RESUMO
BACKGROUND AND OBJECTIVES: There is an urgent need to discover blood-based biomarkers of multiple sclerosis (MS) to better define the underlying biology of relapses and monitor disease progression. The main goal of this study is to search for candidate biomarkers of MS relapses associated with circulating extracellular vesicles (EVs), an emerging tool for biomarker discovery. METHODS: EVs, purified from unpaired plasma and CSF samples of RRMS patients by size-exclusion chromatography (SEC), underwent proteomic analysis to discover novel biomarkers associated with MS relapses. The candidate biomarkers of disease activity were detected by comparison approach between plasma- and CSF-EV proteomes associated with relapses. Among them, a selected potential biomarker was evaluated in a cohort of MS patients, using a novel and highly reproducible flow cytometry-based approach in order to detect low abundant EV subsets in a complex body fluid such as plasma. RESULTS: The proteomic profiles of both SEC-purified plasma EVs (from 6 patients in relapse and 5 patients in remission) and SEC-purified CSF EVs (from 4 patients in relapse and 3 patients in remission) revealed a set of proteins associated with MS relapses significant enriched in the synaptic transmission pathway. Among common proteins, excitatory amino-acid transporter 2, EAAT2, responsible for the majority of the glutamate uptake in CNS, was worthy of further investigation. By screening plasma samples from 110 MS patients, we found a significant association of plasma EV-carried EAAT2 protein (EV-EAAT2) with MS relapses, regardless of disease-modifying therapies. This finding was confirmed by investigating the presence of EV-EAAT2 in plasma samples collected longitudinally from 10 RRMS patients, during relapse and remission. Moreover, plasma EV-EAAT2 levels correlated positively with Expanded Disability Status Scale (EDSS) score in remitting MS patients but showed a negative correlation with age in patients with secondary progressive (SPMS). CONCLUSION: Our results emphaticize the usefulness of plasma EVs as a source of accessible biomarkers to remotely analyse the CNS status. Plasma EV-EAAT2 showed to be a promising biomarker for MS relapses. Further studies are required to assess the clinical relevance of this biomarker also for disability progression independent of relapse activity and transition from RRMS towards SPMS.
Assuntos
Transportador 2 de Aminoácido Excitatório , Vesículas Extracelulares , Esclerose Múltipla , Proteômica , Humanos , Vesículas Extracelulares/metabolismo , Masculino , Feminino , Adulto , Proteômica/métodos , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Esclerose Múltipla/sangue , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/líquido cefalorraquidiano , Esclerose Múltipla Recidivante-Remitente/sangue , Estudos de CoortesRESUMO
Polybrominated diphenyl ethers (PBDEs) are persistent organic chemicals implied as flame retardants. Humans are mainly exposed to BDE-47, -99, and -209 congeners by diet. PBDEs are metabolic disruptors with the liver as the main target organ. To investigate their mode of action at a human-relevant concentration, we exposed HepG2 cells to these congeners and their mixture at 1 nM, analyzing their transcriptomic and proteomic profiles. KEGG pathways and GSEA Hallmarks enrichment analyses evidenced that BDE-47 disrupted the glucose metabolism and hypoxia pathway; all the congeners and the MIX affected lipid metabolism and signaling Hallmarks regulating metabolism as mTORC1 and PI3K/AKT/MTOR. These results were confirmed by glucose secretion depletion and increased lipid accumulation, especially in BDE-47 and -209 treated cells. These congeners also affected the EGFR/MAPK signaling; further, BDE-47 enriched the estrogen pathway. Interestingly, BDE-209 and the MIX increased ERα gene expression, whereas all the congeners and the MIX induced ERß and PPARα. We also found that PBDEs modulated several lncRNAs and that HNRNAP1 represented a central hub in all the four interaction networks. Overall, the PBDEs investigated affected glucose and lipid metabolism with different underlying modes of action, as highlighted by the integrated omics analysis, at a dietary relevant concentration. These results may support the mechanism-based risk assessment of these compounds in relation to liver metabolism disruption.
Assuntos
Éteres Difenil Halogenados , Metabolismo dos Lipídeos , Humanos , Éteres Difenil Halogenados/toxicidade , Células Hep G2 , Glucose , Transcriptoma , Proteômica , Fosfatidilinositol 3-Quinases/metabolismo , DietaRESUMO
Saposin (Sap) C is an essential cofactor for the lysosomal degradation of glucosylceramide (GC) by glucosylceramidase (GCase) and its functional impairment underlies a rare variant form of Gaucher disease (GD). Sap C promotes rearrangement of lipid organization in lysosomal membranes favoring substrate accessibility to GCase. It is characterized by six invariantly conserved cysteine residues involved in three intramolecular disulfide bonds, which make the protein remarkably stable to acid environment and degradation. Five different mutations (i.e. p.C315S, p.342_348FDKMCSKdel, p.L349P, p.C382G and p.C382F) have been identified to underlie Sap C deficiency. The molecular mechanism by which these mutations affect Sap C function, however, has not been delineated in detail. Here, we characterized biochemically and functionally four of these gene lesions. We show that all Sap C mutants are efficiently produced, and exhibit lipid-binding properties, modulatory behavior on GCase activity and subcellular localization comparable with those of the wild-type protein. We then delineated the structural rearrangement of these mutants, documenting that most proteins assume diverse aberrant disulfide bridge arrangements, which result in a substantial diminished half-life, and rapid degradation via autophagy. These findings further document the paramount importance of disulfide bridges in the stability of Sap C and provide evidence that accelerated degradation of the Sap C mutants is the underlying pathogenetic mechanism of Sap C deficiency.
Assuntos
Doença de Gaucher/genética , Doença de Gaucher/metabolismo , Lisossomos/metabolismo , Mutação , Saposinas/genética , Saposinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Expressão Gênica , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estabilidade Proteica , Transporte Proteico , Proteólise , Saposinas/química , Saposinas/deficiência , Alinhamento de SequênciaRESUMO
Chlorpyrifos (CPF) is a widely used pesticide inducing adverse neurodevelopmental and reproductive effects. However, knowledge of the underlying mechanisms is limited, particularly in the hypothalamus. We investigated the mode of action of CPF at human relevant concentrations (1 nM-100 nM) in immortalized mouse hypothalamic GnRH neurons (GT1-7), an elective model for studying disruption of the hypothalamus-pituitary-gonads (HPG) axis. We firstly examined cell vitality, proliferation, and apoptosis/necrosis. At not-cytotoxic concentrations, we evaluated neuron functionality, gene expression, Transmission Electron Microscopy (TEM) and proteomics profiles, validating results by immunofluorescence and western blotting (WB). CPF decreased cell vitality with a dose-response but did not affect cell proliferation. At 100 nM, CPF inhibited gene expression and secretion of GnRH; in addition, CPF reduced the immunoreactivity of the neuronal marker Map2 in a dose-dependent manner. The gene expression of Estrogen Receptor α and ß (Erα, Erß), Androgen Receptor (Ar), aromatase and oxytocin receptor was induced by CPF with different trends. Functional analysis of differentially expressed proteins identified Autophagy, mTOR signaling and Neutrophil extracellular traps (NETs) formation as significant pathways affected at all concentrations. This finding was phenotypically supported by the TEM analysis, showing marked autophagy and damage of mitochondria, as well as by protein analysis demonstrating a dose-dependent decrease of mTOR and its direct target pUlk1 (Ser 757). The bioinformatics network analysis identified a core module of interacting proteins, including Erα, Ar, mTOR and Sirt1, whose down-regulation was confirmed by WB analysis. Overall, our results demonstrate that CPF is an inhibitor of the mTOR pathway leading to autophagy in GnRH neurons; a possible involvement of the Erα/Ar signaling is also suggested. The evidence for adverse effects of CPF in the hypothalamus in the nanomolar range, as occurs in human exposure, increases concern on potential adverse outcomes induced by this pesticide on the HPG axis.
Assuntos
Autofagia , Clorpirifos , Hormônio Liberador de Gonadotropina , Neurônios , Transdução de Sinais , Serina-Treonina Quinases TOR , Autofagia/efeitos dos fármacos , Animais , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Clorpirifos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Inseticidas/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Hipotálamo/metabolismo , Hipotálamo/efeitos dos fármacos , Linhagem Celular , Apoptose/efeitos dos fármacosRESUMO
Parabens are preservatives found in cosmetics, processed foods, and medications. The harmful repercussions on the central nervous system by one of the most common parabens, propylparaben (PrP), are yet unknown, especially during development. In this study, the neurodevelopmental effects of PrP and long-term neurotoxicity were investigated in the zebrafish model, using an integrated approach. Zebrafish embryos were exposed to two different concentrations of PrP (10 and 1000 µg/L), then larvae were examined for their behavioral phenotypes (open-field behavior, startle response, and circadian rhythmicity) and relevant brain markers (cyp19a1b, pax6a, shank3a, and gad1b). Long-term behavioral and cognitive impacts on sociability, cerebral functional asymmetry and thigmotaxis were also examined on juveniles at 30 dpf and 60 dpf. Moreover, proteomics and gene expression analysis were assessed in brains of 60 dpf zebrafish. Interestingly, thigmotaxis was decreased by the high dose in larvae and increased by the low dose in juveniles. The expression of shank3a and gad1b genes was repressed by both PrP concentrations pointing to possible effects of PrP on neurodevelopment and synaptogenesis. Proteomics analysis evidenced alterations related to brain development and lipid metabolism. Overall, the results demonstrated that early-life exposure to PrP promotes developmental and persistent neurobehavioral alterations in the zebrafish model, affecting genes and protein levels possibly associated with brain diseases.
Assuntos
Parabenos , Peixe-Zebra , Animais , Parabenos/toxicidade , Parabenos/metabolismo , Larva , Conservantes FarmacêuticosRESUMO
The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Fenótipo , Proteômica , Células de Schwann/patologia , Fator de Crescimento Transformador beta/genética , Invasividade NeoplásicaRESUMO
We prospectively studied the pharmacokinetics (PK) and clinical outcomes of intravenous busulfan (Bu) in 71 children with preexisting liver damage who underwent hematopoietic stem cell transplantation for thalassemia. Intravenous Bu was administered every 6 hours as part of a conditioning regimen with PK-based dose adjustment to target a conservative area under the concentration-versus-time curve (AUC) range (900-1350 microMol*min). The first-dose Bu clearance (CL) was significantly higher than the subsequent daily CL that remained unchanged in the ensuing days. One-third of patients required dose escalation based on dose 1 AUC, whereas dose reduction was needed in the subsequent days. At doses 5, 9, and 13, 78%, 81%, and 87% of patients, respectively, achieved the target range of AUC. A population PK analysis confirmed that the first-dose CL was 20% higher and that body weight was the most important covariate to explain PK variability. Patients with variant GSTA1*B had a 10% lower Bu CL than wild-type. These results suggest that the disease-specific behavior of intravenous Bu PK should be considered for PK-guided dose adjustment in patients with thalassemia, and the use of a conservative AUC range resulted in low toxicity, good engraftment, and good survival rate.
Assuntos
Bussulfano/administração & dosagem , Bussulfano/farmacocinética , Transplante de Células-Tronco Hematopoéticas , Talassemia/metabolismo , Talassemia/terapia , Adolescente , Adulto , Sequência de Bases , Bussulfano/farmacologia , Criança , Pré-Escolar , Primers do DNA/genética , Intervalo Livre de Doença , Monitoramento de Medicamentos , Feminino , Genótipo , Glutationa Transferase/genética , Sobrevivência de Enxerto , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/farmacocinética , Imunossupressores/farmacologia , Lactente , Injeções Intravenosas , Masculino , Taxa de Depuração Metabólica , Estudos Prospectivos , Talassemia/tratamento farmacológico , Resultado do Tratamento , Adulto JovemRESUMO
This work describes the activity of 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and of its newly identified carboxylic acid metabolite on the human malaria parasite Plasmodium falciparum. NBDHEX has been previously identified as a potent cytotoxic agent against murine and human cancer cells as well as towards the protozoan parasite Giardia duodenalis. We show here that NBDHEX is active in vitro against all blood stages of P. falciparum, with the rare feature of killing the parasite stages transmissible to mosquitoes, the gametocytes, with a 4-fold higher potency than that on the pathogenic asexual stages. This activity importantly translates into blocking parasite transmission through the Anopheles vector in mosquito experimental infections. A mass spectrometry analysis identified covalent NBDHEX modifications in specific cysteine residues of five gametocyte proteins, possibly associated with its antiparasitic effect. The carboxylic acid metabolite of NBDHEX retains the gametocyte preferential inhibitory activity of the parent compound, making this novel P. falciparum transmission-blocking chemotype at least as a new tool to uncover biological processes targetable by gametocyte selective drugs. Both NBDHEX and its carboxylic acid metabolite show very limited in vitro cytotoxicity on VERO cells. This result and previous evidence that NBDHEX shows an excellent in vivo safety profile in mice and is orally active against human cancer xenografts make these molecules potential starting points to develop new P. falciparum transmission-blocking agents, enriching the repertoire of drugs needed to eliminate malaria.
RESUMO
The discovery of new biomarkers for Alzheimer's disease (AD) is essential for an accurate diagnosis, to conceive new strategies of treatments, and for monitoring the efficacy of potential disease-modifying therapies in clinical trials. proNGF levels in the cerebrospinal fluid (CSF) represent a promising diagnostic biomarker for AD, but its validation was hampered by the absence of a reliable immunoassay. In the literature, proNGF is currently measured in postmortem brain tissue by semiquantitative immunoblot. Here we describe the development and validation of a new method to measure proNGF in the CSF of living patients. This method, based on molecular size separation by capillary electrophoresis, is automated and shows a 40-fold increase in sensitivity with respect to the proNGF immunoblot, largely used in literature, and is robust, specific, and scalable to high-throughput. We have measured proNGF in the cerebrospinal fluid of 84 living patients with AD, 13 controls, and 15 subjective memory complaints (SMC) subjects. By comparing the proNGF levels in the three groups, we found a very significant difference between proNGF levels in AD samples compared with both controls and SMC subjects, while no significant difference was found between SMC and controls. Because of the development of this new immunoassay, we are ready to explore the potentiality of proNGF as a new biomarker for AD or subgroups thereof, as well as for other neurodegenerative diseases.
RESUMO
Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/-2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.
RESUMO
The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II-IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0-I, II and III-IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0-I) from late (III-IV) stages' CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III-IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV' FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.
RESUMO
Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disease most commonly caused by autosomal dominant mutations in the SPG4 gene encoding the microtubule-severing protein spastin. We hypothesise that SPG4-HSP is attributable to reduced spastin function because of haploinsufficiency; thus, therapeutic approaches which elevate levels of the wild-type spastin allele may be an effective therapy. However, until now, how spastin levels are regulated is largely unknown. Here, we show that the kinase HIPK2 regulates spastin protein levels in proliferating cells, in differentiated neurons and in vivo. Our work reveals that HIPK2-mediated phosphorylation of spastin at S268 inhibits spastin K48-poly-ubiquitination at K554 and prevents its neddylation-dependent proteasomal degradation. In a spastin RNAi neuronal cell model, overexpression of HIPK2, or inhibition of neddylation, restores spastin levels and rescues neurite defects. Notably, we demonstrate that spastin levels can be restored pharmacologically by inhibiting its neddylation-mediated degradation in neurons derived from a spastin mouse model of HSP and in patient-derived cells, thus revealing novel therapeutic targets for the treatment of SPG4-HSP.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Paraplegia Espástica Hereditária/metabolismo , Espastina/metabolismo , Animais , Proteínas de Transporte/fisiologia , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Mutação , Neuritos/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteólise , Paraplegia Espástica Hereditária/fisiopatologia , Espastina/fisiologia , Sinapses/metabolismo , UbiquitinaçãoRESUMO
Natural killer (NK) cells contribute to immunosurveillance and first-line defense in the control of tumor growth and metastasis diffusion. NK-cell-derived extracellular vesicles (NKEVs) are constitutively secreted and biologically active. They reflect the protein and genetic repertoire of originating cells, and exert antitumor activity in vitro and in vivo. Cancer can compromise NK cell functions, a status potentially reflected by their extracellular vesicles. Hence, NKEVs could, on the one hand, contribute to improve cancer therapy by interacting with tumor and/or immune cells and on the other hand, sense the actual NK cell status in cancer patients. Here, we investigated the composition of healthy donors' NKEVs, including NK microvesicles and exosomes, and their interaction with uncompromised cells of the immune system. To sense the systemic NK cell status in cancer patients, we developed an immune enzymatic test (NKExoELISA) that measures plasma NK-cell-derived exosomes, captured as tsg101+CD56+ nanovesicles. NKEV mass spectrometry and cytokine analysis showed the expression of NK cell markers, i.e., NKG2D and CD94, perforin, granzymes, CD40L, and other molecules involved in cytotoxicity, homing, cell adhesion, and immune activation, together with EV markers tsg101, CD81, CD63, and CD9 in both NK-derived exosomes and microvesicles. Data are available via Proteome Xchange with identifier PXD014894. Immunomodulation studies revealed that NKEVs displayed main stimulatory functions in peripheral blood mononuclear cells (PBMCs), inducing the expression of human leukocyte antigen DR isotype (HLA-DR) and costimulatory molecules on monocytes and CD25 expression on T cells, which was maintained in the presence of lipopolysaccharide (LPS) and interleukin (IL)-10/transforming growth factor beta (TGFß), respectively. Furthermore, NKEVs increased the CD56+ NK cell fraction, suggesting that effects mediated by NKEVs might be potentially exploited in support of cancer therapy. The measurement of circulating NK exosomes in the plasma of melanoma patients and healthy donors evidenced lower levels of tsg101+CD56+ exosomes in patients with respect to donors. Likewise, we detected lower frequencies of NK cells in PBMCs of these patients. These data highlight the potential of NKExoELISA to sense alterations of the NK cell immune status.
Assuntos
Vesículas Extracelulares/patologia , Imunoensaio/métodos , Células Matadoras Naturais/patologia , Leucócitos Mononucleares/imunologia , Melanoma/imunologia , Antígeno CD56/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Vigilância Imunológica , Imunomodulação , Melanoma/diagnóstico , Monitorização Imunológica , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Mapas de Interação de Proteínas , Proteômica , Fatores de Transcrição/metabolismoRESUMO
Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.
Assuntos
Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/patologia , Junções Intercelulares/metabolismo , Placofilinas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células CACO-2 , Linhagem Celular Tumoral , Células Cultivadas , Centrifugação com Gradiente de Concentração , Fatores de Iniciação em Eucariotos/metabolismo , Resposta ao Choque Térmico , Humanos , Queratinócitos/citologia , Estresse Oxidativo , Proteína I de Ligação a Poli(A)/metabolismo , Ligação Proteica , Transporte Proteico , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
A chemical plant located in Brescia, an industrial city in North-Western Italy, produced polychlorobiphenyls (PCBs) during a 30-50 year period, causing widespread pollution of the surrounding agricultural area. This area contains several small farms, which principally produce veal meat for private consumption of the farmers' families. The pollution went undiscovered for many years, during which period contaminated food was regularly consumed. This paper reports the polychlorodibenzodioxin (PCDD), polychlorodibenzofuran (PCDF) and PCB levels of a serum sample pooled from the consumers of contaminated food, compared to six population groups of the city of Brescia. Four of these groups were selected in order to represent, respectively, the local general population and the residents of three zones of the polluted area, while the last two groups represented, respectively, the present and the former workers of the plant. One human milk sample from one of the consumers of contaminated food was also analyzed. Results show that the consumers of the contaminated food and the former workers of the plant display considerably higher levels than all other groups. The levels of general population and of all other groups were generally similar both to each other and to the range of literature values for unexposed populations. The respective contribution of PCDDs, PCDFs, mono-ortho and non-ortho PCBs (dioxin-like PCBs) to (Toxicity Equivalents) TEQ of the population groups of this study were also compared to literature data: the two groups with a high contamination level, together with the human milk sample, displayed a higher incidence of mono-ortho PCBs and a lower contribution of PCDD, possibly correlated with the source of contamination.
Assuntos
Benzofuranos/sangue , Exposição Ambiental/estatística & dados numéricos , Bifenilos Policlorados/sangue , Dibenzodioxinas Policloradas/análogos & derivados , Grupos Populacionais/estatística & dados numéricos , Idoso , Dibenzofuranos Policlorados , Feminino , Contaminação de Alimentos , Humanos , Itália , Masculino , Manufaturas/toxicidade , Pessoa de Meia-Idade , Exposição Ocupacional/estatística & dados numéricos , Dibenzodioxinas Policloradas/sangueRESUMO
Dystroglycan (DG) is a membrane receptor, belonging to the dystrophin-glycoprotein complex (DGC) and formed by two subunits, α-dystroglycan (α-DG) and ß-dystroglycan (ß -DG). The C-terminal domain of α-DG and the N-terminal extracellular domain of ß -DG are connected, providing a link between the extracellular matrix and the cytosol. Under pathological conditions, such as cancer and muscular dystrophies, DG may be the target of metalloproteinases MMP-2 and MMP-9, contributing to disease progression. Previously, we reported that the C-terminal domain α-DG (483-628) domain is particularly susceptible to the catalytic activity of MMP-2; here we show that the α-DG 621-628 region is required to carry out its complete digestion, suggesting that this portion may represent a MMP-2 anchoring site. Following this observation, we synthesized an α-DG based-peptide, spanning the (613-651) C-terminal region. The analysis of the kinetic and thermodynamic parameters of the whole and the isolated catalytic domain of MMP-2 (cdMMP-2) has shown its inhibitory properties, indicating the presence of (at least) two binding sites for the peptide, both located within the catalytic domain, only one of the two being topologically distinct from the catalytic active groove. However, the different behavior between whole MMP-2 and cdMMP-2 envisages the occurrence of an additional binding site for the peptide on the hemopexin-like domain of MMP-2. Interestingly, mass spectrometry analysis has shown that α-DG (613-651) peptide is cleavable even though it is a very poor substrate of MMP-2, a feature that renders this molecule a promising template for developing a selective MMP-2 inhibitor.
Assuntos
Distroglicanas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Humanos , Cinética , Camundongos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em Tandem , TermodinâmicaRESUMO
This study deals with a PCB, PCDD and PCDF contamination in Brescia, a city in the North-West of Italy, affecting an area with about 11000 inhabitants. The area is close to an industrial plant that produced, in total, some 31,000 ton of PCB. A relevant part of the polluted area is agricultural soil, where cattle were fed with polluted forage and farmers were consuming their own products, so that contamination led eventually to human exposure. Total levels of PCDD/Fs varied from 8 to 592 pgTE(WHO)/g for soil samples and when the dioxin-like PCBs (dl-PCBs) are included, the levels varied from 14.6 to 1033.7 pgTE(WHO)/g. In several cases, the legal limit was exceeded by more than one order of magnitude, with the highest contamination in some agricultural areas and in the surrounding zones. For the forage samples, total levels of PCDD/Fs varied from 0.29 to 2.04 pgTE(WHO)/g and, when dl-PCBs are included, this range increased from 2.04 to 4.75 pgTE(WHO)/g. PCB contamination of the forage through vapor condensation seemed to be relevant. The toxic contribution of dl-PCBs is always relevant and must be considered for risk management. The main component of the contamination source is probably a heavy PCB mixture, such as Aroclor 1262. The study dealt generally with the contamination transfer of PCBs, PCDDs and PCDFs from soil up to humans across the food chain. Results on soils and forages are shown, while measurements concerning the contamination of the animals fed with contaminated forage, and the exposure of the farmers (through human serum analyses), as compared to general population, will be reported in a dedicated paper.
Assuntos
Benzofuranos/análise , Exposição Ambiental , Cadeia Alimentar , Contaminação de Alimentos/análise , Bifenilos Policlorados/análise , Dibenzodioxinas Policloradas/análogos & derivados , Poluentes do Solo/análise , Agricultura , Cidades , Dibenzofuranos Policlorados , Geografia , Humanos , Resíduos Industriais , Itália , Dibenzodioxinas Policloradas/análise , Medição de RiscoRESUMO
Giardiasis, a parasitic diarrheal disease caused by Giardia duodenalis, affects one billion people worldwide. Treatment relies only on a restricted armamentarium of drugs. The disease burden and the increase in treatment failure highlight the need for novel, safe and well characterized drug options. The antitumoral compound NBDHEX is effective in vitro against Giardia trophozoites and inhibits glycerol-3-phosphate dehydrogenase. Aim of this work was to search for additional NBDHEX protein targets. The intrinsic NBDHEX fluorescence was exploited in a proteomic analysis to select and detect modified proteins in drug treated Giardia. In silico structural analysis, intracellular localization and functional assays were further performed to evaluate drug effects on the identified targets. A small subset of Giardia proteins was covalently bound to the drug at specific cysteine residues. These proteins include metabolic enzymes, e.g. thioredoxin reductase (gTrxR), as well as elongation factor 1B-γ (gEF1Bγ), and structural proteins, e.g. α-tubulin. We showed that NBDHEX in vitro binds to recombinant gEF1Bγ and gTrxR, but only the last one could nitroreduce NBDHEX leading to drug modification of gTrxR catalytic cysteines, with concomitant disulphide reductase activity inhibition and NADPH oxidase activity upsurge. Our results indicate that NBDHEX reacts with multiple targets whose roles and/or functions are specifically hampered. In addition, NBDHEX is in turn converted to reactive intermediates extending its toxicity. The described NBDHEX pleiotropic action accounts for its antigiardial activity and encourages the use of this drug as a promising alternative for the future treatment of giardiasis.
Assuntos
Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Giardia lamblia/efeitos dos fármacos , Giardia lamblia/fisiologia , Oxidiazóis/farmacologia , Proteoma/efeitos dos fármacos , Ligação Proteica , Proteômica , Proteínas de Protozoários/análiseRESUMO
Ricotta cheese is a typical Italian product, made with whey from various species, including cow, buffalo, sheep, and goat. Ricotta cheese nominally manufactured from the last three species may be fraudulently produced using the comparatively cheaper cow whey. Exposing such food frauds requires a reliable analytical method. Despite the extensive similarities shared by whey proteins of the four species, a mass spectrometry-based analytical method was developed that exploits three species-specific peptides derived from ß-lactoglobulin and α-lactalbumin. This method can detect as little as 0.5% bovine whey in ricotta cheese from the other three species. Furthermore, a tight correlation was found (R(2)>0.99) between cow whey percentages and mass spectrometry measurements throughout the 1-50% range. Thus, this method can be used for forensic detection of ricotta cheese adulteration and, if properly validated, to provide quantitative evaluations.
Assuntos
Queijo/análise , Contaminação de Alimentos/análise , Espectrometria de Massas/métodos , Soro do Leite/química , Animais , Búfalos , Bovinos , Feminino , Cabras , Lactalbumina/análise , Lactoglobulinas/análise , OvinosRESUMO
AIMS: The biochemistry underlying the physiological, adaptive, and toxic effects of carbon monoxide (CO) is linked to its affinity for reduced transition metals. We investigated CO signaling in the vasculature, where hemoglobin (Hb), the CO most important metal-containing carrier is highly concentrated inside red blood cells (RBCs). RESULTS: By combining NMR, MS, and spectrophotometric techniques, we found that CO treatment of whole blood increases the concentration of reduced glutathione (GSH) in RBC cytosol, which is linked to a significant Hb deglutathionylation. In addition, this process (i) does not activate glycolytic metabolism, (ii) boosts the pentose phosphate pathway (PPP), (iii) increases glutathione reductase activity, and (iv) decreases oxidized glutathione concentration. Moreover, GSH concentration was partially decreased in the presence of 2-deoxyglucose and the PPP antagonist dehydroepiandrosterone. Our MS results show for the first time that, besides Cys93, Hb glutathionylation occurs also at Cys112 of the ß-chain, providing a new potential GSH source hitherto unknown. INNOVATION: This work provides new insights on the signaling and antioxidant-boosting properties of CO in human blood, identifying Hb as a major source of GSH release and the PPP as a metabolic mechanism supporting Hb deglutathionylation. CONCLUSIONS: CO-dependent GSH increase is a new RBC process linking a redox-inactive molecule, CO, to GSH redox signaling. This mechanism may be involved in the adaptive responses aimed to counteract stress conditions in mammalian tissues.