Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands. Our results indicate that Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.     

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.     

Fluorescence properties of a tryptophan residue in an aromatic core of the protein subunit of ribonuclease P from Escherichia coli

Escherichia coli ribonuclease P (RNase P), a ribonucleoprotein complex which primarily functions in tRNA biosynthesis, is composed of a catalytic RNA subunit, M1 RNA, and a protein cofactor, C5 protein. The fluorescence emission spectrum of the single tryptophan residue-containing C5 protein exhibits maxima at 318 nm and 332 nm. Based on a comparison of the emission spectra of wild-type C5 protein and some of its mutant derivatives, we have determined that the 318 nm maximum could be the result of a complex formed in the excited state as a result of hydrophobic interactions between Trp109, Phe18 and Phe73. The analogous tryptophan fluorescence emission spectra of wild-type C5 protein and the barstar mutant W38F/W44F, taken together with the detailed structural information available for barstar, provide a possible explanation for the unusual emission spectrum of C5 protein.     

Electrophysiological studies on oxindole, a neurodepressant tryptophan metabolite

1. The aim of the present work was to investigate the electrophysiological effects of oxindole, a tryptophan metabolite present in rat blood and brain, and recently proposed as a contributing factor in the pathogenesis of hepatic encephalopathy. 2. Using rat hippocampal slices in vitro and extra- or intracellular recordings, we evaluated oxindole effects on the neurotransmission of the CA1 region following orthodromic stimulation of the Schaffer collaterals. 3. Oxindole (0.3-3 mM) decreased the amplitude of population spikes extracellularly recorded at the somatic level and of the fEPSPs recorded at the dendritic level. In intracellular recordings, oxindole (0.1-3 mM) did not affect the resting membrane potential or the neuronal input resistance, but reduced the probability of firing action potentials upon either synaptic or direct activation of the pyramidal cells. 4. Oxindole (0.3-3 mM) increased the threshold and the latency of firing action potentials elicited by depolarizing steps without changing the duration or the peak amplitude of the spikes. It also significantly increased the spike frequency adaptation induced by long lasting (400 ms) depolarizing stimuli. 5. In separate experiments, performed by measuring AMPA or NMDA-induced responses in cortical slices, oxindole (1-3 mM) did not modify glutamate receptor agonist responses. 6. Our results show that concentrations of oxindole which may be reached in pathological conditions, significantly decrease neuronal excitability by modifying the threshold of action potential generation.     

On defining the rules for interactions between the T cell receptor and its ligand: a critical role for a specific amino acid residue of the T cell receptor beta chain

The specificity of T cell-mediated immune responses is primarily determined by the interaction between the T cell receptor (TCR) and the antigenic peptide presented by the major histocompatibility complex (MHC) molecules. To refine our understanding of interactions between the TCR and the antigenic peptide of vesicular stomatitis virus (VSV) presented by the class I MHC molecule H-2Kb, we constructed a TCR alpha chain transgenic mouse in a TCR alpha-deficient background to define specific structural features in the TCR beta chain that are important for the recognition of the VSV/H-2Kb complex. We found that for a given peptide, a peptide-specific, highly conserved amino acid could always be identified at position 98 of the complementarity-determining region 3 (CDR3) loop of TCR beta chains. Further, we demonstrated that substitutions at position 6, but not position 1, of the VSV peptide induced compensatory changes in the TCR in both the amino acid residue at position 98 and the length of the CDR3beta loop. We conclude that the amino acid residue at position 98 of the CDR3beta loop is a key residue that plays a critical role in determining the specificity of TCR-VSV/H-2Kb interactions and that a specific length of the CDR3beta loop is required to facilitate such interactions. Further, these findings suggest that the alpha and beta chains of TCRs interact with amino acid residue(s) toward the N and C termini of the VSV peptide, respectively, providing functional evidence for the orientation of a TCR with its peptide/MHC ligand as observed in the crystal structures of TCR/peptide/MHC complexes.     

A rapid and efficient purification method for recombinant annexin V for biophysical studies

Annexin V binds in a calcium-dependent manner to acidic phospholipids and exhibits ion channel activity in vitro. We are investigating mutants of annexin V by single channel measurements, X-ray crystallography and electron microscopy in order to understand the structure-function relationships of the ion channel activity. We describe here a method to obtain very pure recombinant annexin V required for such studies. The initial step is the mild opening of the bacterial cells by an osmotic shock. In the purification procedure, use is made of the reversible calcium-mediated binding of annexin V to liposomes. In the last purification step the protein is subjected to ion-exchange chromatography and elutes as a single peak free of any detectable contaminants.     

A possible role for acetylcholine in the dialogue between thymocytes and thymic stroma

In this article we will review data suggesting that acetylcholine takes part in the mutual interplay between developing T cells and thymic epithelium, and thereby may influence the generation of the T-cell repertoire. In the first part we will recapitulate our findings according to which cholinergic agonists affect thymocyte apoptosis via a nicotinergic effect on thymic epithelial cells. In the second part we will present evidence that acetylcholine within the thymus is mainly derived from the thymocytes themselves, and that the production and release of this neurotransmitter is dependent on activation of thymic lymphocytes.     

Neurotrophin-Trk receptor interactions in neoplasia: a possible role in interstitial and perineural invasion in ductal pancreatic cancer

Autocrine and paracrine influences of growth factors play a critical role in the regulation of the growth, survival, differentiation, and invasion potential of tumor cells. These influences on the neoplastic phenotype are particularly important in those cancers in which tumor-stroma interactions constitute important components of tumor development, as exemplified by prostatic, breast, and pancreatic carcinomas. The neurotrophins and their corresponding Trk and p75(NGFR) receptor subtypes are families of growth factors and receptors that have received relatively little attention with respect to neoplasia. This review attempts to summarize their biochemical properties, their role in neuronal and non-neuronal systems, and their involvement in the development of a variety of cancers, particularly those in which perineural invasion and/or metastasis to the CNS are a part of the pathophysiological presentation. In this regard, we have focused on our studies of neurotrophin-Trk/p75(NGFR) expression and interactions in pancreatic ductal carcinoma (PDAC) and their potential role in the perineural invasive phenotype characteristic of this cancer.     

CD44 plays a role in adhesive interactions between glioma cells and extracellular matrix components

To evaluate how individuals infected with the human immunodeficiency virus (HIV) became aware of their infection, when they first suspected they were infected with HIV and factors associated with suspecting HIV infection, we surveyed 227 patients at an urban outpatient HIV clinic. Though nearly all patients acknowledged risk factors for HIV, 60% reported that they did not suspect that they were infected until they received a positive HIV antibody test result. Non-white patients were less likely to suspect HIV infection prior to testing than white subjects (p < 0.03). Subjects not suspecting infection more often received HIV testing through a screening program or during a medical encounter (p = 0.02) and were less likely to be told by others that they might be infected (p = 0.001) than patients suspecting infection prior to testing. Forty-eight percent of subjects who suspected HIV infection prior to testing waited one year or more before obtaining their HIV antibody test. Interventions to reduce faulty personal HIV risk perception are needed to promote earlier HIV diagnosis.     

Dopamine inactivates tryptophan hydroxylase and forms a redox-cycling quinoprotein: possible endogenous toxin to serotonin neurons

Exposure of tryptophan hydroxylase (TPH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, to dopamine under mild oxidizing conditions (iron + H2O2) or in the presence of tyrosinase results in a concentration-dependent inactivation of the enzyme. Dopamine, iron, H2O2, or tyrosinase alone does not alter TPH activity. Similarly, N-acetyldopamine oxidized with one equivalent of sodium periodate causes a concentration-dependent inactivation of TPH as well. TPH is protected from dopamine-induced inactivation by reduced glutathione, ascorbic acid, and dithiothreitol but not by the radical scavengers DMSO, mannitol, or superoxide dismutase. Parallel studies with [3H]dopamine reveal a high negative correlation between inhibition of catalysis and incorporation of tritium into the enzyme. Those reducing agents and antioxidants that protect TPH from inactivation are effective in preventing the labeling of TPH by [3H]dopamine. Acid hydrolysis and HPLC with electrochemical detection (HPLC-EC) analysis of inactivated TPH revealed the formation of cysteinyl-dopamine residues within the enzyme. Exposure of dopamine-modified TPH to redox-cycling staining after SDS-PAGE confirmed the formation of a quinoprotein. These results indicate that dopamine-quinones covalently modify cysteinyl residues in TPH, leading directly to the loss of catalytic activity, and establish that TPH could be a target for dopamine-quinones in vivo after drugs (e.g., neurotoxic amphetamines) that cause dopamine-dependent inactivation of TPH. Redox cycling of a TPH-quinoprotein could also participate in the serotonin neuronal toxicity caused by these same drugs.     

Rapid depletion of plasma tryptophan: a review of studies and experimental methodology

The body mass is one of the major indicators which determine the clinical condition of newborns, influence the rate of neonatal mortality and further development of newborns. In order to prevent malformations it is necessary to identify factors which impair the fetus development and cause fetal hypotrophy. The main objective of the study was to find out whether mothers' occupational work affected the body mass of infants born at term. The study covered a group of 1015 women randomly sampled among those who had delivered their babies in the Polish Mother Memorial Hospital in Lód?, during the years 1992-94. In this group the percentage of working women accounted for 69%. The comparison of the body mass in infants born to women employed and not employed during pregnancy did not indicate differences. However, it was found that male infants born to employed mothers showed a lower body mass than those born to not employed mothers. The effect of some factors modifying infants' body mass was different in the case of not employed women than in those employed. In the group of not employed women a significantly lower body mass in infants was observed in the following subgroups: mothers under 24 years of age, spontaneous abortion of one of previous pregnancies, consumption of large quantity of caffeine (equivalent of > two cups of coffee), and poor economic status. Smoking during pregnancy decreased the body mass of infants in both groups of women. In the group of working women, chronic diseases before pregnancy and diseases involving fever during pregnancy proved to be factors affecting the body mass of infants. The indicators of perinatal medical care (the beginning of medical care and the number of visits during pregnancy) in working women were better than in those not working. None of factors characterizing occupational work affected significantly the body mass of infants. Slightly lower infants' body mass was observed only in those born to mothers working overtime (> 9 hrs daily), involved in hard physical work or working in the environment with harmful chemical and physical factors. According to the data obtained, a negative effect of occupational work on the fetus development should not be overestimated. Nevertheless, the performance of occupational work may aggravate a negative effect of woman's bad health condition on the fetus development.     

Incorporation of tryptophan analogues into staphylococcal nuclease, its V66W mutant, and Delta 137-149 fragment: spectroscopic studies

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been biosynthetically incorporated into Staphylococcal nuclease, its V66W mutant, and the Delta 137-149 fragment of the latter mutant. The guanidine-HCl induced unfolding and thermal unfolding of these proteins were studied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three proteins have tryptophan residues at positions 140 (in wild type) and 66 (in the Delta 137-149 fragment of V66W) and at both positions (in V66W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wild-type nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-Azatryptophan is found to destabilize all three proteins. 4-Fluorotryptophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.     

Mutagenesis of the conserved residue Glu259 of Gsalpha demonstrates the importance of interactions between switches 2 and 3 for activation

We previously reported that substitution of Arg258 within the switch 3 region of Gsalpha impaired activation and increased basal GDP release due to loss of an interaction between the helical and GTPase domains (Warner, D. R., Weng, G., Yu, S., Matalon, R., and Weinstein, L. S. (1998) J Biol. Chem. 273, 23976-23983). The adjacent residue (Glu259) is strictly conserved in G protein alpha-subunits and is predicted to be important in activation. To determine the importance of Glu259, this residue was mutated to Ala (Gsalpha-E259A), Gln (Gsalpha-E259Q), Asp (Gsalpha-E259D), or Val (Gsalpha-E259V), and the properties of in vitro translation products were examined. The Gsalpha-E259V was studied because this mutation was identified in a patient with Albright hereditary osteodystrophy. S49 cyc reconstitution assays demonstrated that Gsalpha-E259D stimulated adenylyl cyclase normally in the presence of GTPgammaS but was less efficient with isoproterenol or AlF4-. The other mutants had more severely impaired effector activation, particularly in response to AlF4-. In trypsin protection assays, GTPgammaS was a more effective activator than AlF4- for all mutants, with Gsalpha-E259D being the least severely impaired. For Gsalpha-E259D, the AlF4--induced activation defect was more pronounced at low Mg2+ concentrations. Gsalpha-E259D and Gsalpha-E259A purified from Escherichia coli had normal rates of GDP release (as assessed by the rate GTPgammaS binding). However, for both mutants, the ability of AlF4- to decrease the rate of GTPgammaS binding was impaired, suggesting that they bound AlF4- more poorly. GTPgammaS bound to purified Gsalpha-E259D irreversibly in the presence of 1 mM free Mg2+, but dissociated readily at micromolar concentrations. Sucrose density gradient analysis of in vitro translates demonstrated that all mutants except Gsalpha-E259V bind to beta gamma at 0 degreesC and were stable at higher temperatures. In the active conformation Glu259 interacts with conserved residues in the switch 2 region that are important in maintaining both the active state and AlF4- in the guanine nucleotide binding pocket. Although both Gsalpha Arg258 and Glu259 are critical for activation, the mechanisms by which these residues affect Gsalpha protein activation are distinct.