Selective recognition and detection of biomacromolecules utilizing chemical property of amino Acid or Peptide

Recently, the fluorometric detection of biomacromolecules has attracted much attention. In this paper, we report the development of two new techniques utilizing the chemical properties of amino acids or peptides: 1) a fluorescence assay for serine/threonine kinase activity; and 2) "turn-on" fluorescent probes for protein labeling, which could be useful for bioimaging. To develop the novel kinase assay, we utilized the chemical reactivity of phosphorylated serine or threonine. Phosphorylated peptide on resin was successfully labeled fluorescently via base-mediated beta-elimination, followed by Michael addition with novel coumarin derivatives. Protein kinase A and casein kinase I activities were detectable with our method. Also, this method was confirmed to be applicable for kinase inhibitor screening. For the development of the novel protein labeling technique, the selective interaction between "His-tag (His(6))" and "metal ion nitrilotriacetic acid (NTA) complex" was utilized. This interaction is useful for protein purification and immobilization. We designed fluorescent probes composed of a fluorophore and Ni2+ or Co2+-NTA complex. These probes were found to be weakly fluorescent as expected. When His-tag peptide was added, these probes became brightly fluorescent. On the other hand, these probes remained non fluorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH). These probes will be powerful tools for the bioimaging of target proteins.     

Covalent binding of acrylonitrile to specific rat liver glutathione S-transferases in vivo.

Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.     

Proteomic analysis of rat striatal synaptosomes during acrylamide intoxication at a low dose rate.

We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical presynaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry. ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins. Synaptosomes were prepared from striatum of ACR-intoxicated rats (21 mg/kg/day x 7, 14, or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light (12C9--control) or heavy (13C9--ACR) ICAT reagent. Results show that ACR intoxication caused a progressive reduction in the ICAT labeling of many nerve terminal proteins. A label-free mass spectrometric approach (multidimensional protein identification) was used to show that the observed reductions in ICAT incorporation were not due to general changes in protein abundance and that ACR formed adducts with cysteine residues on peptides which also exhibited reduced ICAT incorporation. The decrease in labeling was temporally correlated to the development of neurological toxicity and confirmed previous findings that cysteine adducts of ACR accumulate as a function of exposure. The accumulation of adduct is consistent with the cumulative neurotoxicity induced by ACR and suggests that cysteine adduct formation is a necessary neuropathogenic step. Furthermore, our analyses identified specific proteins (e.g., v-ATPase, dopamine transporter, N-ethylmaleimide-sensitive factor) that were progressively and significantly adducted by ACR and might, therefore, be neurotoxicologically relevant targets.     

Optimization and Applications of CDAP Labeling for the Assignment of Cysteines

Purpose The aim of the study is to provide a methodology for assigning unpaired cysteine residues in proteins formulated in a variety of different conditions to identify structural heterogeneity as a potential cause for protein degradation.Methods 1-Cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was employed for cyanylating free cysteines in proteins and peptides. Subsequent basic cleavage of the peptide bond at the N-terminal side of the cyanylated cysteines provided direct information about their location.Results CDAP was successfully employed to a wide variety of labeling conditions. CDAP was reactive between pH 2.0 and 8.0 with a maximum labeling efficiency at pH 5.0. Its reactivity was not affected by excipients, salt or denaturant. Storing CDAP in an organic solvent increased its intrinsic stability. It was demonstrated that CDAP can be employed as a thiol-directed probe to investigate structural heterogeneity of proteins by examining the accessibility of unpaired cysteine residues.Conclusion CDAP is a unique cysteine-labeling reagent because it is reactive under acidic conditions. This provides an advantage over other sulfhydryl labeling reagents as it avoids potential thiol-disulfide exchange. Optimization of the cyanylation reaction allowed the utilization of CDAP as a thiol-directed probe to investigate accessibility of sulfhydryl groups in proteins under various formulation conditions to monitor structural heterogeneity.     

纳米颗粒跟踪分析和纳米流式细胞仪对脂肪间充质干细胞的细胞外囊泡检测结果评价

目的 探讨纳米颗粒跟踪分析（nanoparticle tracking analysis,NTA）和纳米流式细胞仪（nano-flow cytometry,nanoFCM）对对照品微球和脂肪间充质干细胞（adipose mesenchymal stem cells,AdMSCs）的细胞外囊泡（extracellular vesicles,EVs）的检测能力,为EVs的鉴定和质量控制提供依据。方法 首先使用对照品微球来验证NTA和nanoFCM的检测能力。随后,对于聚乙二醇6000沉淀-超速离心法制备的AdMSCs-EVs,通过Western blotting和电子透射显微镜进行一般特征鉴定后,使用NTA和nanoFCM来检测其粒径分布和PKH67染色后的荧光颗粒占比。结果 NTA和nanoFCM在颗粒浓度方面的检测能力相近,但是nanoFCM的精度更高,粒径区分度更好。用NTA检测EVs的检测结果显示NTA粒径分布较广,nanoFCM粒径分布较窄;NTA的PKH67的荧光检测阳性率显著低于nanoFCM。结论 NanoFCM拥有较高的精度,但也存在着一定应用局限性。NTA的粒径区分度相对较差,但是可检测的粒径范围更广。因此,NTA可以满足EVs的粒径分布检测,对荧光检测具有较高灵敏度的nanoFCM更适合分析EVs的表面标志物比例。     

Isolation,characterization, and physiology of bacteria able to degrade nitrilotriacetate

Until recently, only three obligately aerobic bacteria (affiliated with the genus Pseudo-monas) that can grow with nitrilotriacetate (NTA) as their only source of carbon, nitrogen, and energy have been isolated and studied in pure culture. By employing a different isolation strategy than was used previously, several nonpseudomonads were isolated in pure culture from both soil and wastewater that are able to utilize NTA under aerobic growth conditions. Additionally, a denitrifying bacterium was isolated from river sediment that is able to utilize NTA in the absence of oxygen. These isolates have been characterized with respect to their cell morphology and physiology. The data collected so far do not allow classification of both the gram-negative and the gram-positive strains isolated, and the taxonomic position of the isolates remains obscure. However, properties like C₁ utilization, production of acetoïn, and nonmotility clearly indicate that the gram-negative strains do not belong to the genus Pseudomonas. Information is presented on the regulation of NTA-metabolizing enzymes in isolate TE 1 suggesting that these enzymes are inducible in this bacterium.     

The influence of 2,3-butanedione monoxime on dichlorvos-induced enzymatic changes in buffalo calves.

The effects of administration of 2,3-butanedione monoxime (2,3-BM) or atropine alone and in combination were determined on the blood enzymatic activities of dichlorvos-exposed buffalo calves. Dichlorvos given po at 160 mg/kg body weight produced pronounced inhibition of erythrocyte acetylcholinesterase (AChE) and elevation in serum aminotransferases and phosphatases within 30 min. 2,3-BM administered alone or in conjunction with atropine to dichlorvos-exposed calves significantly reactivated erythrocyte AChE activity whereas atropine was ineffective. The effect of 2,3-BM plus atropine on other enzymatic activities was comparatively greater than that of either drug alone. The results indicated that combined treatment with 2,3-BM and atropine was most effective in reversing dichlorvos-induced enzymatic alterations.     

In vivo detection and characterization of protein adducts resulting from bioactivation of haloethene cysteine S-conjugates by 19F NMR: chlorotrifluoroethene and tetrafluoroethene.

Several haloalkenes are selective nephrotoxins. The bioactivation of nephrotoxic haloalkenes involves hepatic glutathione S-conjugate formation, peptidase-catalyzed metabolism of the glutathione S-conjugates to the corresponding cysteine S-conjugates, uptake of cysteine S-conjugates by the kidneys, and renal cysteine conjugate beta-lyase-catalyzed beta-elimination of a thiol. The haloalkyl and haloalkenyl thiols thus released are unstable and yield reactive intermediates whose interactions with cellular constituents are though to contribute to the observed toxicity of S-conjugates. Tetrafluoroethene and chlorotrifluoroethene are metabolized to the cysteine S-conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), respectively. Administration of TFEC (1.0 mmol/kg) or CTFC (1.0 mmol/kg) to rats resulted in acylation of renal proteins, as demonstrated with 19F nuclear magnetic resonance spectroscopy. Single, broad resonances near 41 or 56 ppm were found in spectra of renal proteins from TFEC- or CTFC-treated rats, respectively, and these resonances were not lost on dialysis. Renal protein incubated with 2-chloro-1,1,2-trifluoroethyl-2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiol, showed the same 19F NMR spectrum as was found with CTFC-treated rats. In vitro incubation of various N alpha-blocked amino acids with this proreactive intermediate indicated that only lysine is stably adducted, whereas histidine is transiently acylated. In each case, proteolysis of modified protein converted a single broad NMR resonance to a doublet with little change in chemical shift and with clearly resolved, characteristic H-F couplings. The single, stable amino acid adduct formed with renal proteins of rats given CTFC or TFEC was N epsilon-(chlorofluorothioacetyl)lysine and N epsilon-(difluorothioacetyl)lysine, respectively.     

Effects of detergent formula chelating agents on the metabolism and toxicity of cadmium in mice.

Chelating agents like NTA (nitrilotriacetic acid) STPP (sodiumtripolyphosphate, Na5P3O10) and EDTA (ethylenediaminetetraacetic acid) are used as components of detergents. An increased toxicity of some metal compounds when combined with NTA has led to decreased use of this chelating agent in relation to STPP. In the present studies short-term and long-term effects of these chelating agents on cadmium toxicity in mice were investigated. I: In the short-term study, mice subcutaneously exposed to CdCl2 (3.2 mg Cd/kg b. wt.) in combination with STPP (32 mg/kg b. wt.) demonstrated a markedly higher mortality compared to animals given CdCl2 alone. This increase in mortality was similar to the one encountered when CdCl2 (3.2 mg Cd/kg b. wt.) and NTA (32 mg/kg b. wt.) were combined. Animals exposed subcutaneously to CdCl2 + STPP or CdCl2 + NTA showed histological evidence of liver necrosis 24 hrs after exposure not seen in animals given the same dose of CdCl2 alone and also had markedly lower cadmium concentrations in the livers compared to only Cd-exposed animals. II: In the long-term study, mice were exposed orally to CdSO4 (50p.p.m Cd) alone or in combination with STPP (500 p.p.m.), NTA (500 p.p.m.) or EDTA (50 p.p.m.) by continuous administration via the drinking water for 18 months. A decreased total excretion of urine proteins was seen in all Cd-treated animals irrespectively of the combination with various chelating agents. The conclusion of the present work was the NTA and STPP given by subcutaneous injection to mice markedly increased the toxicity of cadmium but that neither NTA, STPP nor EDTA given orally altered the toxicity of cadmium during a period of long-term exposure of 18 months.     

Effect of N-nitroso-n-butyl-(4-hydroxybutyl)amine exposure on the changes in mineral disposition caused by trisodium nitrilotriacetate

Male Fischer 344 rats were used to determine effect of consumption of 0.5% N-nitroso-n-butyl-(4-hydroxybutyl)amine (BNN) in the drinking-water for 2 wk on the response to 0.02, 0.2 and 2.0% trisodium nitrilotriacetate (Na3 NTA . H2O) in the diet in terms of urinary mineral excretion, bladder mass and bladder mineral concentrations. The primary objective of the study was to determine whether exposure of rats to an initiating dose of a bladder carcinogen (BBN) alters the threshold dose of Na3NTA . H2O required to alter urinary or bladder mineral concentrations or the dose-response to NTA. Such alterations are considered to be necessary precursors for changes in bladder morphology in rats fed NTA in chronic toxicity studies (Anderson, Bishop & Campbell, CRC Crit. Rev. Toxicol. 1985, 15, 1). The results demonstrated that BBN exposure caused an increase in bladder mass and bladder-tissue Zn concentration. However, BBN pretreatment did not have any effect on Na3NTA . H2O metabolism, the threshold dose of Na3NTA . H2O required to attain the necessary conditions for induction of bladder toxicity by NTA, or the dose-response relationships for NTA's effects on any parameter examined. From these data, it is concluded that it is unlikely that NTA would show a different threshold or dose-response for bladder tumour promotion than for its tumorigenicity at this site, which has been demonstrated previously (National Cancer Institute, DHEW Publication No. (NIH) 77-806, 1977).     

Functional analysis of the extracellular cysteine residues in the human organic anion transporting polypeptide, OATP2B1

Organic anion transporting polypeptide (OATP) superfamily member 2B1 (OATP2B1) mediates the uptake of steroid hormone precursors and selected drugs in the placenta, liver, mammary gland, brain, and intestine. This action is modulated by sulfhydryl reagents. Common to all OATPs is a large extracellular loop between transmembrane domains IX and X with 10 conserved cysteines. To elucidate the structure-function relationship of this cysteine rich ectodomain, a truncated OATP2B1 lacking 10 extracellular cysteines (OATP2B1(Delta489-557)) and 10 OATP2B1 mutants containing individual Cys-to-Ala substitutions were generated and expressed in CHO-K1 cells. The immunolocalization, cell-surface expression, transport activity, and free cysteine labeling with N-biotinoylaminoethylmethane-thiosulfonate of mutant proteins and wild-type OATP2B1 were compared. OATP2B1(Delta489-557) accumulated intracellularly. Nine Cys-to-Ala substitutions, C489A, C495A, C504A, C516A, C520A, C539A, C541A, C553A, and C557A, were misprocessed, appearing predominantly as core-glycosylated, 60-kDa proteins and as 180-kDa complexes. Only C493A was a fully glycosylated 75-kDa protein expressed at the cell surface. Thapsigargin partially corrected the misprocessing of mutants. Compared with OATP2B1, C493A and C557A transported estrone-3-sulfate and dehydroepiandrosterone sulfate less efficiently, whereas all other mutants were functionally impaired. MTSEA labeled free cysteines in all Cys-to-Ala mutants but not in OATP2B1, suggesting that all 10 extracellular cysteines are normally disulfide-bonded. Our findings show that the trafficking and function of OATP2B1 is vulnerable to changes in the cysteine residues of extracellular loop IX-X.     

Effects of Detergent Formula Chelating Agents on the Metabolism and Toxicity of Cadmium in Mice

Abstract Chelating agents like NTA (nitrilotriacetic acid) STPP (sodiumtripolyphosphate, Na₅P₃O₁₀) and EDTA (ethylenediaminetetraacetic acid) are used as components of detergents. An increased toxicity of some metal compounds when combined with NTA has led to decreased use of this chelating agent in relation to STPP. In the present studies short-term and long-term effects of these chelating agents on cadmium toxicity in mice were investigated. I: In the short-term study, mice subcutaneously exposed to CdCl₂ (3.2 mg Cd/kg b.wt.) in combination with STPP (32 mg/kg b.wt.) demonstrated a markedly higher mortality compared to animals given CdCl₂ alone. This increase in mortality was similar to the one encountered when CdCl₂ (3.2 mg Cd/kg b.wt.) and NTA (32 mg/kg b.wt.) were combined. Animals exposed subcutaneously to CdCl₂ + STPP or CdCl₂ + NTA showed histological evidence of liver necrosis 24 hrs after exposure not seen in animals given the same dose of CdCl₂ alone and also had markedly lower cadmium concentrations in the livers compared to only Cd-exposed animals. II: In the long-term study, mice were exposed orally to CdSO₄ (50 p.p.m. Cd) alone or in combination with STPP (500 p.p.m.), NTA (500 p.p.m.) or EDTA (50 p.p.m.) by continuous administration via the drinking water for 18 months. A decreased total excretion of urine proteins was seen in all Cd- treated animals irrespectively of the combination with various chelating agents. The conclusion of the present work was that NTA and STPP given by subcutaneous injection to mice markedly increased the toxicity of cadmium but that neither NTA, STPP nor EDTA given orally altered the toxicity of cadmium during a period of long-term exposure of 18 months.     

Mammalian cell transformation induced by chromium(VI) compounds in the presence of nitrilotriacetic acid

We used a soft agar assay on cultured Syrian hamster fibroblasts to determine the ability of nitrilotriacetic acid (NTA) and Cr(VI) compounds to induce malignant cell transformation. Induction of extended anchorage-independent growth was detected in BHK 21/c13 cells by scoring colonies of transformed cells visible to the naked eye 20-25 d after plating in growth medium containing agar. Survival was determined by plating cells in liquid medium without agar and by counting the number of macroscopic colonies after 7-10 d. Mitomycin C and 4-nitroquinoline 1-oxide were used as reference direct transforming agents, with clearly positive results. In our hands no increase of the spontaneous transformation rate of BHK cells was induced by NTA concentrations ranging from 2 X 10(-3) to 10(-2) M, although the survival index was significantly reduced above 4 X 10(-3) M NTA. Two Cr(VI) compounds, K2Cr2O7, which is highly soluble in water, and CaCrO4, which is partially soluble, were tested in the soft agar assay either in the absence or in the presence of NTA. When used alone, both compounds behaved as positive transforming agents. NTA increased 4 or 10 times the cytotoxicity and the transforming activity of CaCrO4 and K2Cr2O7, respectively. As the amounts of soluble Cr(VI) detectable in the K2Cr2O7 and CaCrO4 solutions were not increased in the presence of NTA, a synergistic interaction between NTA and soluble Cr(VI) is inferred.     

Influence of Chelating Agents on Toxicity and Distribution of Cadmium among Proteins of Mouse Liver and Kidney Following Oral or Subcutaneous Exposure

Abstract: Acute toxicity and organ distribution of cadmium was investigated in mice exposed to 1) single subcutaneous doses of ¹⁰⁹Cd-labelled cadmium (3.2 mg (0.028 mmol)/kg b.wt.) alone or in combination with nitrilotriacetic acid, NTA (32 mg (0.167 mmol)/kg b.wt.) or sodium tripolyphosphate, STPP (32 mg (0.087 mmol)/kg b.wt.) and 2) single oral doses of cadmium (60 mg (0.53 mmol)/kg b.wt.) alone or together with NTA (600 mg (3.14 mmol)/kg b.wt.) or STPP (600 mg (1.63 mmol)/kg b.wt.) Whole-body retention of radiolabelled cadmium as well as mortality was registered in all groups during 20–21 days. Five hours after exposure, 3–4 mice in each group were killed and cadmium distribution among proteins in liver and kidney studied by gel chromatography on a G-75 Sephadex column. Organ concentration of cadmium was also determined at sacrifice of all other mice after an observation time of 20–21 days. A markedly increased mortality was observed during the first 24 hours after subcutaneous exposure to Cd+NTA (70%) or Cd + STPP (40%) compared to Cd alone (0%). On the contrary, no mortality was seen after oral exposure to Cd + STPP while oral exposure to Cd alone or with NTA resulted in a mortality of about 45% during the same observation time. Five hours after subcutaneous exposure, liver cadmium concentrations were equal in all mice and bound mainly to a low molecular weight protein (probably metallothionein). In kidney, concentrations were about twice as high in mice given Cd + NTA or Cd + STPP compared to mice given Cd alone. More cadmium was transferred to the kidneys in the presence of chelating agents. This was further supported by the decreased liver/kidney cadmium concentration ratio between 5 hours and 21 days. The increased mortality in mice given Cd + NTA or Cd + STPP may be due to an initially high accumulation of cadmium in liver, which may exceed the upper limit for metallothionein synthesis, whereby toxic damage of the hepatic cells and leakage of cadmium from the liver would occur. Five hours after oral exposure, cadmium retention in organs of mice given Cd+STPP was 4–6 times lower than in mice given Cd alone, while mice given Cd+NTA had slightly higher organ concentrations. The binding of more cadmium to metallothionein in liver of Cd + STPP-exposed mice 5 hours after exposure, may be due to the lower accumulation of cadmium in these mice compared to those given Cd alone or Cd+NTA, where cadmium was bound mainly to high molecular proteins in the liver. After 20 days, mice given Cd+STPP has slightly higher body and organ retention of cadmium compared to mice given Cd alone. This may be due to a binding of cadmium to metallothione in these mice which is known to prolong the retention time of cadmium in tissue.     

Identification of specific tethered inhibitors for caspase-5

The development of highly selective small molecule inhibitors for individual caspases, a class of cysteine-dependent aspartate-specific proteases, has been challenging due to conservation of the active site. Previously, we discovered an allosteric site at the dimer interface of caspases-3, -7, and -1 using disulfide trapping. Here, we show this approach can generate selective tethered ligands and inhibitors for caspase-5, which is remarkable considering its high sequence similarity to caspase-1. Among the 62 hits of a screen of ～15 000 thiol-containing fragments, a naphthyl-thiazole-containing molecule was identified that selectively inhibited and labeled the allosteric cysteine in the p10 subunit of caspase-5, but caused very little inhibition or labeling of caspase-1. Interestingly, some of allosteric tethered compounds to caspase-5 did not inhibit its enzymatic activity, suggesting that thiol-labeling itself is not sufficient to drive inhibition. These studies validate an allosteric site on caspase-5 and provide a useful starting point to develop selective compounds to probe the role of caspase-5 separate from caspase-1 in the innate immune response.     

A review of the environmental and mammalian toxicology of nitrilotriacetic acid

This article provides a review of available information on the chemistry, environmental toxicology, and mammalian toxicology of nitrilotriacetic acid (NTA). The ability of NTA to chelate metal ions such as Mg++ and Ca++ into water soluble complexes makes NTA useful as an additive to boiler water, as a builder in laundry detergents, and as a stabilizer in textile, paper, and pulp processing. Environmental fate studies show NTA biodegrades in wastewater treatment plants, in natural waters, and in soils under a wide variety of conditions. Studies on the environmental effects of NTA indicate that no adverse effects occur in treatment plants or receiving waters at anticipated levels. Monitoring programs have established that only low steady-state concentrations of NTA occur in natural waters as a result of NTA usage. In mammalian systems, NTA is not metabolized and is excreted rapidly by filtration in the kidney. No reproductive, teratogenic, or adverse bone effects have been observed at highly exaggerated doses. In numerous genotoxicity assay systems, both in vivo and in vitro, NTA is nongenotoxic. Chronic oral exposure of rodents to high doses of NTA is associated with tumorigenicity in, and restricted to, the urinary tract. The urinary tract tumors are the consequence of chronic toxicity that is caused by changes in Zn and Ca distributions between the urinary tract tissues and urine at high doses of NTA. Thresholds for the effects of NTA on Zn and Ca distributions are 10(5) to 10(6) greater than the possible maximum human exposure resulting from the low levels of NTA that are known to occur in the environment.     

Protein targets of reactive electrophiles in human liver microsomes

Liver microsomes are widely used to study xenobiotic metabolism in vitro, and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4'-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible, and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB- but not BMCC-induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds.     

Conserved cysteine residues in the extracellular loop of the human P2X(1) receptor form disulfide bonds and are involved in receptor trafficking to the cell surface.

P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2- to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 +/- 2.0 and 95.0 +/- 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MTSEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEA-biotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X(1) receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.     

Control of oxidative posttranslational cysteine modifications: from intricate chemistry to widespread biological and medical applications

Cysteine residues in proteins and enzymes often fulfill rather important roles, particularly in the context of cellular signaling, protein-protein interactions, substrate and metal binding, and catalysis. At the same time, some of the most active cysteine residues are also quite sensitive toward (oxidative) modification. S-Thiolation, S-nitrosation, and disulfide bond and sulfenic acid formation are processes which occur frequently inside the cell and regulate the function and activity of many proteins and enzymes. During oxidative stress, such modifications trigger, among others, antioxidant responses and cell death. The unique combination of nonredox function on the one hand and participation in redox signaling and control on the other has placed many cysteine proteins at the center of drug design and pesticide development. Research during the past decade has identified a range of chemically rather interesting, biologically very active substances that are able to modify cysteine residues in such proteins with huge efficiency, yet also considerable selectivity. These agents are often based on natural products and range from simple disulfides to complex polysulfanes, tetrahydrothienopyridines, α,β -unsaturated disulfides, thiuramdisulfides, and 1,2-dithiole-3-thiones. At the same time, inhibition of enzymes responsible for posttranslational cysteine modifications (and their removal) has become an important area of innovative drug research. Such investigations into the control of the cellular thiolstat by thiol-selective agents cross many disciplines and are often far from trivial.