RESUMEN
Cyanide (CN = HCN + CN(-)) is a renowned poison and neurotoxicant that is prevalent throughout the environment. Despite a plethora of studies conducted over the last half century, relatively little is known of its potential to cause adverse health outcomes at sublethal exposures. CN exposure is normally determined from blood, but because CN is rapidly metabolized and cleared from this compartment (t(1/2) < 1 h), it is common for several half-lives to have passed before blood samples are drawn for analysis. This variable, coupled with a very narrow toxic index and metabolic diversity within the human population, has rendered accurate assessment of CN exposure, and consequently any predictions of possible adverse health outcomes, highly problematic. Prior studies by us showed the potential of Cys-SCN adducts within human serum albumin (HSA) to act as retrospective surrogates of CN exposure. Here, we report the discovery of a stable, SCN adduct at Cys(567) formed by the reaction of CN with the C-terminal Cys(558)Cys(567) disulfide bond of HSA. Treatment of HSA purified from human serum with base in guanidine hydrochloride releases a readily detectable, uniquely modified, C-terminal-19-mer peptide from Cys(567)-SCN moieties in all the samples examined thus far. Inclusion of a HSA-Cys(567)-S(13)C(15)N labeled internal standard permits quantitation of the Cys(567)-SCN adduct by LC-MS/MS in selective reaction monitoring (SRM) of the surrogate peptide with high sensitivity and good precision. Reaction of CN in vitro with the Cys(558)Cys(567) disulfide bond in HSA is specific, rapid, and concentration dependent within a putative, physiologically relevant range. Data from various human sera demonstrate the potential usefulness of this adduct as a biomarker of CN exposure.
Asunto(s)
Cianuros/química , Albúmina Sérica/química , Biomarcadores/química , Cromatografía Líquida de Alta Presión , Cianuros/toxicidad , Cisteína/química , Deuterio/química , Disulfuros/química , Humanos , Péptidos/química , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: To identify ovarian autoantigens associated with ovarian autoantibodies. DESIGN: Hypothesis-generating prospective study. SETTING: Urban infertility referral centers and academic research institution. PATIENT(S): Seventy-four patients with infertility, 19 patients with premature ovarian failure (POF), and 16 healthy control women. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of autoantigens. RESULT(S): To identify major antigens for ovarian autoimmunity, sera from 74 women with unexplained infertility were screened for ovarian autoantibodies (AOAs) by immunoassay and one-dimensional Western blot. The majority of sera had immunoreactions at 50-56 kDa. Six representative positive infertility sera were used to identify antigens between 40 and 60 kD by two-dimensional Western blot and mass spectrometry. Antigens included aldehyde (retinal) dehydrogenases (ALDH1A1, ALDH1A2, and ALDH7A1), protein disulfide isomerase A3, vimentin, α-enolase, phosphoglycerate dehydrogenase, and selenium-binding protein 1 (SBP1). Sixty percent (24 out of 40) of infertility and POF sera were positive for recombinant ALDH1A1, SBP1, or enolase; 80.7% (21 out of 26) of AOA-positive sera had antibodies to one or more of the three antigens, and only 7% (1 out of 14) of AOA-negative sera had antibodies to recombinant proteins. CONCLUSION(S): ALDH1A1 and SBP1 are unique to ovarian autoimmunity associated with infertility and POF, and may provide the basis for specific tests to identify patients with ovarian autoimmunity.
Asunto(s)
Autoantígenos/sangre , Autoinmunidad/fisiología , Infertilidad Femenina/etiología , Ovario/inmunología , Insuficiencia Ovárica Primaria/etiología , Adolescente , Adulto , Autoantígenos/análisis , Estudios de Casos y Controles , Femenino , Humanos , Inmunoensayo/métodos , Infertilidad Femenina/sangre , Infertilidad Femenina/inmunología , Espectrometría de Masas , Persona de Mediana Edad , Ovario/patología , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/inmunología , Adulto JovenRESUMEN
Wild-type thymidylate synthase (WT-TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT-TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis. The known crystal structures for WT-TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT-TS and mutants are mainly dimers.
Asunto(s)
Mutación , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Dimerización , Estabilidad de Enzimas/efectos de los fármacos , Concentración de Iones de Hidrógeno , Ligandos , Espectrometría de Masas , Modelos Moleculares , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Timidilato Sintasa/genética , Tripsina/metabolismo , Ultracentrifugación , Urea/farmacologíaRESUMEN
Cyanide (CN) is a ubiquitous environmental toxicant. The measurement of CN in whole blood is a common exposure assay, but values are error prone because of CN's rapid metabolism and clearance (t1/2 < 1 h) from this compartment. This study was undertaken to determine whether CN forms covalent adduct(s) with plasma proteins that could serve as stable biomarker(s) and potential surrogate(s) of exposure. When added to human blood, plasma, or serum, CN formed covalent adducts with immunoglobulin G (IgG) and serum albumin (HSA) in the plasma fraction. Covalent adducts were not detected in the cellular, primarily erythrocyte, fraction. With human, mouse, and rabbit IgGs, the reaction with CN occurred at intra- and/or interchain disulfide linkages in the heavy and light chains. Digestion of CN-treated HSA with trypsin or the endoproteinase Lys-C at basic pH produced tautomeric 2-iminothiazoline-4-carboxylyl/2-aminothiazolidine-4-carboxylyl (itcCys) N-terminal peptides exclusively, consistent with prior model peptide/protein studies showing that under basic conditions internal S-cyanylated-Cys residues cyclize with concomitant release of the upstream peptide. The most readily detectable reaction of CN with purified HSA was at Cys34, the only Cys of the 35 present not connected as internal cystines. Because CN does not react with free sulfhydryl groups, it is probable that S-cyanylation at Cys34 occurs at those residues that carry GSH, Cys, or other small molecules as mixed disulfides. Relatively less detectable, modified Cys residues were also identified at positions 53, 124, 392, 477, and 487. When 14CN was added to human serum or whole blood at concentrations spanning a putative nontoxic to lethal range, stable adduct formation with HSA occurred in a linear, concentration-dependent reaction that was complete within 2 h. These attributes of the reaction, coupled with a plasma compartment location, suggest that quantitation of CN bound to HSA would provide a much more reliable assessment of exposure than does measurement of CN in blood.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cianuros/química , Secuencia de Aminoácidos , HumanosRESUMEN
Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD65 and GAD67, were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD65 and GAD67 exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the Km of GAD67 and increasing the Vmax of both isoforms. Of the 15 cysteines in GAD65, the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD67, cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD65 and GAD67 are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD65 suggests that alteration of normal redox conditions could affect GAD targeting.
Asunto(s)
Cisteína/metabolismo , Glutamato Descarboxilasa/metabolismo , Isoenzimas/metabolismo , Secuencia de Aminoácidos , Animales , Dimerización , Disulfuros/química , Disulfuros/metabolismo , Electroforesis en Gel de Poliacrilamida , Glutamato Descarboxilasa/química , Isoenzimas/química , Cinética , Datos de Secuencia Molecular , Peso Molecular , Oxidación-Reducción , Conformación Proteica , Desnaturalización Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Heavy metals are well known to be able to induce immunotoxicity, but comparative metal studies related to apoptosis have not been conducted. In the present study, the effects of arsenic, cadmium, gold, lead, manganese, and mercury on thymocytes from BALB/c mice were analyzed. Thymic cells were cultured for 3-24 h in vitro in the absence or presence of metal, and markers of apoptosis or cell death, including annexin V binding, DNA loss/oligonucleosomal fragmentation, 7-amino-actinomycin D uptake (loss of impermeance), changes of the mitochondrial membrane potential (JC-1 fluorescence), and Western analysis of cellular thiols, were assayed. Mercury (Hg) was the only metal shown to be consistently toxic with the dose and times utilized. Cadmium (Cd) was the only other metal tested that also produced some significant level of DNA loss; however, the induction of apoptosis by Cd was not as consistent as that observed with Hg. When Hg was added with 2-mercaptoethanol (2-ME), Hg produced greater toxicity. Endogenous DNA synthesis by thymocytes was immediately inhibited by Hg and Hg + 2-ME. The Hg + 2-ME-induced apoptosis appeared to be associated with altered levels of cellular thiols, in that glutathione (GSH) depletion was significant in comparison to the non-metal control and Hg alone. The increased Hg-induced toxicity in the presence of 2-ME likely was due to the ability of 2-ME to enhance (10- to 20-fold) the cellular uptake of Hg. Western analysis with biotin maleimide demonstrated that Hg + 2-ME and to a lesser extent the positive control dexamethasone eliminated many reactive thiols; the major thiol-reactive protein still reactive with the maleimide probe had an approximate Molecular Mass of 45 kD. Surprisingly, Hg alone enhanced the expression of this thiol-expressing protein, which by Mass Spectrometry (MS)/MS analysis was shown to be beta-actin. Hg also produced the appearance of yet to be identified new proteins. Based on the results with Hg + 2-ME, it is suggested that numerous protein thiols participate in maintenance of cell survival and their loss is associated with apoptosis. The increased expression of new thiol-reactive proteins or thiol-reactive proteins with altered electrophoretic profiles needs to be further investigated. However, the enhanced toxicity attributed to Hg + 2-ME suggests that increased intracellular oxidative stress, observed as increased depletion of GSH, is responsible for the accelerated cell death.
Asunto(s)
Apoptosis , ADN/efectos de los fármacos , Metales Pesados/toxicidad , Compuestos de Sulfhidrilo/metabolismo , Linfocitos T/efectos de los fármacos , Animales , Células Cultivadas , ADN/biosíntesis , ADN/metabolismo , Fragmentación del ADN , Sinergismo Farmacológico , Citometría de Flujo , Glutatión/análisis , Glutatión/metabolismo , Masculino , Mercaptoetanol/química , Mercaptoetanol/farmacología , Cloruro de Mercurio/química , Cloruro de Mercurio/toxicidad , Mercurio/análisis , Metales Pesados/química , Ratones , Ratones Endogámicos BALB C , Compuestos de Sulfhidrilo/análisis , Linfocitos T/química , Linfocitos T/metabolismo , Factores de TiempoRESUMEN
Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa. We found previously that an element in the proximal promoter region of CYP2A3 (the nasal predominant transcriptional activating (NPTA) element), which is similar to a nuclear factor 1 (NFI)-binding site, is critical for transcriptional activation of CYP2A3 in vitro. We proposed that this element might be important for tissue-selective CYP2A3 expression. The goals of the present study were to characterize NPTA-binding proteins and to obtain more definitive evidence for the role of NFI in the transcriptional activation of CYP2A3. The NPTA-binding proteins were isolated by DNA-affinity purification from rat olfactory mucosa. Mass spectral analysis indicated that isoforms corresponding to all four NFI genes were present in the purified NPTA-binding fraction. Further analysis of NPTA-binding proteins led to the identification of a novel NFI-A isoform, NFI-A-short, which was derived from alternative splicing of the NFI-A transcript. Transient transfection assay showed that NFI-A2, an NFI isoform previously identified in the olfactory mucosa, transactivated the CYP2A3 promoter, whereas NFI-A-short, which lacks the transactivation domain, counteracted the activation. Chromatin immunoprecipitation assays indicated that NFI proteins are associated with the CYP2A3 promoter in vivo, in rat olfactory mucosa, but essentially not in the liver where the CYP2A3 promoter is hypermethylated and CYP2A3 is not expressed. These data strongly support a role for NFI transcription factors in the transcriptional activation of CYP2A3.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Regulación de la Expresión Génica , Oxigenasas de Función Mixta/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transcripción Genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/metabolismo , Codón , Citocromo P-450 CYP2A6 , Citosina/metabolismo , Metilación de ADN , Dimerización , Genes Reporteros , Vectores Genéticos , Humanos , Hígado/metabolismo , Luciferasas/metabolismo , Masculino , Espectrometría de Masas , Modelos Genéticos , Datos de Secuencia Molecular , Factores de Transcripción NFI , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Distribución Tisular , Activación Transcripcional , TransfecciónRESUMEN
Mycoplasma arthritidis-derived mitogen (MAM), a bacterial superantigen, has been crystallized in complex with its human receptor, major histocompatibility complex (MHC) class II antigen, by the hanging-drop vapor-diffusion method. Crystals were obtained under three conditions, with ammonium sulfate, phosphate salt and PEG 8000 as the precipitant. The crystals grown under these conditions all belong to space group I222, with the same unit-cell parameters: a = 137.4, b = 178.2, c = 179.6 A. Diffraction data were collected to 3.3 and 3.4 A resolution from crystals of native and selenomethionylated MAM-MHC complexes, respectively. Self- and cross-rotation function calculations suggest the presence of two complex molecules in the asymmetric unit, resulting in a V(M) of 4.0 and a solvent content of 69%. An interpretable electron-density map was produced using a combination of molecular replacement and SAD phasing.
Asunto(s)
Complejo Mayor de Histocompatibilidad , Mitógenos/química , Mycoplasma arthritidis/metabolismo , Antígenos , Antígenos Bacterianos/química , Cromatografía , Cristalografía por Rayos X/métodos , Difusión , Electrones , Escherichia coli/metabolismo , Glutatión Transferasa/metabolismo , Humanos , Cinética , Espectrometría de Masas , Péptidos/química , Polietilenglicoles/química , Proteínas , Proteínas Recombinantes/química , Selenometionina/química , Superantígenos , Factores de TiempoRESUMEN
Recombinant human bile salt-stimulated lipase (hBSSL) was expressed in and secreted by Pichia pastoris, an organism exploited for the large-scale production of recombinant (glyco)proteins by bioprocessing technology. The 76.3-kDa glycoprotein was associated with 75-80 Man and a small amount of GlcNAc. hBSSL has one N-glycosylation site at Asn187, which was 38-40% occupied with a Man(10)GlcNAc(2) structure defined previously in Pichia as the oligosaccharide-lipid form of Man(9)GlcNAc(2) trimmed of the middle-arm terminal alpha 1,2-Man and elongated with Man alpha 1,2Man alpha 1,6-disaccharide attached to the lower-arm core alpha 1,3-Man (Trimble et al. [1991], J. Biol. Chem., 266, 22807-22817). The C-terminal 192 residues of hBSSL contain 16 Pro-rich 11-amino-acid repeats, which include 32 Ser/Thr residues as potential O-glycosylation sites. Using hBSSL as a platform to study Pichia's O-glycosylation capabilities, we found that nearly all of these sites were occupied by mannose-containing O-glycans, whose structures, after beta-elimination and purification, were assigned by (1)H NMR and, in some cases, by linkage-specific exoglycosidases and methylation analysis. The most abundant O-glycan was alpha 1,2-mannobiitol (55%), followed by alpha 1,2-mannotriitol (16%) and mannitol (10%) and a lesser amount was alpha 1,2-mannotetraitol. Unexpectedly, Man(5) and Man(6) O-glycans were present, which had the structure Man beta 1,2Man beta 1,2Man alpha 1,2(Man alpha 1,2)(1,2)mannitol. Also a small amount of a phosphorylated Man(6) O-glycan was characterized by MALDI-TOF MS postsource decay analysis as having the reducing-end mannitol disubstituted with a glycosidically linked phosphorylated Man and an unbranched Man(4) polymer elongated from a different mannitol carbon. This is the first report of the synthesis of beta-Man- and phosphate-containing O-linked constituents on glycoproteins synthesized by P. pastoris.