Probing the structure of the cytoplasmic domain of the aspartate receptor by targeted disulfide cross-linking

Applying the technique of targeted disulfide cross-linking to the cytoplasmic domain of the aspartate receptor of Salmonella typhimurium indicates a generally alpha-helical conformation of the linker region, and a close juxtaposition and a parallel alignment at the interface between the two subunits in the linker region. This conclusion is supported by the results from the Fourier transform of the hydrophobicity values of the amino acid sequences. Aspartate binding in the periplasmic domain causes a closer juxtaposition of the two subunits in the cytoplasmic domain, as indicated by the more rapid disulfide cross-linking on addition of aspartate.     

Characterization of a mammalian peroxiredoxin that contains one conserved cysteine

A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor. The conserved Cys47-SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys-SOH. In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys-SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys-SOH of 1-Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1-Cys Prx and consequently could not support peroxidase activity. Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function. However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein. Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase.     

The regulation of the binding affinity of the luteinizing hormone/choriogonadotropin receptor by sodium ions is mediated by a highly conserved aspartate located in the second transmembrane domain of G protein-coupled receptors

Sequence alignment shows that there is a highly conserved aspartate in the second transmembrane helix of virtually all G protein-coupled receptors. A previous study on the alpha 2-adrenergic receptor demonstrated that substitution of this acidic residue for the corresponding amide slightly decreases the affinity of the receptor for agonists and completely abolishes the effect of Na+ on the affinity for agonists. Since we have previously shown that Na+ modulates the binding affinity of the LH/CG receptor for ovine LH (oLH) [but not for human CG (hCG)], the experiments described here were designed to determine if the corresponding residue (D383) of the rat LH/CG receptor also mediates this Na+ effect. We used site-directed mutagenesis to create an LH/CG receptor mutant in which D383 was substituted by N. The wild type and mutant receptor [designated rLHR(D383N)] were expressed in human embryonic kidney 293 cells, and the transfected cells were tested for their ability to bind hCG and oLH in medium containing Na+ or an isoosmolar concentration of an appropriate sodium substitute. The results presented here show that this single point mutation of the LH/CG receptor leads to a slight reduction in affinity for hCG and oLH but completely abolishes the effects of Na+ removal on the affinity for oLH. Thus, regardless of the presence or absence of Na+, cells expressing rLHR(D383N) bind oLH with a low affinity comparable to that of the wild type receptor assayed in the presence of Na+. We also measured the ability of hCG and oLH to increase cAMP accumulation in cells expressing the wild type and mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.     

Fatty acylation of the rat and human asialoglycoprotein receptors. A conserved cytoplasmic cysteine residue is acylated in all receptor subunits

Functional rat or human asialoglycoprotein receptors (ASGP-Rs) are hetero-oligomeric integral membrane glycoproteins. Rat ASGP-R contains three subunits, designated rat hepatic lectins (RHL) 1, 2, and 3; human ASGP-R contains two subunits, HHL1 and HHL2. Both receptors are covalently modified by fatty acylation (Zeng, F.-Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387; Zeng, F.-Y., Oka, J. A., and Weigel, P. H. (1996) Biochem. Biophys. Res. Commun. 218, 325-330). We report here that the single Cys residue in the cytoplasmic domain of each RHL or HHL subunit is fatty acylated. The degree of acylation is >/=90% per subunit. Deacylation of affinity-purified ASGP-Rs with hydroxylamine results in the spontaneous formation of dimers through reversible disulfide bonds, indicating that deacylation concomitantly generates free thiol groups. Reaction of hydroxylamine-treated ASGP-R with [14C]iodoacetamide resulted in the specific incorporation of radioactivity into all RHL and HHL subunits, verifying that fatty acids are attached via thioester linkages. To identify the Cys residue involved in the thioester linkages, 14C-carboxyamidomethylated RHL subunits were separated by SDS-polyacrylamide gel electrophoresis and digested in-gel with trypsin, and the resulting peptides were separated by reverse-phase high performance liquid chromatography. Amino acid sequence of radioactive peptides revealed that Cys35 in RHL1 and Cys54 in RHL2 and RHL3 were radiolabeled and, therefore, are fatty acylation sites. Fatty acylation of HHL subunits was analyzed by site-directed mutagenesis. Metabolic labeling of Cos7 cells transfected with wild type HHL1 cDNA resulted in substantial incorporation of [3H]palmitate into purified HHL1. Incorporation of [3H]palmitate into a C36S mutant of HHL1 was negligible ( approximately 1%) compared with wild type. This result also shows that Cys57 within the transmembrane domain of HHL1 is not normally palmitoylated. We conclude that Cys35 in RHL1, Cys54 in RHL2 and RHL3, and Cys36 in HHL1 are fatty acylated. Cys57 in HHL1 and probably Cys56 in RHL1 are not palmitoylated.     

Integrin receptor signaling: the propagation of an alpha-helix model

Improper assessment and treatment of asthma attacks have been identified as causes of increased morbidity and mortality: several pneumological societies have therefore created and published guidelines for facilitating decision making and for preventing unnecessary failures of therapy. The objective of this study was to examine emergency department compliance with such guidelines in our hospital, comparing the performance of pneumologists and other specialists. We reviewed the records of 117 patients treated for acute asthma attacks in 1994 (87 women and 30 men, mean age 46 years); 37 patients were treated by pneumologists and 80 by other specialists. The two physician groups differed significantly with respect to initial assessment of severity, particularly in the recording of vital signs (p < 0.05) and in the examination of some signs such as the use of accessory musculature (38% versus 10%, for pneumologists and other specialists, respectively) or the presence of cyanosis (81% versus 55%). Other factors associated with risk of death were noted only occasionally. Peak flow meters were used with only 5 patients, all examined by pneumologists; on the other hand, arterial blood samples for gasometric measurements were taken from 97%, although only 24% met the criteria stipulated in the guidelines. Treatment evaluated against the guidelines was incorrect in 24%, with no significant differences between pneumologists and other specialists. We conclude that: 1) the emergency clinical assessment and treatment of patients presenting with acute asthma attack is inadequate for a large proportion of patients, as the recommendations of consensual guidelines are habitually ignored, and 2) although there are differences in the management of these patients by pneumologists and other emergency room specialists, the former do not generally do a better job of following the guidelines.     

A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst

tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.     

Surface of the olfactory region in man observed by scanning electron microscope

This report describes the surface of the regio olfactoria of a 25-year-old man as observed in the scanning electron-microscope. Corresponding to the distal ends of the receptor cells, the surface displays various planes: (a) Upper-most is a film of mucus of varying thickness. (b) The olfactory hairs, which are arranged closely together, mostly in a parallel fashion in layers, dip into this mucus film. The long, distal parts of the olfactory hairs are of a rather uniform shape and form, together with the mucus film, a single functional unit. (c) Now follows a layer consisting of the short parts of the olfactory hairs. They branch from the olfactory vesiculae, mostly in a stellar fashion (5-12 on one vesicula) and arch themselves, after a short course, into the mucus film. (d) There then follows the plane of the olfactory vesiculae, mostly bulbous, partly club- and pyramidal in shape. (e) After that comes the plane of the microvilli receptors, which have about 100 or more villi on one cell. (f) Right at the bottom is the apical surface of the supporting cells, different in size, showing microvilli in a regularly arranged pattern. Because of the various planes of the surface of the olfactory region and because of the close situation of the long, distal parts of the olfactory hairs and their rather uniform shape, one could assume, from observations in the scanning electron microscope, that the olfactory-active molecules react immediately with the functional unit of mucus film and long distal parts of the olfactory hairs, so that for the different sense of smell, a quite different structure of the olfactory-active molecules must be assumed as a different shape of the distal ends of the olfactory receptors.     

Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.     

Mutations of conserved cysteine residues in the CWLC motif of the oncoretrovirus SU protein affect maturation and translocation

The envelope glycoprotein complex is composed of two polypeptides, an external heavily glycosylated polypeptide (SU) and a membrane-spanning protein (TM). Together they form a heterodimer on the surface of the virion. These proteins are synthesized in the form of a polyprotein precursor which is glycosylated and proteolytically processed during its maturation in the secretory pathway. A highly conserved stretch of four amino acids, CWLC, has been identified in most known oncoretroviral SU proteins, about two-thirds of the distance from the amino terminus. To study the significance of this sequence for the structure and/or function of SU, cysteine to serine mutations were made in reticuloendotheliosis virus strain A. Initial studies showed that substitution of either one or both cysteines resulted in the production of noninfectious virus. Furthermore, immunoprecipitations and pulse-chase analysis demonstrated that the mutants yielded envelope polyprotein precursors which were stable. However, the polyprotein precursors were not proteolytically processed into SU and TM, and immunoprecipitations indicate that the immature polyproteins form aggregates, suggesting that the mutations interfere with proper folding. Although not proteolytically processed, at least one of the mutant glycoproteins appeared to be efficiently transported to the cell surface. These studies indicate that changing either cysteine residue abrogates viral infectivity by affecting folding, inhibiting normal maturation of the envelope glycoproteins.     

Citrate-67Ga scanning of the maxillofacial region

One hundred forty nine subjects have their echocardiography recorded by Echoview apparatus, Picker firm. The echocardiographic parameters found in 72 patients in pure or almost pure mitral stenosis were confronted to those of 77 healthy subjects. The high sensitivity of the echocardiographic method is stressed upon in the differentiation of mitral stenosis. The most important echocardiographic criterion of mitral stenosis is the slowed down speed of the early diastolic movement, EF of the anterior mitral cusp and the shifting of the posterior mitral cusp forward from the line, passing through the point of the mitral closing. Confirmed diagnosis of mitral stenosis could only be obtained by the combination of those two echocardiographic changes. Echocardiographic significance is stressed in the establishment of the thickness of the two mitral cusps, increased number of component echolines and amplitude decrease in anterior mitral cusp opening in the assessment of the presence of mitral fibrosclerosis and calcinosis.     

The cytoplasmic fragment of the aspartate receptor displays globally dynamic behavior

A number of cloned soluble fragments if the bacterial chemotaxis transmembrane receptors retain partial function. Prior studies of a fragment corresponding to the cytoplasmic domain (c-fragment) of the Escherichia coli aspartate receptor have correlated the signaling state of mutant receptors with the oligomerization state of the c-fragments: equilibria of smooth-swimming mutants are shifted toward oligomeric states; tumble mutants are shifted toward monomeric states [Long, D. G., & Weis, R. M. (1992) Biochemistry 31, 9904-9911]. We have applied several experimental probes of local and global structural flexibility to two signaling states, the wild-type (monomeric) and S461L smooth mutant (predominantly dimeric) c-fragments. Featureless near-UV CD spectra are observed, which indicate that the single Trp residue is in a symmetric environment (most likely averaged by fluctuations) and suggest that the C-termini of both proteins are highly mobile. Both proteins undergo extremely rapid proteolysis and enhance ANS fluorescence, which indicates that many sites are accessible to trypsin cleavage and hydrophobic sites are accessible to ANS binding. The global nature of the flexibility is demonstrated by 1H NMR studies. Lack of chemical shift dispersion suggests that fluctuations average the environments of side chains and backbone protons. Rapid exchange of 99% of the observable amide protons suggests that these fluctuations give high solvent accessibility to nearly the entire backbone. This evidence indicates that both monomeric and dimeric c-fragments are globally flexible proteins, with properties similar to "molten-globule" states. The significance of this flexibility depends on whether it is retained in functioning receptors: the c-fragment structure may lack important tertiary contacts, protein-protein interactions, or topological constraints needed to stabilize a nondynamic native structure, or the cytoplasmic domain of the native receptor may retain flexibility which may be modulated in the mechanism of transmembrane signaling.     

Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.     

Palmitoylated cysteine 341 modulates phosphorylation of the beta2-adrenergic receptor by the cAMP-dependent protein kinase

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.     

The pore-lining region of shaker voltage-gated potassium channels: comparison of beta-barrel and alpha-helix bundle models

Although there is a large body of site-directed mutagenesis data that identify the pore-lining sequence of the voltage-gated potassium channel, the structure of this region remains unknown. We have interpreted the available biochemical data as a set of topological and orientational restraints and employed these restraints to produce molecular models of the potassium channel pore region, H5. The H5 sequence has been modeled either as a tetramer of membrane-spanning beta-hairpins, thus producing an eight-stranded beta-barrel, or as a tetramer of incompletely membrane-spanning alpha-helical hairpins, thus producing an eight-staved alpha-helix bundle. In total, restraints-directed modeling has produced 40 different configurations of the beta-barrel model, each configuration comprising an ensemble of 20 structures, and 24 different configurations of the alpha-helix bundle model, each comprising an ensemble of 24 structures. Thus, over 1300 model structures for H5 have been generated. Configurations have been ranked on the basis of their predicted pore properties and on the extent of their agreement with the biochemical data. This ranking is employed to identify particular configurations of H5 that may be explored further as models of the pore-lining region of the voltage-gated potassium channel pore.