated with blood donation.
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15 MeSH Terms
Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.
Shi M, Bennett TA, Cimino DF, Maestas DC, Foutz TD, Gurevich VV, Sklar LA, Prossnitz ER
(2003) Biochemistry 42: 7283-93
MeSH Terms: Animals, Arrestin, Calcium, GTP-Binding Protein alpha Subunit, Gi2, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Proteins, Heterotrimeric GTP-Binding Proteins, Humans, Leukemia, Myeloid, Mice, Microscopy, Confocal, N-Formylmethionine Leucyl-Phenylalanine, Phosphorylation, Protein Subunits, Proto-Oncogene Proteins, Receptors, Formyl Peptide, Receptors, Immunologic, Receptors, Peptide, Recombinant Fusion Proteins, Transfection, Tumor Cells, Cultured, U937 Cells, Ultracentrifugation
Show Abstract · Added December 10, 2013
G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
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23 MeSH Terms