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PubMed2003

Methanethiosulfonate-modification alters local anesthetic block in rNav1.4 cysteine-substituted mutants S1276C and L1280C.

We previously showed that lysine substitutions at two residues in segment 6 of domain 3 in voltage-gated Na(+) channel rNav1.4 (S1276K, L1280K) reduced steady-state inactivated local anesthetic block. Here we studied cysteine substitutions at the same residues (S1276C, L1280C). We used whole-cell recordings to determine local anesthetic block (100 microM bupivacaine) before and after cysteine modification with 1.5 mM 2-aminoethyl methanethiosulfonate (MTSEA). Compared with rNav1.4, steady-state resting bupivacaine block at -180 mV was increased in S1276C, while inactivated block at -50 mV was not different in the mutants. After application of MTSEA at -160 mV, rNav1.4 showed enhanced bupivacaine block and a negative shift in V(1/2) of the bupivacaine affinity curve, while L1280C and S1276C showed a decrease in inactivated bupivacaine block after MTSEA. Application of MTSEA at 0 mV produced similar results in rNav1.4 and L1280C, but an opposite effect in S1276C, i.e., enhancement of bupivacaine block, with a large negative shift in V(1/2) of the bupivacaine affinity curve similar to that found in rNav1.4. We conclude that 1) MTSEA modification of 1276C or 1280C decreases inactivated bupivacaine block similar to that found in L1280K and S1276K, 2) residue 1276C is only accessible to MTS-modification in the resting state, and 3) MTSEA may modify a native cysteine in rNav1.4 that produces an allosteric, indirect effect on bupivacaine affinity.

Anesthetics, Local↗

PubMed2004

Redox events in interleukin-1 signaling.

There is increasing evidence that reactive oxygen species (ROS) are mediators in growth factor and cytokine signaling pathways. Mechanisms by which ROS can interfere with signaling cascades may include regulation of protein activities by the modification of essential cysteines. Modification can be performed chemically or enzyme-catalyzed. Enzymes catalyzing a reversible thiol modification within proteins are to be able to react with both, ROS and protein thiols. If hydroperoxides are involved, promising candidates are peroxiredoxins and glutathione peroxidases (GPx), especially the phospholipid hydroperoxide GPx. Interleukin-1, one of the key players in inflammatory response, stimulates the production of ROS itself, but its signaling cascade can also be influenced by ROS and by thiol modifying agents. Targets are located in early, intermediate, and late events in the signaling cascade. We here summarize what is known about the effects of thiol modifying agents, selenium and glutathione peroxidases, on the assembly of the IL-1 receptor signaling complex as an early event, on the activation of NF-kappa B as an intermediate event, and on the expression of cell adhesion molecules as a late event in IL-1 signaling.

Animals↗

PubMed2002

Transport function of the renal type IIa Na+/P(i) cotransporter is codetermined by residues in two opposing linker regions.

Two highly similar regions in the predicted first intracellular (ICL-1) and third extracellular loop (ECL-3) of the type IIa Na+/P(i) cotransporter (NaPi-IIa) have been shown previously to contain functionally important sites by applying the substituted cysteine accessibility method (SCAM). Incubation in methanethiosulfonate (MTS) reagents of mutants that contain novel cysteines in both loops led to full inhibition of cotransport activity. To elucidate further the role these regions play in defining the transport mechanism, a double mutant (A203C-S460C) was constructed with novel cysteines in each region. The effect of cysteine modification by different MTS reagents on two electrogenic transport modes (leak and cotransport) was investigated. MTSEA (2-aminoethyl MTS hydrobromide) and MTSES (MTS ethylsulfonate) led to full inhibition of cotransport and increased the leak, whereas incubation in MTSET (2-[trimethylammonium]ethyl MTS bromide) inhibited only cotransport. The behavior of other double mutants with a cysteine retained at one site and hydrophobic or hydrophilic residues substituted at the other site, indicated that most likely only Cys-460 was modifiable, but the residue at Ala-203 was critical for conferring the leak and cotransport mode behavior. Substrate interaction with the double mutant was unaffected by MTS exposure as the apparent P(i) and Na+ affinities for P(i)-induced currents and respective activation functions were unchanged after cysteine modification. This suggested that the modified site did not interfere with substrate recognition/binding, but prevents translocation of the fully loaded carrier. The time-dependency of cotransport loss and leak growth during modification of the double cysteine mutant was reciprocal, which suggested that the modified site is a kinetic codeterminant of both transport modes. The behavior is consistent with a kinetic model for NaPi-IIa that predicts mutual exclusiveness of both transport modes. Together, these findings suggest that parts of the opposing linker regions are associated with the NaPi-IIa transport pathway.

Amino Acid Substitution↗

PubMed2003

Iron regulatory protein 2 as iron sensor. Iron-dependent oxidative modification of cysteine.

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an iron-binding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.

Amino Acids↗

PubMed1995

Evidence for voltage-dependent S4 movement in sodium channels.

The mutation R1448C substitutes a cysteine for the outermost arginine in the fourth transmembrane segment (S4) of domain 4 in skeletal muscle sodium channels. We tested the accessibility of this cysteine residue to hydrophilic methanethiosulfonate reagents applied to the extracellular surface of cells expressing these mutant channels. The reagents irreversibly increase the rate of inactivation of R1448C, but not wild-type, channels. Cysteine modification is voltage dependent, as if depolarization extends this residue into the extracellular space. The rate of cysteine modification increases with depolarization and has the voltage dependence and kinetics expected for the movement of a voltage sensor controlling channel gating.

Adult↗

PubMed1997

The conversion from the dehydrogenase type to the oxidase type of rat liver xanthine dehydrogenase by modification of cysteine residues with fluorodinitrobenzene.

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348-7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys754 and Lys771 by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3H-dinitrophenyl compounds. After modification with 3H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys535, Cys992 and Cys1324. These residues are conserved among the all known mammalian enzymes, but Cys992 and Cys1324 are not conserved in the chicken enzyme. Cys1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys535 and Cys992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion.

Amino Acid Sequence↗

PubMed2004

Thiol modification of cysteine 327 in the eighth transmembrane domain of the light subunit xCT of the heteromeric cystine/glutamate antiporter suggests close proximity to the substrate binding site/permeation pathway.

We measured sensitivity to thiol modification of the heteromeric glutamate/cystine transporter 4F2hc-xCT expressed in Xenopus oocytes. p-Chloromercuribenzoate (pCMB) and p-chloromercuribenzenesulfonate (pCMBS) rapidly blocked transport activity. Cys(327), located in the middle of the eighth transmembrane domain of the light subunit (xCT), was found to be the main target of inactivation. Cysteine, an impermeant reducing reagent, reversed pCMB and pCMBS effects only when applied from the extracellular medium. l-Glutamate and l-cystine, but not l-arginine, protected from the inactivation with an IC(50) similar to the K(m). Protection was not temperature-dependent, suggesting that it did not depend on large substrate-induced conformational changes. Mutation of Cys(327) to Ala and Ser slightly modified the K(m) and a C327L mutant abolished transport function without compromising transporter expression at the plasma membrane. The results indicate that Cys(327) is a functionally important residue accessible to the aqueous extracellular environment and is structurally linked to the permeation pathway and/or the substrate binding site.

Amino Acid Sequence↗

PubMed1992

Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.

Animals↗

PubMed2004

Modification of cysteine residue in transthyretin and a synthetic peptide: analyses by electrospray ionization mass spectrometry.

Cysteine and cystine in protein are modified to various derivatives in vitro and in vivo. By electrospray ionization mass spectrometry (ESI-MS), we previously found derivatives of serum transthyretin (TTR) in which cysteine residue at position 10 was changed to glycine residue and sulfocysteine residue. The change, cysteine to glycine, was unique and the origin of the sulfonic acid group was controversial. In the present paper, we show the molecular masses of various TTR derivatives including these two, and the modification process was studied using a synthetic peptide with the same sequence as cysteine containing part of TTR, i.e., SKCPLMVK. After incubation of the peptide at pH 8.3, various derivatives were generated, which showed changes of cysteine residue to glycine, dehydroalanine, S-thiocysteine, and S-sulfocysteine residues, which were confirmed by molecular mass and collision-induced dissociation spectra. Dehydroalanine may react with other amino acids and contribute to form cross-linking fibril, causing amyloidosis.

Cysteine↗

PubMed1998

Nitric oxide inhibition of the rat olfactory cyclic nucleotide-gated cation channel.

The effects of nitric oxide (NO) and other cysteine modifying agents were examined on cyclic nucleotide-gated (CNG) cation channels from rat olfactory receptor neurons. The NO compounds, S-nitroso-cysteine (SNC) and 3-morpholino-sydnonomine (SIN-1), did not activate the channels when applied for up to 10 min. The cysteine alkylating agent, N-ethylmaleimide (NEM), and the oxidising agent, dithionitrobensoate (DTNB), were also without agonist efficacy. Neither SNC nor DTNB altered the cAMP sensitivity of the channels. However, 2-min applications of SIN-1, SNC and DTNB inhibited the cAMP-gated current to approximately 50% of the control current level. This inhibition showed no spontaneous reversal for 5 min but was completely reversed by a 2-min exposure to DTT. The presence of cAMP protected the channels against NO-induced inhibition. These results indicate that inhibition is caused by S-nitrosylation of neighboring sulfhydryl groups leading to sulfhydryl bond formation. This reaction is favored in the closed channel state. Since recombinantly expressed rat olfactory alpha and beta CNG channel homomers and alpha/beta heteromers are activated and not inhibited by cysteine modification, the results of this study imply the existence of a novel subunit or tightly bound factor which dominates the effect of cysteine modification in the native channels. As CNG channels provide a pathway for calcum influx, the results may also have important implications for the physiological role of NO in mammalian olfactory receptor neurons.

Animals↗

PubMed1997

Stabilising and destabilising modifications of cysteines in the E. coli outer membrane porin protein OmpC.

Three sulfhydryl labels were used to modify two mutated sites, R37C and R74C in the eyelet of the outer membrane porin OmpC. Modification of R37C with the neutral groups Aldrithiol and bimane increases thermal stability but the negatively charged iodoacetate causes a decrease in thermal stability. The effects of substitution at R74C were less significant. Bimane labelling increases the voltage sensitivity and decreases the single channel conductance at R37C asymmetrically with smaller channels being recorded at cis negative voltages. Negatively charged acetate does not affect the voltage gating.

Bacterial Outer Membrane Proteins↗

PubMed2006

Transcriptional regulation via cysteine thiol modification: a novel molecular strategy for chemoprevention and cytoprotection.

Chemoprevention refers to the use of defined nontoxic chemical regimens to inhibit, reverse, or retard the process of multistage carcinogenesis that involves multiple signal transduction events. Identification of signaling molecules associated with carcinogenesis as prime targets of chemopreventive agents has become an area of great interest. Recent studies have implicated cysteine thiols present in various transcription factors, such as NF-kappaB, AP-1, and p53 as redox sensors in transcriptional regulation of many genes essential for maintaining cellular homeostasis. Some chemopreventive and cytoprotective agents have been found to target cysteine thiols present in key transcription factors or their regulators, thereby suppressing aberrant over-activation of carcinogenic signal transduction or restoring/normalizing or even potentiating cellular defense signaling. The focus of this review is the oxidation or covalent modification of thiol groups present in key representative redox-sensitive transcription factors and their regulating molecules as a unique strategy for molecular target-based chemoprevention and cytoprotection.

Cysteine↗

PubMed1996

Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups.

L-Threonine dehydrogenase (TDH) from Escherichia coli is rapidly inactivated and develops a new absorbance peak at 347 nm when incubated with N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K, WRK). The cofactors, NAD+ or NADH (1.5 mM), provide complete protection against inactivation; L-threonine (60 mM) is approximately 50% as effective. Tryptic digestion of WRK-modified TDH followed by HPLC fractionation (pH 6.2) yields four 340-nm-absorbing peptides, two of which are absent from enzyme incubated with WRK and NAD+. Peptide I has the sequence TAICGTDVH (TDH residues 35-43), whereas peptide II is TAICGTDVHIY (residues 35-45). Peptides not protected are TMLDTMNHGGR (III, residues 248-258) and NCRGGRTHLCR (IV, residues 98-108). Absorbance spectra of these WRK-peptides were compared with WRK adducts of imidazole, 2-hydroxyethanethiolate, and acetate. Peptides III and IV have pH-dependent lambda max values (340-350 nm), consistent with histidine modification. Peptide I has pH-independent lambda max (350 nm) indicating that a thiol is modified. WRK, therefore, does not react specifically with carboxyl groups in this enzyme, but rather modifies Cys-38 in the active site of TDH; modification of His-105 and His-255 does not affect enzyme activity. These results are the first definitive proof of WRK modifying cysteine and histidine residues of a protein and show that enzyme inactivation by WRK associated with the appearance of new absorptivity at 340-350 nm does not establish modification of aspartate or glutamate residues, as has been assumed in numerous earlier reports.

Alcohol Oxidoreductases↗

PubMed2005

Reactivity of zinc finger cysteines: chemical modifications within labile zinc fingers in estrogen receptor.

Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monobromobimane. Although they are slower to react, the sterically bulkier reagents, monobromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions.

Binding Sites↗

PubMed2005

MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3.

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.

Amino Acid Motifs↗

PubMed2006

Modification of cysteine 111 in human Cu,Zn-superoxide dismutase.

Human Cu,Zn-superoxide dismutase (hSOD1) has 4 cysteines per subunit. Cys57 and Cys148 are involved in an intrasubunit disulfide bond, while Cys6 and Cys111 are free. Cys6 is buried within the protein while Cys111 is on the surface, near the dimer interface. We examined by liquid chromatography-mass spectrometry the commercially purchased hSOD1 isolated from erythrocytes as well as hSOD1s isolated from human erythrocytes, brain, and hSOD1 expressed in Sf9, yeast, and E. coli. Our goal was to ascertain whether the Cys111 modification occurred naturally in vivo. Only the Sigma erythrocyte hSOD1 appeared to contain a trisulfide crosslink between the Cys111 residues. Thus it failed to react with N-ethylmaleimide, showed absorbtion at 325 nm that was eliminated by 2-mercaptoethanol, and had a mass 30 units more than expected for the native dimer. We examined the possibility that different purification methods might cause this modification in erythrocyte hSOD1. None of the procedures examined for hSOD1 purification produced such a trisulfide. In disagreement with Liu et al. [Biochemistry, 2000, 39, 8125-8132], complete derivitization of both Cys111s of hSOD1 from Sf9 cells with N-ethylmaleimide, 4-vinylpyridine, and by 5,5'-dithiobis(2-nitrobenzoic acid) were readily achieved; indicating that steric hindrance was not a problem.

Animals↗

PubMed2005

Sulfhydryl modification of cysteine mutants of a neuronal glutamate transporter reveals an inverse relationship between sodium dependent conformational changes and the glutamate-gated anion conductance.

In the central nervous system, glutamate transporters remove the neurotransmitter from the synaptic cleft. The electrogenic transport of glutamate is coupled to the electrochemical sodium, proton and potassium gradients. Moreover, these transporters mediate a sodium- and glutamate-dependent uncoupled chloride conductance. In contrast to the wild type, the uptake of radiolabeled substrate of the G283C mutant is inhibited by [2-(trimethylammonium)ethyl]methanethiosulfonate, a membrane impermeant sulfhydryl reagent. In the wild type and the unmodified mutant, substrate-induced currents are inwardly rectifying and reflect the sum of the coupled electrogenic flux and the anion conductance. However, the sulfhydryl-modified G283C mutant exhibits currents that are non-rectifying and reverse at the equilibrium potential for chloride. These properties are similar to those of the I421C mutant after sulfhydryl modification. Importantly, in contrast to I421C, the modification of G283C does not cause an increase of the magnitude of the anion conductance and a decrease of the apparent substrate affinity. Moreover, in the G283C/I421C double mutant the phenotype of I421C is dominant. Sulfhydryl modification of I421C, but not of G283C, abolishes the sodium dependent transient currents. The results indicate the existence of multiple transitions between the coupled transport cycle and anion conducting states.

Amino Acid Transport System X-AG↗

PubMed2001

Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity.

The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues. In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport. Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed. Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied. Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate). In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein. With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity. Cys(188) exhibited an apparent pK(a) of 7.6. The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface. The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity. However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity. These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity. Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP. Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding.

Animals↗