N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.

The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

Recycling of the high valence states of heme proteins by cysteine residues of thimet-oligopeptidase

The peptidolytic enzyme THIMET-oligopeptidase (TOP) is able to act as a reducing agent in the peroxidase cycle of myoglobin (Mb) and horseradish peroxidase (HRP). The TOP-promoted recycling of the high valence states of the peroxidases to the respective resting form was accompanied by a significant decrease in the thiol content of the peptidolytic enzyme. EPR (electron paramagnetic resonance) analysis using DBNBS spin trapping revealed that TOP also prevented the formation of tryptophanyl radical in Mb challenged by H2O2. The oxidation of TOP thiol groups by peroxidases did not promote the inactivating oligomerization observed in the oxidation promoted by the enzyme aging. These findings are discussed towards a possible occurrence of these reactions in cells.

Machine learning methods for prediction of disulphide bonding states of cysteine residues in proteins

The goal of this thesis work is to develop a computational method based on machine learning techniques for predicting disulfide-bonding states of cysteine residues in proteins, which is a sub-problem of a bigger and yet unsolved problem of protein structure prediction. Improvement in the prediction of disulfide bonding states of cysteine residues will help in putting a constraint in the three dimensional (3D) space of the respective protein structure, and thus will eventually help in the prediction of 3D structure of proteins. Results of this work will have direct implications in site-directed mutational studies of proteins, proteins engineering and the problem of protein folding. We have used a combination of Artificial Neural Network (ANN) and Hidden Markov Model (HMM), the so-called Hidden Neural Network (HNN) as a machine learning technique to develop our prediction method. By using different global and local features of proteins (specifically profiles, parity of cysteine residues, average cysteine conservation, correlated mutation, sub-cellular localization, and signal peptide) as inputs and considering Eukaryotes and Prokaryotes separately we have reached to a remarkable accuracy of 94% on cysteine basis for both Eukaryotic and Prokaryotic datasets, and an accuracy of 90% and 93% on protein basis for Eukaryotic dataset and Prokaryotic dataset respectively. These accuracies are best so far ever reached by any existing prediction methods, and thus our prediction method has outperformed all the previously developed approaches and therefore is more reliable. Most interesting part of this thesis work is the differences in the prediction performances of Eukaryotes and Prokaryotes at the basic level of input coding when ‘profile’ information was given as input to our prediction method. And one of the reasons for this we discover is the difference in the amino acid composition of the local environment of bonded and free cysteine residues in Eukaryotes and Prokaryotes. Eukaryotic bonded cysteine examples have a ‘symmetric-cysteine-rich’ environment, where as Prokaryotic bonded examples lack it.

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts

A family of RNA m5C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m5C 967 MTase, Fmu, as an initial probe. The RNA m5C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m5C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m5U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m5C MTase uses a different Cys as a catalytic nucleophile than the DNA m5C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m5C MTases remains unknown.

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Ubiquitination and cysteine nitrosylation during aging and Alzheimer's disease.

Protein oxidation and ubiquitination of brain proteins are part of mechanisms that modulate protein function or that inactivate proteins and target misfolded proteins to degradation. In this study, we focused on brain aging and on mechanism involved in neurodegeneration such as events occurring in Alzheimer's disease (AD). The goal was to identify differences in nitrosylated proteins - at cysteine residues, and in the composition of ubiquinated proteins between aging and Alzheimer's samples by using a proteomic approach. A polyclonal anti-S-nitrosyl-cysteine, a mono- and a polyclonal anti-ubiquitin antibody were used for the detection of modified or ubiquitinated proteins in middle-aged and aged human entorhinal autopsy brains tissues of 14 subjects without neurological signs and 8 Alzheimer's patients. Proteins were separated by one- and two-dimensional gel electrophoresis and analyzed by Coomassie blue and immuno-blot staining. We identified that the glial fibrillary acidic and tau proteins are more ubiquitinated in brain tissues of Alzheimer's patients. Furthermore, glial fibrillary proteins were also found in nitrosylated state and further characterized by 2D Western blots and identified. Since reactive astrocytes localized prominently around senile plaques one can speculate that elements of plaques such as beta-amyloid proteins may activate surrounding glial elements and proteins.

Methionine residues as endogenous antioxidants in proteins

Cysteine and methionine are the two sulfur-containing residues normally found in proteins. Cysteine residues function in the catalytic cycle of many enzymes, and they can form disulfide bonds that contribute to protein structure. In contrast, the specific functions of methionine residues are not known. We propose that methionine residues constitute an important antioxidant defense mechanism. A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, available as an efficient oxidant scavenger. Reduction back to methionine by methionine sulfoxide reductases would allow the antioxidant system to function catalytically. The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system. Eight of the 16 methionine residues could be oxidized with little effect on catalytic activity of the enzyme. The oxidizable methionine residues were found to be relatively surface exposed, whereas the intact residues were generally buried within the core of the protein. Furthermore, the susceptible residues were physically arranged in an array that guarded the entrance to the active site.

Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

Resolution and characterisation of multiple isoforms of bovine kappa-casein by 2-DE following a reversible cysteine-tagging enrichment strategy

Visualisation of multiple isoforms of kappa-casein on 2-D gels is restricted by the abundant alpha- and beta-caseins that not only limit gel loading but also migrate to similar regions as the more acidic kappa-casein isoforms. To overcome this problem, we took advantage of the absence of cysteine residues in alpha(S1)- and beta-casein by devising an affinity enrichment procedure based on reversible biotinylation of cysteine residues. Affinity capture of cysteine-containing proteins on avidin allowed the removal of the vast majority of alpha(S1)- and beta-casein, and on subsequent 2-D gel analysis 16 gel spots were identified as kappa-casein by PMF. Further analysis of the C-terminal tryptic peptide along with structural predictions based on mobility on the 2-D gel allowed us to assign identities to each spot in terms of genetic variant (A or B), phosphorylation status (1, 2 or 3) and glycosylation status (from 0 to 6). Eight isoforms of the A and B variants with the same PTMs were observed. When the casein fraction of milk from a single cow, homozygous for the B variant of kappa-casein, was used as the starting material, 17 isoforms from 13 gel spots were characterised. Analysis of isoforms of low abundance proved challenging due to the low amount of material that could be extracted from the gels as well as the lability of the PTMs during MS analysis. However, we were able to identify a previously unrecognised site, T-166, that could be phosphorylated or glycosylated. Despite many decades of analysis of milk proteins, the reasons for this high level of heterogeneity are still not clear.

S-nitrosothiols Regulate Nitric Oxide Production And Storage In Plants Through The Nitrogen Assimilation Pathway.

Nitrogen assimilation plays a vital role in plant metabolism. Assimilation of nitrate, the primary source of nitrogen in soil, is linked to the generation of the redox signal nitric oxide (NO). An important mechanism by which NO regulates plant development and stress responses is through S-nitrosylation, that is, covalent attachment of NO to cysteine residues to form S-nitrosothiols (SNO). Despite the importance of nitrogen assimilation and NO signalling, it remains largely unknown how these pathways are interconnected. Here we show that SNO signalling suppresses both nitrate uptake and reduction by transporters and reductases, respectively, to fine tune nitrate homeostasis. Moreover, NO derived from nitrate assimilation suppresses the redox enzyme S-nitrosoglutathione Reductase 1 (GSNOR1) by S-nitrosylation, preventing scavenging of S-nitrosoglutathione, a major cellular bio-reservoir of NO. Hence, our data demonstrates that (S)NO controls its own generation and scavenging by modulating nitrate assimilation and GSNOR1 activity.

Bp-13 Pla2: Purification And Neuromuscular Activity Of A New Asp49 Toxin Isolated From Bothrops Pauloensis Snake Venom.

A new PLA2 (Bp-13) was purified from Bothrops pauloensis snake venom after a single chromatographic step of RP-HPLC on μ-Bondapak C-18. Amino acid analysis showed a high content of hydrophobic and basic amino acids and 14 half-cysteine residues. The N-terminal sequence showed a high degree of homology with basic Asp49 PLA2 myotoxins from other Bothrops venoms. Bp-13 showed allosteric enzymatic behavior and maximal activity at pH 8.1, 36°-45°C. Full Bp-13 PLA2 activity required Ca(2+); its PLA2 activity was inhibited by Mg(2+), Mn(2+), Sr(2+), and Cd(2+) in the presence and absence of 1 mM Ca(2+). In the mouse phrenic nerve-diaphragm (PND) preparation, the time for 50% paralysis was concentration-dependent (P < 0.05). Both the replacement of Ca(2+) by Sr(2+) and temperature lowering (24°C) inhibited the Bp-13 PLA2-induced twitch-tension blockade. Bp-13 PLA2 inhibited the contractile response to direct electrical stimulation in curarized mouse PND preparation corroborating its contracture effect. In biventer cervicis preparations, Bp-13 induced irreversible twitch-tension blockade and the KCl evoked contracture was partially, but significantly, inhibited (P > 0.05). The main effect of this new Asp49 PLA2 of Bothrops pauloensis venom is on muscle fiber sarcolemma, with avian preparation being less responsive than rodent preparation. The study enhances biochemical and pharmacological characterization of B. pauloensis venom.