Selective nitration of Tyr99 in calmodulin as a marker of cellular conditions of oxidative stress

We examined the possible role of methionines as oxidant scavengers that prevent the peroxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. The possible role of methionines in scavenging peroxynitrite (ONOO-) was assessed in wild-type CaM and following substitution of all nine methionines in CaM with leucines. We find that peroxynitrite selectively nitrates Tyr99 at physiological pH, resulting in the formation of between 0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite per CaM was varied between 2.5 and 1.5. In wild-type CaM there is a corresponding oxidation of between 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent of nitration by peroxynitrite was unchanged. These results indicate that Tyr99 is readily nitrated by peroxynitrite and that methionine side chains do not function as an antioxidant in scavenging peroxynitrite. Thus, separate reactive species are involved in the oxidation of methionine and nitration of Tyr99 whose relative concentrations are determined by solution conditions. The sensitivity of Tyr99 in CaM to nitration suggests that CaM-dependent signaling pathways are sensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker of peroxynitrite-induced changes in cellular function.     

Dissociation of protein kinase-mediated regulation of metabotropic glutamate receptor 7 (mGluR7) interactions with calmodulin and regulation of mGluR7 function

Presynaptic metabotropic glutamate receptors (mGluRs) often act as feedback inhibitors of synaptic transmission and serve important roles in defining the activity of glutamatergic synapses. Recent investigations have begun to identify novel interactions of presynaptic mGluRs, especially mGluR7, with multiple protein kinases and putative regulatory proteins that probably serve to further shape the overall activity of glutamatergic synapses. In the present study, we report that in addition to protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) can inhibit calmodulin (CaM) interactions with the carboxyl-terminal tail of mGluR7. These actions are mediated by PKC-, PKA-, or PKG-dependent phosphorylation of mGluR7 at a single serine residue, Ser(862), in the carboxyl terminus of the receptor. Mutation of this residue inhibits kinase-mediated phosphorylation of the mGluR7 carboxyl terminus and reverses kinase-mediated inhibition of CaM binding to mGluR7. However, PKC-mediated inhibition of the functional coupling of mGluR7 to G protein-coupled inward rectifier potassium (GIRK) currents in a heterologous expression system is not affected by mutating Ser(862). Furthermore, mutation of Ser(862) to glutamate to mimic receptor phosphorylation and inhibit CaM interactions with mGluR7 does not affect receptor function. These studies demonstrate that the ability of these second messenger-dependent kinases to inhibit mGluR7-mediated activation of GIRK current is not dependent on the phosphorylation of Ser(862) or the regulation of CaM binding to mGluR7. Furthermore, our studies suggest that CaM binding is not required for mGluR7-mediated activation of GIRK current.     

The Kinetics of Relaxin Oxidation by Hydrogen Peroxide

In this study, hydrogen peroxide was used to study the oxidation of rhRlx under various conditions. Oxidation of rhRlx occurred at both of the two methionines on the B chain, Met B(4) and Met B(25), as expected from the three-dimensional structure of the molecule, which shows that these two residues are located on the surface of the molecule and exposed to solvent. The reaction produced three different oxidized forms of rhRlx containing either Met B(4) sulfoxide, Met B(25) sulfoxide, or both residues oxidized. The corresponding sulfone was not formed under these conditions. The oxidation at the two methionines proceeded independently from each other but Met B(25) was oxidized at a significantly faster rate than Met B(4). The fact that the rate of oxidation at Met B(25) was identical to the rate of oxidation of free methionine and that of two model peptides mimicking the residues around Met B(4) and Met B(25) suggests that the lower reactivity at Met B(4) was due to steric hindrance, and at least in this case, neighboring groups do not influence the oxidation kinetics of methionine residues. The reaction was independent of pH, ionic strength, and buffer concentration in the range studied. The enthalpy of activation for the reaction was approximately 10–14 kcal mol^–1, with an entropy of activation of the order of –30 cal K^–1 mol^–1. These data are consistent with previously published mechanisms for organic sulfide oxidation by alkyl hydroperoxides.     

Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca²⁺-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.     

Evaluation of recombinant human interferon alpha-2b structure and stability by in-gel tryptic digestion, H/D exchange and mass spectrometry

Stability and structure of recombinant interferon alpha-2b (rHuINF alpha-2b) was studied by mass spectrometry (MALDI-TOF and Q-TOF MS), chromatography (LC-UV-FLD-DAD, LC-MS) and CD spectroscopy. Besides analysis of the substance according to Ph. Eur. methods, two additional mass spectrometric methods were developed. The aim of both methods was to estimate structure-stability relationship connected to methionine oxidation or protein degradation. Preservation or degradation of protein structure was confirmed by H/D exchange in four separate experiments. Kinetics of deuterium incorporation into macromolecule was monitored over 2670 min. Isoforms of rHuINF alpha-2b were separated by 2D gel electrophoresis. In-gel digestion with trypsin and mass spectrometric analysis, performed on four separated isoforms at the mass corresponding to the mass of rHuINF alpha-2b with oxidized methionines, confirmed oxidation of all methionines to a different extent. Another four isoforms observed in 2D gel are most likely dimers of the same macromolecules with scrambled disulphide bridges. Oxidation and dimerisation are consequences of protein interaction with oxidizing reagents in polyacrilamide gel.     

Differential regulation of prostaglandin F(2alpha) receptor isoforms by protein kinase C

Prostaglandin F(2alpha) receptors (FP) are G protein-coupled receptors that bind prostaglandin F(2alpha) (PGF(2alpha)), resulting in the activation of an inositol phosphate (IP) second messenger pathway. Alternative mRNA splicing generates two FP receptor isoforms. These isoforms, designated FP(A) and FP(B), are otherwise identical except for their carboxyl termini. FP(B) is essentially a truncated version of FP(A) that lacks the 46 carboxyl-terminal amino acids, including four putative protein kinase C (PKC) phosphorylation sites. Until now, functional differences between these FP receptor isoforms have not been identified. We now report that pretreatment with the PKC inhibitor bisindolylmaleimide I enhanced PGF(2alpha)-stimulated IP accumulation in transfected cells stably expressing the FP(A) isoform but not in cells stably expressing the FP(B) isoform. Whole-cell phosphorylation experiments showed a strong agonist-dependent phosphorylation of the FP(A) isoform but little or no phosphorylation of the FP(B). Pretreatment of cells with bisindolylmaleimide I decreased PGF(2alpha)-stimulated phosphorylation of the FP(A) isoform consistent with a PKC-dependent phosphorylation. In vitro phosphorylation of an FP(A) carboxyl-terminal fusion protein by recombinant PKCalpha showed that the carboxyl terminus of the FP(A) is a substrate for PKC. These results suggest that PKC-dependent phosphorylation is responsible for differential regulation of second messenger signaling by FP prostanoid receptor isoforms.     

Oxidative Degradation of Antiflammin 2

Purpose. To study the oxidation of the methionine residue of antiflammin 2 (HDMNKVLDL, AF2) as a function of pH, buffer concentration, ionic strength, and temperature using different concentrations of hydrogen peroxide and to determine the accessibility of methionine residue to oxidation. Methods. Reversed-phase high-performance liquid chromatography (RPHPLC) was used as the main analytical method in determining the oxidation rates of AF2. Calibration curves for AF2 and the oxidation product, methionine sulfoxide of AF2 (Met(O)-3-AF2), were constructed for each measurement using standard materials. Fast Atom Bombardment Mass Spectroscopy (FABMS) was used to characterize the product. Results. Met(O)-3-AF2 was the only oxidation product detected at pH 3.0 to 8.0. The oxidation rates were independent of buffer concentrations, ionic strength, and pH from 3.0 to 7.0. However, there was an acceleration of the rates at basic pHs, and small amounts of degradation products other than Met(O)-3-AF2 were observed in this alkaline region. Conclusions. Oxidation of methionine in AF2 does not cause the biological inactivation reported by other laboratories since this drug is relatively stable under neutral conditions in the absence of oxiding agent.     

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3‘-BROMOINDOLEMINE

SELECTIVE CLEAVAGE AT THE SINGLE TRYPTOPHAN RESIDUE IN BOVINE SOMATOTROPIN BY 2-(2-NITROPHENYLSULFENYL)-3-METHYL-3′-BROMOINDOLEMINE The single tryptophan residue at position 86 in bovine somatotropin has been cleaved by the use of 2-(2-nitrophenylsulfenyl)-3-methyl-3′-bromoindolemine. This reaction was carried out with native bovine growth hormone as well as its reduced and alkylated derivative. When performed on the intact hormone, followed by the reduction and alkylation of the disulfides to liberate the two fragments, the cleavage reaction yielded the fragment containing residues 87–191 in a monomeric state. The other fragment, containing residues 1–86, could only be obtained in a highly aggregated form admixed with other material. When the cleavage reaction was carried out on the reduced and alkylated hormone, both the N-terminal fragment of reduced-carbamidomethylated bovine somatotropin as a monomer and the C-terminal fragment as a dimer were obtained in 35–40% yield. Since the cleavage reaction also oxidized methionine to methionine sulfoxide, the sulfoxides were reduced back to the methionine with N-methylmercapto acetamide. In this manner the following new derivatives and fragments of bovine somatotropin have been prepared: completely reduced and alkylated hormone, reduced and alkylated hormone with its methionines and tryptophan oxidized, reduced and alkylated hormone with its tryptophan oxidized, the N-terminal fragment of the hormone containing residues 1–86 with its methionine and tryptophan oxidized, the C-terminal fragment containing residues 87–191, both with its methionines oxidized and in their native state. The chemical and physical properties of these derivatives and fragments have been determined. Biological activity in the rat tibia was found to be negligible for all derivatives and fragments.     

Synthesis of Met‐enkephalin by solution‐phase peptide synthesis methodology utilizing para‐toluene sulfonic acid as N‐terminal masking of l‐methionine amino acid

The Met‐enkephalin, Tyr‐Gly‐Gly‐Phe‐Met, was synthesized by the solution‐phase synthesis (SPS) methodology employing ‐OBzl group as carboxyls' protection, while the t‐Boc groups were employed for the N‐terminal α‐amines' protection for the majority of the amino acids of the pentapeptide sequence. The l ‐methionine (l ‐Met) amino acid was used as PTSA.Met‐OBzl obtained from the simultaneous protection of the α‐amino, and carboxyl group with para‐toluene sulfonic acid (PTSA) and as‐OBzl ester, respectively in a C‐terminal start of the 2 + 2 + 1 fragments condensation convergent synthetic approach. The protection strategy provided a short, single‐step, simultaneous, orthogonal, nearly quantitative, robust, and stable process to carry through the protected l ‐methionine and l ‐phenylalanine coupling without any structural deformities during coupling and workups. The structurally confirmed final pentapeptide product was feasibly obtained in good yields through the current approach.     

Calmodulin Binding to Peptides Derived from the i3 Loop of Muscarinic Receptors

Purpose This study was conducted to identify and characterize the structural requirements of a calmodulin-binding motif identified in the third intracellular (i3) loop of muscarinic acetylcholine receptors (M1–M5), a region important for G protein coupling. Methods GST fusion proteins and synthetic peptides derived from the hM1 i3 loop were tested for binding to CaM using a cross-linking gel shift assay and a dansyl-CaM fluorescence assay. Mutagenesis studies further characterized the structural requirements for the interaction and identified critical residues. Results 28-Mer peptides from the C terminus of i3, representing the putative calmodulin domains of M1, M2, and M3, were found capable of interacting with CaM. In addition, smaller peptides defined a 5-amino-acid sequence essential for calmodulin binding. Studies performed with M1 peptides derived from GST fusion proteins, representing larger portions of the i3 C terminus, suggested the presence of a second adjacent CaM binding site. Mutagenesis studies identified two mutants that are unable to bind CaM: a point mutation, E360A, and a deletion mutant, Δ232–358. Conclusion Calmodulin can bind to an M1 region implicated in G protein coupling. This indicates an important role for CaM in the regulation of muscarinic signal transduction.     

Calmodulin antagonists' binding sites on calmodulin

Troponin I inhibited, concentration-dependently, [3H]-N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and [3H]-trifluoperazine (TFP) binding to purified bovine brain calmodulin (CaM). Selective oxidation of methionine residues of CaM by N-chlorosuccinimide resulted in a rapid decrease in [3H]-W-7, [3H]-TFP and [14C]-chlorpromazine binding concomitant with the loss of CaM activity. Carbethoxylation of histidine residues, nitration of tyrosine residues and chemical modification of arginine residues with 1,2-cyclohexanedione produced no significant changes either in [3H]-W-7 binding to CaM or in the ability of CaM to stimulate phosphodiesterase. Our results suggest that the binding sites of these CaM antagonists on CaM may be located between the second and third Ca2+-binding loops.     

Biosynthesis of antibiotic 1233A (F-244) and preparation of [14C]1233A.

The biosynthesis of antibiotic 1233A (F-244) was studied by feeding 13C-labeled precursors to the producing organism, Scopulariopsis sp. F-244. 13C NMR spectroscopy established that 1233A is derived from 4 methionines and 7 acetates. Seven acetates are condensed to form a hexaketide and 4 methyl residues from methionine are introduced into the main skeleton. The beta-lactone is derived from the alpha-carboxylic acid of the hexaketide. Since methionine was efficiently incorporated into 1233A, radiolabeled 1233A was prepared biosynthetically by feeding [14C]methionine to the producer. As a result, [14C]1233A was obtained with high specific radioactivity (27.2 muCi/mumols).     

A tail of two signals: the C terminus of the A(2A)-adenosine receptor recruits alternative signaling pathways

G protein-coupled receptors are endowed with carboxyl termini that vary greatly in length and sequence. In most instances, the distal portion of the C terminus is dispensable for G protein coupling. This is also true for the A(2A)-adenosine receptor, where the last 100 amino acids are of very modest relevance to G(s) coupling. The C terminus was originally viewed mainly as the docking site for regulatory proteins of the beta-arrestin family. These beta-arrestins bind to residues that have been phosphorylated by specialized kinases (G protein-coupled receptor kinases) and thereby initiate receptor desensitization and endocytosis. More recently, it has become clear that many additional "accessory" proteins bind to C termini of G protein-coupled receptors. The article by Sun et al. in the current issue of Molecular Pharmacology identifies translin-associated protein-X as yet another interaction partner of the A(2A) receptor; translin-associated protein allows the A(2A) receptor to impinge on the signaling mechanisms by which p53 regulates neuronal differentiation, but the underlying signaling pathways are uncharted territory. With a list of five known interaction partners, the C terminus of the A(2A) receptor becomes a crowded place. Hence, there must be rules that regulate the interaction. This allows the C terminus to act as coincidence detector and as signal integrator. Despite our ignorance about the precise mechanisms, the article has exciting implications: the gene encoding for translin-associated protein-X maps to a locus implicated in some forms of schizophrenia; A(2A) receptor agonists are candidate drugs for the treatment of schizophrenic symptoms. It is of obvious interest to explore a possible link.     

Comparison Between Light Induced and Chemically Induced Oxidation of rhVEGF

Purpose. The primary objective of this study was to compare the effects of light-and chemical-induced oxidation of recombinant human vascular endothelial growth factor (rhVEGF) and the impact of these reactions on protein formulation. Methods. A liquid formulation of rhVEGF was exposed to fluorescent light (2 × 10⁴ lux for up to 4 weeks), hydrogen peroxide (H₂O₂), or t-butylhydroperoxide (t-BHP) to induce oxidation of rhVEGF. All samples were then treated by tryptic digest and analyzed by reversed phase HPLC to determine the extent of oxidation. Chemically treated samples were also examined by near-UV and far-UV circular dichroism spectroscopy to determine the effect of oxidation on the structure of the protein. Results. Exposure to light for 2 weeks resulted in 8 to 40% oxidation of all 6 methionine residues of rhVEGF (Met3 > Met18 > Met55 > Met78,81 > Met94). This amount of oxidation did not affect the binding activity of rhVEGF to its kinase domain receptor (KDR). Light exposure for 4 weeks increased metsulfoxide formation at Met3 and Met18 by an additional 16%, but did not affect the other residues. This oxidation decreased the receptor binding capacity to 73%, possibly due to the role of Met18 in receptor binding. Chemical oxidation of rhVEGF resulted in a greater extent of oxidation at all 6 methionines. Complete oxidation of Met3, Met18 and Met55 was observed after treatment with H₂O₂, while these residues underwent 40 to 60% oxidation after treatment with t-BHP. The receptor binding capacity was significantly reduced to 25% and 55% after treatment with H₂O₂ and t-BHP, respectively. After chemical oxidation, no changes in the secondary or tertiary structure were observed by far-UV and near-UV CD spectroscopy, respectively. Conclusions. Methionine residues with exposed surface areas greater than 65 Ų and sulfur surface areas greater than 16 Ų were most susceptible to oxidation. Chemical oxidation resulted in higher metsulfoxide formation and decreased binding activity of the protein to KDR than light-induced oxidation. The reduction in KDR binding was not caused by measurable conformational changes in the protein. Photooxidation was dependent on the amount of energy imparted to the protein, while the ability of t-BHP or H₂O₂ to react with methionine was governed by solvent accessibility of the methionine residues and steric limitations of the oxidizing agent. Significant chemical oxidation occurred on sulfurs with minimum surface areas of 16 Ų, while increased photooxidation occurred as a function of increasing surface areas of solvent exposed sulfur atoms. Such differences in the extent of oxidation should be considered during protein formulation since it may help predict potential oxidation problems.     

THE INFLUENCE OF ESTERIFICATION OF CARBOXYL GROUPS OF RIBONUCLEASE-A ON ITS STRUCTURE AND IMMUNOLOGICAL ACTIVITY

The effect of modification of carboxyl groups of Ribonuclease-A^a on the enzymatic activity and the antigenic structure of the protein has been studied. Modification of four of the eleven free carboxyl groups of the protein by esterification in anhydrous methanol/0.1 M hydrochloric acid resulted in nearly 80% loss in enzymatic activity but had very little influence on the antigenic structure of the protein. Further increases in the modification of the carboxyl groups caused a progressive loss in immunological activity, and the fully methylated RNase-A exhibited nearly 30% immunological activity. Concomitant with this change in the antigenic structure of the protein, the ability of the molecule to complement with RNase-S-protein increased, clearly indicating the unfolding of the peptide “tail” from the remainder of the molecule. The susceptibility to proteolysis, accessibility of methionine residues for orthobenzoquinone reaction and the loss in immunological activity of the more extensively esterified derivatives of RNase-A are suggestive of the more flexible conformation of these derivatives as compared with the compact native conformation. The fact that even the fully methylated RNase-A retains nearly 30% of its immunological activity suggested that the modified protein contained antibody recognizable residual native structure, which presumably accommodates some antigenic determinants.     

Structure and immunological properties of thymosin β9 Met,a new analog of thymosin β4 isolated from porcine thymus

During the course for the studies of thymosin β₄ and prothymosin α from porcine thymus, a new analog of thymosin β₄ has been identified. This peptide consists of 41 amino acid residues. The amino terminus is blocked by an acetyl group as revealed by fast atom bombardment mass spectrometric analysis. Amino acid sequence studies disclosed that this peptide is identical to bovine thymosin β₉ except that leucine at position 6 in β₉ is substituted by methionine. Thus, this new peptide has been termed thymosin β₉ Met. The recoveries of β₉Met, β₄, and prothymosin α in porcine tissues have been determined (in μg/g tissue) as follows: thymus (43, 85, 133); spleen (68, 203, 37); liver (10, 31, 27); heart (1.5, 10, 0); kidney (5, 51, 37); brain (0.8, 31, 5). Biologically, thymosin β₉ Met was found to be more active than β₄ in enhancing γ-interferon production in cord blood lymphocytes. However, β₄ appeared to stimulate higher amounts of interleukin 2 and tumor necrotic factor. The significance for the coexistence of two homologous peptides with similar functions in the thymus and a number of other organs is not clear, and deserves further investigation.     

Chemical Pathways of Peptide Degradation. V. Ascorbic Acid Promotes Rather than Inhibits the Oxidation of Methionine to Methionine Sulfoxide in Small Model Peptides

The effect of primary structure and external conditions on the oxidation of methionine to methionine sulfoxide by the ascorbate/Fe³⁺ system was studied in small model peptides. Degradation kinetics and yield of sulfoxide formation were dependent on the concentration of ascorbate and H⁺, with a maximum rate observed at pH 6–7. Phosphate buffer significantly accelerated the peptide degradation compared to Tris, HEPES, and MOPS buffers; however, the formation of sulfoxide was low. The oxidation could not be inhibited by the addition of EDTA. Other side products besides sulfoxide were observed, indicating the existence of various other pathways. The influence of methionine location at the C terminus, at the N terminus, and in the middle of the sequence was investigated. The presence of histidine in the sequence markedly increased the degradation rate as well as the sulfoxide production. The histidine catalysis of methionine oxidation occurred intramolecularly with a maximum enhancement of the oxidation rate and sulfoxide production when one residue was placed between the histidine and the methionine residue.     

Cloning and expression of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2

The human liver cDNA clone UDPGTh2, encoding a liver UDP-glucuronosyltransferase (UDPGT) was isolated from a λ gt 11 cDNA library by hybridization to mouse transferase cDNA clone, UDPGTm1. UDPGTh2 encoded a 529 amino acid protein with an amino terminus membrane-insertion signal peptide and a carboxyl terminus membrane-spanning region. There were three potential asparagine-linked glycosylation sites at residues 67, 68, and 315. In order to obtain UDPGTh2 protein encoded from cloned human liver UDP-glucuronosyltransferase cDNA, the clone was inserted into the pSVL vector (pUDPGTh2) and expressed in COS 1 cells. The presence of a transferase with Mr≈52,000 in transfected cells cultured in the presence of [³⁵S]methionine was shown by immunocomplexed products with goat antimouse transferase IgG and protein A-Sepharose and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The expressed UDPGT was a glycoprotein as indicated by electrophoretic mobility shift in Mr≈3,000–4,000 when expressed in the presence of tunicamycin. The extent of glycosylation was difficult to assess, although one could assume that glycosyl structures incorporated at the level of endoplasmic reticulum were always the core oligosaccharides. Thus, it is likely that at least two moieties inserted can account for the shift of Mr≈3,000–4,000. This study demonstrates the cDNA and deduced amino acid sequence of human liver UDP-glucuronosyltransferase cDNA, UDPGTh2.