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Xi class glutathione transferases (GSTs) are a recently identified group, within this large superfamily of enzymes, specifically endowed with glutathione-dependent reductase activity on glutathionyl-hydroquinone. Enzymes belonging to this group are widely distributed in bacteria, fungi, and plants but not in higher eukaryotes. Xi class GSTs are also frequently found in archaea and here we focus on the enzyme produced by the extreme haloalkaliphilic archaeon Natrialba magadii (NmGHR). We investigated its function and stability and determined its 3D structure in the apo form by X-ray crystallography. NmGHR displays the same fold of its mesophilic counterparts, is enriched in negatively charged residues, which are evenly distributed along the surface of the protein, and is characterized by a peculiar distribution of hydrophobic residues. A distinctive feature of haloalkaliphilic archaea is their preference for γ-glutamyl-cysteine over glutathione as a reducing thiol. Indeed we found that the N. magadii genome lacks a gene coding for glutathione synthase. Analysis of NmGHR structure suggests that the thiol binding site (G-site) of the enzyme is well suited for hosting γ-glutamyl-cysteine.
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Membrane trafficking via the Golgi-localised KDEL receptor activates signalling cascades that coordinate both trafficking and other cellular functions, including autophagy and extracellular matrix degradation. In this study, we provide evidence that membrane trafficking activates KDEL receptor and the Src family kinases at focal adhesions of HeLa cells, where this phosphorylates ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain (ASAP)1 and focal adhesion kinase (FAK). Previous studies have reported extracellular matrix degradation at focal adhesions. Here, matrix degradation was not seen at focal adhesions, although it occurred at invadopodia, where it was increased by KDEL receptor activation. This activation of KDEL receptor at invadopodia of A375 cells promoted recruitment and phosphorylation of FAK on tyrosines 397 and 861. From the functional standpoint, FAK overexpression inhibited steady-state and KDEL-receptor-stimulated extracellular matrix degradation, whereas overexpression of the FAK-Y397F mutant only inhibited KDEL-receptor-stimulated matrix degradation. Finally, we show that the Src and FAK activated downstream of KDEL receptor are part of parallel signalling pathways. In conclusion, membrane-traffic-generated signalling via KDEL receptor activates Src not only at the Golgi complex, but also at focal adhesions. By acting on Src and FAK, KDEL receptor increases invadopodia-mediated matrix degradation.
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Glutathione transferase classical GSH conjugation activity plays a critical role in cellular detoxification against xenobiotics and noxious compounds as well as against oxidative stress. However, this feature is also exploited by cancer cells to acquire drug resistance and improve their survival. As a result, various members of the family were found overexpressed in a number of different cancers. Moreover several GST polymorphisms, ranging from null phenotypes to point mutations, were detected in members of the family and found to correlate with the onset of neuro-degenerative diseases. In the last decades, a great deal of research aimed at clarifying the role played by GSTs in drug resistance, at developing inhibitors to counteract this activity but also at exploiting GSTs for prodrugs specific activation in cancer cells. Here we summarize some of the most important achievements reached in this lively area of research.
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AIM: To evaluate the antibacterial and antibiofilm mechanisms of usnic acid (USN) against methicillin-resistant Staphylococcus aureus from cystic fibrosis patients. MATERIALS & METHODS: The effects exerted by USN at subinhibitory concentrations on S. aureus Sa3 strain was evaluated by proteomic, real-time PCR and electron microscopy analyses. RESULTS & CONCLUSION: Proteomic analysis showed that USN caused damage in peptidoglycan synthesis, as confirmed by microscopy. Real-time PCR analysis showed that antibiofilm activity of USN is mainly due to impaired adhesion to the host matrix binding proteins, and decreasing lipase and thermonuclease expression. Our data show that USN exerts anti-staphylococcal effects through multitarget inhibitory effects, thus confirming the rationale for considering it 'lead compound' for the treatment of cystic fibrosis infections.
Assuntos
Antibacterianos/farmacologia , Benzofuranos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Adesinas Bacterianas/efeitos dos fármacos , Antibacterianos/administração & dosagem , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzofuranos/administração & dosagem , Proteínas de Transporte/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , DNA Bacteriano , Regulação para Baixo , Lipase/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Propídio/metabolismo , Mapas de Interação de Proteínas , Proteômica/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Infecções Estafilocócicas/microbiologia , Fatores de Tempo , Virulência/efeitos dos fármacos , Virulência/genéticaRESUMO
Aging, amyloid deposition, and tau-related pathology are key contributors to the onset and progression of Alzheimer's disease (AD). However, AD is also associated with brain hypometabolism and deficits of mitochondrial bioenergetics. Plasma acylcarnitines (ACCs) are indirect indices of altered fatty acid beta-oxidation, and ketogenesis has been found to be decreased on aging. Furthermore, in elderly subjects, alterations in plasma levels of specific ACCs have been suggested to predict conversion to mild cognitive impairment (MCI) or AD. In this study, we assayed plasma profiles of ACCs in a cohort of healthy elderly control, MCI subjects, and AD patients. Compared with healthy controls or MCI subjects, AD patients showed significant lower plasma levels of several medium-chain ACCs. Furthermore, in AD patients, these lower concentrations were associated with lower prefrontal gray matter volumes and the presence of cognitive impairment. Interestingly, lower levels of medium-chain ACCs were also found to be associated with lower plasma levels of 2-hydroxybutyric acid. Overall, these findings suggest that altered metabolism of medium-chain ACCs and impaired ketogenesis can be metabolic features of AD.
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Envelhecimento/sangue , Envelhecimento/psicologia , Doença de Alzheimer/patologia , Doença de Alzheimer/parasitologia , Carnitina/análogos & derivados , Cognição/fisiologia , Disfunção Cognitiva/sangue , Disfunção Cognitiva/psicologia , Substância Cinzenta/patologia , Corpos Cetônicos/sangue , Idoso , Envelhecimento/patologia , Doença de Alzheimer/sangue , Carnitina/sangue , Disfunção Cognitiva/patologia , Estudos de Coortes , Feminino , Humanos , Hidroxibutiratos/sangue , MasculinoRESUMO
The analytical tools allow the detection of bioactive compounds, diagnostic agents and chemotherapeutics. Recently, new methods have been developed to analyze pharmaceutical samples and ingredients. In this attempt, analytical parameters, e.g., the lack of trueness, robustness and sensitivity, play a pivotal role to quantify and analyze molecules, both for diagnostic applications as well as therapeutic treatments. Spectrophotometers and spectrofluorometers are apparatus for easy and rapid quantification of molecular probes and chemotherapeutics into cells, plasma and tissues. However, they lack accuracy and precision. Conversely, HPLC provides the maximum resolution to detect and separate fluorescent probes and chemotherapeutics after their incubation in cells, plasma and tissues. The aim of this work was to develop an HPLC method that easily detects molecular and fluorescent probes, e.g., Nile Red, in biological samples. To improve the robustness of the method, Nile Red was analyzed before and after loading into niosomes made from Tween 20 and 21, respectively. A significant difference was further obtained by comparing the entrapment efficacy percentage of niosomes made from Tween 21 (42.23%) and Tween 20 (53.25%). The comparison between HPLC and spectrofluorometer assays showed differences between the two methods in terms of limit of detection, linearity and accuracy. The resulting data demonstrated that the HPLC-FLD provides a limit of detection for Nile Red of 0.1 ng/mL, and a good linearity up to 62.5 ng/mL. The HPLC-FLD analysis showed a limit of quantification value for a total mass of Nile Red 1200-folds better than data previously reported in studies; and 312-folds better than the spectrofluorometer analysis. Additionally, results show that the HPLC-FLD increases the sensitivity for biological samples compared to the spectrofluorometer. The Nile Red-loaded niosomes were also incubated at different times with HEK-293 cells. In vitro results demonstrated that the HPLC-FLD apparatus detects Nile Red-loaded niosomes at higher concentrations into HEK-293 cells than the spectrofluorometer. The intracellular uptake of Nile Red was increased at 120 and 24 min using niosomes made from Tween 20 and 21, respectively, and its intracellular accumulation shows a time-dependent internalization over 120 min of incubation time.
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Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/química , Lipossomos/química , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Química Farmacêutica , Células HEK293 , Humanos , Oxazinas/química , Polissorbatos , Reprodutibilidade dos Testes , TensoativosRESUMO
Multiple Sclerosis (MuS) is a disease caused due to an autoimmune attack against myelin components in which non proteic mediators may play a role. Recent research in metabolomics and lipidomics has been driven by rapid advances in technologies such as mass spectrometry and computational methods. They can be used to study multifactorial disorders like MuS, highlighting the effects of disease on metabolic profiling, regardless of the multiple trigger factors. We coupled MALDI-TOF-MS untargeted lipidomics and targeted LC-MS/MS analysis of acylcarnitines and aminoacids to compare cerebrospinal fluid metabolites in 13 MuS subjects and in 12 patients with Other Neurological Diseases (OND). After data processing and statistical evaluation, we found 10 metabolites that significantly (p < 0.05) segregate the two clinical groups. The most relevant result was the alteration of phospholipids levels in MuS and the correlation between some of them with clinical data. In particular lysophosphatidylcholines (m/z = 522.3 Da, 524.3 Da) and an unidentified peak at m/z = 523.0 Da correlated to the Link index, lysophosphatidylinositol (m/z = 573.3 Da) correlated to EDSS and phosphatidylinositol (m/z = 969.6 Da) correlated to disease duration. We also found high levels of glutamate in MuS. In conclusion, our integrated mass spectrometry approach showed high potentiality to find metabolic alteration in cerebrospinal fluid. These data, if confirmed in a wider clinical study, could open the door for the discovery of novel candidate biomarkers of MuS.
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Metabolômica/métodos , Esclerose Múltipla/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Carnitina/líquido cefalorraquidiano , Ácido Glutâmico/líquido cefalorraquidiano , Humanos , Lipídeos/líquido cefalorraquidiano , MetabolomaRESUMO
OBJECTIVE: To investigate enzymatic reactive aldehyde-scavenging enzyme capacity together with lipid peroxidation as expression of oxidative stress in atherosclerotic plaques of cigarette smokers and nonsmokers. METHODS: We have assessed specific enzymatic activities of class 1, 2, and 3 aldehyde dehydrogenase (ALDH1, ALDH2, and ALDH3, respectively), glutathione S-transferase (isozyme A4-4, GSTA4-4), and aldose reductase (AR), namely the major reactive aldehyde-scavenging enzymes, together with lipid peroxidation, i.e., fluorescent damage products of lipid peroxidation (FDPL), in carotid atherosclerotic plaques surgically removed from 17 cigarette smokers and 17 nonsmokers. RESULTS: The enzymatic activities of ALDH1 plus ALDH2, ALDH3, GSTA4-4, and AR were significantly lower in the atherosclerotic plaques of smokers than in those of nonsmokers, while plaque FDPL levels were significantly higher in the smokers than in the nonsmokers. The amount of cigarette smoking was correlated inversely with the aforementioned plaque enzymatic activities and directly with plaque FDPL content. Plaque FDPL levels were inversely correlated with plaque enzymatic activities in smokers and nonsmokers. The degree of carotid atherosclerotic stenosis, as expression of atherosclerosis severity, was correlated inversely with plaque enzymatic activities and directly with plaque FDPL levels in smokers and nonsmokers; moreover, the degree of carotid stenosis was directly correlated with the amount of cigarette smoking. CONCLUSION: atherosclerotic lesions of cigarette smokers are endowed with a depressed enzymatic reactive aldehyde-scavenging capacity eventually favoring oxidative stress and the severity of atherosclerosis.
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Aldeído Desidrogenase/análise , Aldeído Redutase/análise , Artérias Carótidas/enzimologia , Doenças das Artérias Carótidas/enzimologia , Glutationa Transferase/análise , Placa Aterosclerótica , Fumar/efeitos adversos , Idoso , Família Aldeído Desidrogenase 1 , Aldeído-Desidrogenase Mitocondrial , Biomarcadores/análise , Artérias Carótidas/patologia , Artérias Carótidas/cirurgia , Doenças das Artérias Carótidas/diagnóstico , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/cirurgia , Regulação para Baixo , Feminino , Humanos , Isoenzimas/análise , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Prognóstico , Retinal Desidrogenase/análise , Índice de Gravidade de DoençaRESUMO
Proteomics and metabolomics investigations of body fluids present several challenges for biomarker discovery of several diseases. The search for biomarkers is actually conducted in different body fluids, even if the ideal biomarker can be found in an easily accessible biological fluid, because, if validated, the biomarker could be sought in the healthy population. In this regard, tears could be considered an optimum material obtained by noninvasive procedures. In the past years, the scientific community has become more interested in the study of tears for the research of new biomarkers not only for ocular diseases. In this review, we provide a discussion on the current state of biomarkers research in tears and their relevance for clinical practice, and report the main results of clinical proteomics studies on systemic and eye diseases. We summarize the main methods for tear samples analyses and report recent advances in "omics" platforms for tears investigations. Moreover, we want to take stock of the emerging field of metabolomics and lipidomics as a new and integrated approach to study protein-metabolites interplay for biomarkers research, where tears represent a still unexplored and attractive field.
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Biomarcadores/metabolismo , Oftalmopatias/diagnóstico , Oftalmopatias/metabolismo , Proteínas do Olho/análise , Proteoma/análise , Proteômica/métodos , Lágrimas/química , Animais , Humanos , Metabolômica/métodosRESUMO
p63 is an important regulator of epithelial development expressed in different variants containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem-cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development; however, very little is known about the role played by p63 in regulating the cancer stem phenotype. Here we investigate the cellular signals regulated by TAp63 and ΔNp63 in a model of epithelial cancer stem cells. To this end, we used colon cancer stem cells, overexpressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical-labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy at both protein and metabolite levels. The overall data show that TAp63 overexpressing cells are more glycolytic-active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass-spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with data set identifiers PXD000769 and PXD000768.
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Células-Tronco Neoplásicas/metabolismo , Mapas de Interação de Proteínas/fisiologia , Isoformas de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Humanos , Marcação por Isótopo , Metabolômica , Células-Tronco Neoplásicas/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Isoformas de Proteínas/química , Proteoma/análise , Proteoma/metabolismo , Proteômica , Fatores de Transcrição/química , Proteínas Supressoras de Tumor/químicaRESUMO
Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by Stenotrophomonas maltophilia. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by S. maltophilia. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain - significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that S. maltophilia modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of S. maltophilia infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by S. maltophilia.
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Fibrose Cística/metabolismo , Pneumopatias/metabolismo , Pneumopatias/microbiologia , Stenotrophomonas maltophilia/patogenicidade , Oligoelementos/metabolismo , Virulência/fisiologia , Animais , Cobalto/metabolismo , Cobre/metabolismo , Ferro/metabolismo , Magnésio/metabolismo , Masculino , Manganês/metabolismo , Camundongos , Fósforo/metabolismo , Rutênio/metabolismo , Zinco/metabolismoRESUMO
Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.
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Dipodascus/enzimologia , Glutationa Transferase/metabolismo , Fator 1 de Elongação de Peptídeos/metabolismo , Dinitroclorobenzeno/metabolismo , Dipodascus/citologia , Micélio/citologia , Micélio/enzimologia , Espectrometria de Massas em Tandem , Leveduras/citologia , Leveduras/enzimologiaRESUMO
Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.
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Anti-Infecciosos/farmacologia , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli/fisiologia , Animais , Armas Biológicas , Surtos de Doenças , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Infecções por Escherichia coli/prevenção & controle , Europa (Continente)/epidemiologia , HumanosRESUMO
BACKGROUND: Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. CONCLUSION/SIGNIFICANCE: This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.
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Células-Tronco Adultas/química , Polpa Dentária/citologia , Ligamento Periodontal/citologia , Proteoma/análise , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Polpa Dentária/metabolismo , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/fisiologia , Ligamento Periodontal/metabolismo , Adulto JovemRESUMO
Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.
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Expressão Gênica , Genoma Mitocondrial , Projeto Genoma Humano/organização & administração , Mitocôndrias/genética , Proteoma , Mapeamento Cromossômico , Cromossomos Humanos , Perfilação da Expressão Gênica , Genoma Humano , Humanos , ItáliaRESUMO
Primary open angle glaucoma (POAG) is one of the main causes of irreversible blindness worldwide. The pathogenesis of POAG is still unclear. Alteration and sclerosis of trabecular meshwork with changes in aqueous humor molecular composition seem to play the key role. Increased intraocular pressure is widely known to be the main risk factor for the onset and progression of the disease. Unfortunately, the early diagnosis of POAG still remains the main challenge. In order to provide insight into the patho-physiology of glaucoma, here we report a shotgun proteomics approach to tears of patients with POAG naïve to therapy. Our proteomics results showed 27 differential tear proteins in POAG vs. CTRL comparison (25 up regulated proteins in the POAG group and two unique proteins in the CTRL group), 16 of which were associated with inflammatory response, free radical scavenging, cell-to-cell signaling and interaction. Overall the protein modulation shown in POAG tears proves the involvement of biochemical networks linked to inflammation. Among all regulated proteins, a sub-group of 12 up-regulated proteins in naïve POAG patients were found to be down-regulated in medically controlled POAG patients treated with prostanoid analogues (PGA), as reported in our previous work (i.e., lipocalin-1, lysozyme C, lactotransferrin, proline-rich-protein 4, prolactin-inducible protein, zinc-alpha-2-glycoprotein, polymeric immunoglobulin receptor, cystatin S, Ig kappa chain C region, Ig alpha-2 chain C region, immunoglobulin J chain, Ig alpha-1 chain C region). In summary, our findings indicate that the POAG tears protein expression is a mixture of increased inflammatory proteins that could be potential biomarkers of the disease, and their regulation may be involved in the mechanism by which PGA are able to decrease the intraocular pressure in glaucoma patients.
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Proteínas do Olho/análise , Glaucoma de Ângulo Aberto/diagnóstico , Glaucoma de Ângulo Aberto/metabolismo , Lágrimas/química , Adulto , Idoso , Biomarcadores/análise , Feminino , Glaucoma de Ângulo Aberto/tratamento farmacológico , Humanos , Inflamação , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , Prostaglandinas/uso terapêutico , Prostaglandinas Sintéticas/uso terapêutico , Proteômica/métodos , Malha Trabecular/metabolismoRESUMO
Cystic fibrosis (CF) is an autosomal recessive disorder associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and defective chloride transport across the epithelial cell membranes. Abnormal epithelial ion transport is the primary cause of persistent airway infections and chronic inflammation in CF patients. In order to gain further insight into the mechanisms of epithelial dysfunctions linked to CFTR mutations, we performed and integrated proteomic and ionomic analysis of human bronchial epithelial IB3-1 cells and compared them with a CFTR-complemented isogenic cell line (C38). Aside from changes that were consistent with known effects related to CFTR mutations, such as differences in glycolytic and gluconeogenic pathways and unfolded protein responses, differential proteomics highlighted significant alteration of protein expression and, in particular, of the 14-3-3 signalling pathway that is known to be involved in cellular calcium (Ca) homeostasis. Of note, restoring chloride efflux by acting on Ca cellular homeostasis has been shown to be a promising therapeutic intervention for CF. Ionomic analysis showed significant changes in the IB3-1 element profile compared with C38 cells and in particular we observed an increase of intracellular Ca that significantly correlates with intracellular zinc (Zn) levels, suggesting a synergistic role of Ca and Zn influx. This finding is particularly intriguing because Zn has been reported to be effective in CF treatment increasing Ca influx. Taken together, our proteomic and ionomic data reveal that CFTR mutation sets in motion endogenous mechanisms counteracting impaired chloride transport mainly acting on epithelial ion transport and increasing intracellular Ca, suggesting potential links between protein expression and this response.
Assuntos
Cálcio/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Transporte de Íons/genética , Proteínas 14-3-3/metabolismo , Linhagem Celular Transformada , Fibrose Cística/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Homeostase , Humanos , Proteômica , Transdução de Sinais/genética , Zinco/metabolismoRESUMO
Transthyretin (TTR) is a homotetrameric protein of the CNS that plays a role of as the major thyroxine (T4) carrier from blood to cerebrospinal fluid (CSF). T4 physiologically helps oligodendrocyte precursor cells to turn into myelinating oligodendrocytes, enhancing remyelination after myelin sheet damage. We investigated post-translational oxidative modifications of serum and CSF TTR in multiple sclerosis subjects, highlighting high levels of S-sulfhydration and S-sulfonation of cysteine in position ten only in the cerebral TTR, which correlate with an anomalous TTR protein folding as well as with disease duration. Moreover, we found low levels of free T4 in CSF of multiple sclerosis patients, suggestive of a potential role of these modifications in T4 transport into the brain.
Assuntos
Esclerose Múltipla/líquido cefalorraquidiano , Pré-Albumina/líquido cefalorraquidiano , Processamento de Proteína Pós-Traducional , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oxirredução , Pré-Albumina/química , Pré-Albumina/isolamento & purificação , Isoformas de Proteínas/líquido cefalorraquidiano , Isoformas de Proteínas/química , Isoformas de Proteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tiroxina/líquido cefalorraquidianoRESUMO
Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex-binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex-binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.
Assuntos
Nucléolo Celular/metabolismo , DNA Ribossômico/genética , Quadruplex G , Proteínas Nucleares/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA Ribossômico/química , DNA Ribossômico/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Oligonucleotídeos/química , Porfirinas/química , Porfirinas/farmacologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Metal dyshomeostasis plays a crucial role in promoting several neurodegenerative diseases including Alzheimer's disease (AD), a condition that has been linked to deregulation of brain levels of Al, Fe, Mn, Cu, and Zn. Thus, quantitative multi-element profiling of brain tissues from AD models can be of great value in assessing the pathogenic role of metals as well as the value of therapeutic interventions aimed at restoring metal homeostasis in the brain. In this study, we employed low resolution inductively coupled plasma mass spectrometry (ICP-MS) to evaluate levels of ultra-trace, trace, and major elements in brains and cerebella of 3xTg-AD mice, a well characterized transgenic (Tg) AD model. This method is based on alternated cool and hot plasma ICP-MS. The essay fulfilled analytical requirements for the quantification of 14 elements in the Central Nervous System (CNS) of our Tg model. Quantification of Li, Al, Cr, and Co, a procedure that requires a pre-concentration step, was validated by high resolution ICP-MS. Changes in element profiles occurring in 3xTg-AD mice were compared to the ones observed in wild type (WT) mice. We also investigated variations in element profiles in 3xTg-AD mice receiving a long-term (17 months) dietary supplementation of Zn. Our data indicate that, compared to WT animals, 3xTg-AD mice displayed signs of altered brain metal homeostasis. We also found that long-term Zn administration promoted decreased brain levels of some metals (K, Ca, and Fe) and restored levels of Al, Cr, and Co to values found in WT mice.