RESUMO
Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.
Assuntos
Infecções por Coxsackievirus , Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Humanos , Linfócitos T CD8-Positivos , Anticorpos , Epitopos , Peptídeos , AntiviraisRESUMO
The metaproteomic approach is an attractive way to describe a microbiome at the functional level, allowing the identification and quantification of proteins across a broad dynamic range as well as the detection of post-translational modifications. However, it remains relatively underutilized, mainly due to technical challenges that should be addressed, including the complexity of extracting proteins from heterogeneous microbial communities. Here, we show that a ChipFilter microfluidic device coupled to a liquid chromatography tandem mass spectrometry (LC-MS/MS) setup can be successfully used for the identification of microbial proteins. Using cultures of Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae, we have shown that it is possible to directly lyse the cells and digest the proteins in the ChipFilter to allow the identification of a higher number of proteins and peptides than that by standard protocols, even at low cell density. The peptides produced are overall longer after ChipFilter digestion but show no change in their degree of hydrophobicity. Analysis of a more complex mixture of 17 species from the gut microbiome showed that the ChipFilter preparation was able to identify and estimate the amounts of 16 of these species. These results show that ChipFilter can be used for the proteomic study of microbiomes, particularly in the case of a low volume or cell density. The mass spectrometry data have been deposited on the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD039581.
Assuntos
Consórcios Microbianos , Microfluídica , Cromatografia Líquida , Proteômica , Espectrometria de Massas em Tandem , Escherichia coli/genética , Saccharomyces cerevisiae/genética , PeptídeosRESUMO
The RAC1-WAVE-Arp2/3 signaling pathway generates branched actin networks that power lamellipodium protrusion of migrating cells. Feedback is thought to control protrusion lifetime and migration persistence, but its molecular circuitry remains elusive. Here, we identify PPP2R1A by proteomics as a protein differentially associated with the WAVE complex subunit ABI1 when RAC1 is activated and downstream generation of branched actin is blocked. PPP2R1A is found to associate at the lamellipodial edge with an alternative form of WAVE complex, the WAVE Shell Complex, that contains NHSL1 instead of the Arp2/3 activating subunit WAVE, as in the canonical WAVE Regulatory Complex. PPP2R1A is required for persistence in random and directed migration assays and for RAC1-dependent actin polymerization in cell extracts. PPP2R1A requirement is abolished by NHSL1 depletion. PPP2R1A mutations found in tumors impair WAVE Shell Complex binding and migration regulation, suggesting that the coupling of PPP2R1A to the WAVE Shell Complex is essential to its function.
Assuntos
Actinas , Pseudópodes , Actinas/metabolismo , Movimento Celular/fisiologia , Pseudópodes/metabolismo , Transdução de Sinais , Citoplasma/metabolismo , Fatores de Transcrição/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismoRESUMO
Procalcitonin (PCT) is a widely used biomarker for rapid sepsis diagnosis and antibiotic stewardship. Variability of results in commercial assays has highlighted the need for standardization of PCT measurements. An antibody-free candidate reference measurement procedure (RMP) based on the isotope dilution mass spectrometry and protein calibration approach was developed and validated to quantify PCT in human serum. The method allows quantification of PCT from 0.25 to 13.74 µg/L (R > 0.998) with extension up to 132 µg/L after dilution of samples with PCT concentration above 13.74 µg/L. Intraday bias was between -3.3 and +5.7%, and interday bias was between -3.0 and -0.7%. Intraday precision was below 5.1%, and interday precision was below 4.0%. The candidate RMP was successfully applied to the absolute quantification of PCT in five frozen human serum pools. A recombinant PCT used as a primary calibrator was characterized by high-resolution mass spectrometry and amino acid analysis to establish traceability of the results to the SI units. This candidate RMP is fit to assign target values to secondary certified reference materials (CRMs) for further use in external quality assessment schemes to monitor the accuracy and comparability of the commercially available immunoassay results and to confirm the need for improving the harmonization of PCT assays. The candidate RMP will also be used to evaluate whether the correlation between the candidate RMP and immunoassays is sufficiently high. Overall, this candidate RMP will support reliable sepsis diagnosis and guide treatment decisions, patient monitoring, and outcomes.
Assuntos
Pró-Calcitonina , Sepse , Calibragem , Humanos , Espectrometria de Massas , Proteínas Recombinantes , Padrões de Referência , Sepse/diagnósticoRESUMO
The effects of Auranofin (AF) on protein expression and protein oxidation in A2780 cancer cells were investigated through a strategy based on simultaneous expression proteomics and redox proteomics determinations. Bioinformatics analysis of the proteomics data supports the view that the most critical cellular changes elicited by AF treatment consist of thioredoxin reductase inhibition, alteration of the cell redox state, impairment of the mitochondrial functions, metabolic changes associated with conversion to a glycolytic phenotype, induction of ER stress. The occurrence of the above cellular changes was extensively validated by performing direct biochemical assays. Our data are consistent with the concept that AF produces its effects through a multitarget mechanism that mainly affects the redox metabolism and the mitochondrial functions and results into severe ER stress. Results are discussed in the context of the current mechanistic knowledge existing on AF.
Assuntos
Auranofina , Neoplasias Ovarianas , Auranofina/farmacologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Oxirredução , Proteoma/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismoAssuntos
Peptídeos , Pró-Calcitonina , Calibragem , Cromatografia Líquida , Humanos , Espectrometria de MassasRESUMO
Sepsis represents a global health priority because of its high mortality and morbidity. The key to improving prognosis remains an early diagnosis to initiate appropriate antibiotic treatment. Procalcitonin (PCT) is a recognized biomarker for the early indication of bacterial infections and a valuable tool to guide and individualize antibiotic treatment. To meet the increasing demand for PCT testing, numerous PCT immunoassays have been developed and commercialized, but results have been questioned. Many comparison studies have been carried out to evaluate analytical performance and comparability of results provided by the different commercially available immunoassays for PCT, but results are conflicting. External Quality Assessment Schemes (EQAS) for PCT constitute another way to evaluate results comparability. However, when making this comparison, it must be taken into account that the variety of EQA materials consist of different matrices, the commutability of which has not yet been investigated. The present study gathers results from all published comparison studies and results from 137 EQAS surveys to describe the current state-of-the-art harmonization of PCT results. Comparison studies globally highlight a significant variability of measurement results that nonetheless seem to have a moderate impact on medical decision-making. For their part, EQAS for PCT provides highly discrepant estimates of the interlaboratory CV. Due to differences in commutability of the EQA materials, the results from different peer groups could not be compared. To improve the informative value of the EQA data, the existing limitations such as non-harmonized conditions and suboptimal and/or unknown commutability of the EQA materials have to be overcome. The study highlights the need for commutable reference materials that could be used to properly evaluate result comparability and possibly standardize calibration, if necessary. Such an initiative would further improve the safe use of PCT in clinical routine.
Assuntos
Pró-Calcitonina , Sepse , Calibragem , Humanos , Imunoensaio , Controle de Qualidade , Sepse/diagnósticoRESUMO
The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092-5.222 µg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method's accuracy. The bias ranged from -2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 µg/L (bias -1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.
Assuntos
Calcitonina/química , Espectrometria de Massas/métodos , Pró-Calcitonina/química , Soro/química , Sequência de Aminoácidos , Calibragem , Cromatografia Líquida/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
Communities of bacteria called biofilms are characterized by reduced diffusion, steep oxygen, and redox gradients and specific properties compared to individualized planktonic bacteria. In this study, we investigated whether signaling via nitrosylation of protein cysteine thiols (S-nitrosylation), regulating a wide range of functions in eukaryotes, could also specifically occur in biofilms and contribute to bacterial adaptation to this widespread lifestyle. We used a redox proteomic approach to compare cysteine S-nitrosylation in aerobic and anaerobic biofilm and planktonic Escherichia coli cultures and we identified proteins with biofilm-specific S-nitrosylation status. Using bacterial genetics and various phenotypic screens, we showed that impairing S-nitrosylation in proteins involved in redox homeostasis and amino acid synthesis such as OxyR, KatG, and GltD altered important biofilm properties, including motility, biofilm maturation, or resistance to oxidative stress. Our study therefore revealed that S-nitrosylation constitutes a physiological basis underlying functions critical for E. coli adaptation to the biofilm environment.
Assuntos
Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Aminoácidos/metabolismo , Cisteína/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Homeostase , Mutação , Oxirredução , Fenótipo , Proteoma , Proteômica/métodos , Compostos de Sulfidrila/metabolismoRESUMO
Procalcitonin (PCT) is an important biomarker for sepsis diagnosis and management. To date, there is no higher-order reference measurement procedure (RMP) and certified reference material to achieve global standardization of results and results traceability to the SI units. Although efforts have been made to harmonize PCT results, a number of comparison studies and external quality assessment (EQA) schemes show conflicting results regarding results comparability and to date, equivalence of PCT results across the assays remains questionable in absence of studies relying on commutable EQA materials. In this context, the IFCC initiated activities to fill these gaps through the creation of the working group on standardization of PCT assays that gathers experts from National Metrology Institutes, calibration laboratories, clinicians, biologists, EQA providers and assay manufacturers. Among the activities, a higher order RMP and commutable reference materials are under development to build a robust reference measurement system (RMS). A commutability study is being organized to identify EQA materials that are fit for purpose to reliably estimate the current comparability of PCT results. This work will make it possible to evaluate the necessity and the feasibility for establishing and maintaining a new RMS for PCT assays, if deemed necessary.
Assuntos
Laboratórios , Pró-Calcitonina , Calibragem , Estudos de Viabilidade , Humanos , Padrões de ReferênciaRESUMO
Sample preparation is a crucial step in bottom-up proteomics. Analytical performances of bottom-up proteomics can be improved by the miniaturization of sample preparation. Many microfluidic devices have been designed in the field of proteomics, but many of them are not capable of handling complex samples and do not integrate the processing and digestion steps. We propose a ChipFilter Proteolysis (CFP) microfluidic device as a proteomics reactor for the miniaturization of protein sample processing and digestion steps, whose design is closely related to the experimental setup of filter-aided sample processing, even if no denaturing surfactant is required. The microchip has two reaction chambers of 0.6 µL volume separated by a protein filtration membrane in regenerated cellulose (10kD cutoff) that will concentrate or retain large polypeptides and will release small molecules. Cell lysis, protein concentration, and rapid chemical or enzymatic treatment can be performed in the ChipFilter. Complex proteomic samples like yeast protein extract or whole human cells proteome have been successfully analyzed with our microchip. Compared with the membrane-based commercial ultracentrifugation cartridge, our microfluidic device offered a better proteome coverage with 10 times less starting material and 8 times faster protocol duration.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Humanos , Proteólise , Proteoma , Manejo de EspécimesRESUMO
The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology.
Assuntos
Adenosina Trifosfatases/metabolismo , Apoptose , Óxido Nítrico/metabolismo , Processamento de Proteína Pós-Traducional , Fator 1 Associado a Receptor de TNF/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Animais , Sítios de Ligação/genética , Cisteína/genética , Cisteína/metabolismo , Humanos , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Fator 1 Associado a Receptor de TNF/química , Fator 1 Associado a Receptor de TNF/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genéticaRESUMO
The purpose of the present study is to analyze the serum proteome of patients receiving Radiation Therapy (RT) at different stages of their treatment to discovery candidate biomarkers of the radiation-induced skin lesions and the molecular pathways underlying the radiation signatures. Six stages of RT treatment were monitored from patients treated because of brain cancer: before starting the treatment, during the treatment (four time points), and at 4 weeks from the last RT dose. Serum samples were analyzed by a proteomics approach based on the Data Independent Acquisition (DIA) mass spectrometry (MS). RT induced clear changes in the expression levels of 36 serum proteins. Among these, 25 proteins were down- or up-regulated significantly before the emergence of skin lesions. Some of these were still deregulated after the completion of the treatment. Few days before the appearance of the skin lesions, the levels of some proteins involved in the wound healing processes were down-regulated. The pathway analysis indicated that after partial body irradiation, the expression levels of proteins functionally involved in the acute inflammatory and immune response, lipoprotein process and blood coagulation, were deregulated.
Assuntos
Proteínas Sanguíneas/metabolismo , Proteoma/metabolismo , Proteômica , Lesões por Radiação/sangue , Radioterapia/efeitos adversos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Protein tyrosine phosphatase, nonreceptor type 2 (PTPN2) is mainly expressed in hematopoietic cells, where it negatively regulates growth factor and cytokine signaling. PTPN2 is an important regulator of hematopoiesis and immune/inflammatory responses, as evidenced by loss-of-function mutations of PTPN2 in leukemia and lymphoma and knockout mice studies. Benzene is an environmental chemical that causes hematological malignancies, and its hematotoxicity arises from its bioactivation in the bone marrow to electrophilic metabolites, notably 1,4-benzoquinone, a major hematotoxic benzene metabolite. Although the molecular bases for benzene-induced leukemia are not well-understood, it has been suggested that benzene metabolites alter topoisomerases II function and thereby significantly contribute to leukemogenesis. However, several studies indicate that benzene and its hematotoxic metabolites may also promote the leukemogenic process by reacting with other targets and pathways. Interestingly, alterations of cell-signaling pathways, such as Janus kinase (JAK)/signal transducer and activator of transcription (STAT), have been proposed to contribute to benzene-induced malignant blood diseases. We show here that 1,4-benzoquinone directly impairs PTPN2 activity. Mechanistic and kinetic experiments with purified human PTPN2 indicated that this impairment results from the irreversible formation (kinact = 645 m-1·s-1) of a covalent 1,4-benzoquinone adduct at the catalytic cysteine residue of the enzyme. Accordingly, cell experiments revealed that 1,4-benzoquinone exposure irreversibly inhibits cellular PTPN2 and concomitantly increases tyrosine phosphorylation of STAT1 and expression of STAT1-regulated genes. Our results provide molecular and cellular evidence that 1,4-benzoquinone covalently modifies key signaling enzymes, implicating it in benzene-induced malignant blood diseases.
Assuntos
Benzeno , Benzoquinonas/metabolismo , Leucemia , Proteínas de Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 2 , Fator de Transcrição STAT1 , Transdução de Sinais/efeitos dos fármacos , Benzeno/farmacocinética , Benzeno/farmacologia , Células HEK293 , Humanos , Células Jurkat , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 2/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais/genéticaRESUMO
Although CD8+ T-cell-mediated autoimmune ß cell destruction occurs in type 1 diabetes (T1D), the target epitopes processed and presented by ß cells are unknown. To identify them, we combined peptidomics and transcriptomics strategies. Inflammatory cytokines increased peptide presentation in vitro, paralleling upregulation of human leukocyte antigen (HLA) class I expression. Peptide sources featured several insulin granule proteins and all known ß cell antigens, barring islet-specific glucose-6-phosphatase catalytic subunit-related protein. Preproinsulin yielded HLA-A2-restricted epitopes previously described. Secretogranin V and its mRNA splice isoform SCG5-009, proconvertase-2, urocortin-3, the insulin gene enhancer protein ISL-1, and an islet amyloid polypeptide transpeptidation product emerged as antigens processed into HLA-A2-restricted epitopes, which, as those already described, were recognized by circulating naive CD8+ T cells in T1D and healthy donors and by pancreas-infiltrating cells in T1D donors. This peptidome opens new avenues to understand antigen processing by ß cells and for the development of T cell biomarkers and tolerogenic vaccination strategies.
Assuntos
Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Epitopos de Linfócito T/imunologia , Transcriptoma/imunologia , Animais , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Hormônio Liberador da Corticotropina/metabolismo , Citocinas/metabolismo , Antígenos HLA/metabolismo , Humanos , Insulina/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Camundongos , Proteína Secretora Neuroendócrina 7B2/metabolismo , Pró-Proteína Convertase 2/metabolismo , Precursores de Proteínas/metabolismo , Proteômica/métodos , Urocortinas/metabolismoRESUMO
BAG3 protein is an apoptosis inhibitor and is highly expressed in Anaplastic Thyroid Cancer. We investigated the entire set of proteins modulated by BAG3 silencing in the human anaplastic thyroid 8505C cancer cells by using the Stable-Isotope Labeling by Amino acids in Cell culture strategy combined with mass spectrometry analysis. By this approach we identified 37 up-regulated and 54 down-regulated proteins in BAG3-silenced cells. Many of these proteins are reportedly involved in tumor progression, invasiveness and resistance to therapies. We focused our attention on an oncogenic protein, CAV1, and a tumor suppressor protein, SERPINB2, that had not previously been reported to be modulated by BAG3. Their expression levels in BAG3-silenced cells were confirmed by qRT-PCR and western blot analyses, disclosing two novel targets of BAG3 pro-tumor activity. We also examined the dataset of proteins obtained by the quantitative proteomics analysis using two tools, Downstream Effect Analysis and Upstream Regulator Analysis of the Ingenuity Pathways Analysis software. Our analyses confirm the association of the proteome profile observed in BAG3-silenced cells with an increase in cell survival and a decrease in cell proliferation and invasion, and highlight the possible involvement of four tumor suppressor miRNAs and TP53/63 proteins in BAG3 activity.
RESUMO
The redox conditions that reign inside a cell have a determining effect on a number of biological processes. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are key redox players and have been linked to a number of pathologies. They have also been shown to play an important regulating role in cell signaling events. On the proteome level, thiol groups of cysteinyl side chains constitute the major targets of ROS and RNS. A number of analytical techniques based on mass spectrometry have been developed to characterize the cysteine redoxome, often facing a number of technical challenges, mostly related to the lability, heterogeneity, and low abundance of the oxidized forms. Furthermore, any posttranslational modification (PTM) quantification method needs to take the parent protein's expression level into account. While taking all these limitations into consideration, we have developed a quantitative analytical strategy named OxiTMT, based on chemical labeling with iodoacetyl isobaric tandem mass tags (iodoTMT). OxiTMT allowed the generation of quantitative redox data that could be normalized by the protein's expression profile in up to three different conditions. The method was tested on Escherichia coli with or without an oxidative treatment. Results showed the method to be adequate for the analysis of cysteine PTMs with a good coverage of the cysteine redoxome, especially for the low abundant oxidized species. Some of the challenges that face reporter ion quantification of PTMs by mass spectrometry were also assessed. This study serves as a proof of concept of the established protocol and consequent data treatment step. The use of tandem mass tags opens the ways towards the application of the method to the study of tissues and sera. Graphical abstract OxiTMT workflow.
Assuntos
Cisteína/análise , Proteínas de Escherichia coli/química , Escherichia coli/química , Espectrometria de Massas/métodos , Proteômica/métodos , Acetilação , Sequência de Aminoácidos , Halogenação , Oxirredução , Processamento de Proteína Pós-Traducional , Proteoma/químicaRESUMO
Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.
Assuntos
Cadeias gama de Imunoglobulina/genética , Polimorfismo Genético , Proteômica/métodos , Alelos , Criança , Pré-Escolar , Bases de Dados de Proteínas , Feminino , Humanos , Alótipos Gm de Imunoglobulina , Masculino , Espectrometria de Massas , Peptídeos/metabolismo , Análise de Sequência de DNARESUMO
Dithiocarbamates (DTCs) are important industrial chemicals used extensively as pesticides and in a variety of therapeutic applications. However, they have also been associated with neurotoxic effects and in particular with the development of Parkinson-like neuropathy. Although different pathways and enzymes (such as ubiquitin ligases or the proteasome) have been identified as potential targets of DTCs in the brain, the molecular mechanisms underlying their neurotoxicity remain poorly understood. There is increasing evidence that alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. Interestingly, recent studies with N,N-diethyldithiocarbamate suggest that brain glycogen phosphorylase (bGP) and glycogen metabolism could be altered by DTCs. Here, we provide molecular and mechanistic evidence that bGP is a target of DTCs. To examine this system, we first tested thiram, a DTC pesticide known to display neurotoxic effects, observing that it can react rapidly with bGP and readily inhibits its glycogenolytic activity (kinact = 1.4 × 105 m-1 s-1). Using cysteine chemical labeling, mass spectrometry, and site-directed mutagenesis approaches, we show that thiram (and certain of its metabolites) alters the activity of bGP through the formation of an intramolecular disulfide bond (Cys318-Cys326), known to act as a redox switch that precludes the allosteric activation of bGP by AMP. Given the key role of glycogen metabolism in brain functions and neurodegeneration, impairment of the glycogenolytic activity of bGP by DTCs such as thiram may be a new mechanism by which certain DTCs exert their neurotoxic effects.
Assuntos
Glicogênio Fosforilase Encefálica/química , Neurotoxinas/química , Tiocarbamatos/química , Glicogênio/metabolismo , Glicogênio Fosforilase Encefálica/genética , Glicogênio Fosforilase Encefálica/metabolismo , Humanos , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/metabolismo , Neurotoxinas/toxicidade , Tiocarbamatos/toxicidadeRESUMO
Brain glycogen and its metabolism are increasingly recognized as major players in brain functions. Moreover, alteration of glycogen metabolism in the brain contributes to neurodegenerative processes. In the brain, both muscle and brain glycogen phosphorylase isozymes regulate glycogen mobilization. However, given their distinct regulatory features, these two isozymes could confer distinct metabolic functions of glycogen in brain. Interestingly, recent proteomics studies have identified isozyme-specific reactive cysteine residues in brain glycogen phosphorylase (bGP). In this study, we show that the activity of human bGP is redox-regulated through the formation of a disulfide bond involving a highly reactive cysteine unique to the bGP isozyme. We found that this disulfide bond acts as a redox switch that precludes the allosteric activation of the enzyme by AMP without affecting its activation by phosphorylation. This unique regulatory feature of bGP sheds new light on the isoform-specific regulation of glycogen phosphorylase and glycogen metabolism.