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1.
Childs Nerv Syst ; 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32617710

RESUMO

BACKGROUND: Many efforts have been performed in the last decade to accomplish the genomic and proteomic characterization of pediatric adamantinomatous craniopharyngioma with the purpose to elucidate the molecular mechanisms underlying the onset and development of this pediatric brain tumor, its high recurrence rate, and, although classified as a histologically benign neoplasm, its aggressive behavior. METHODS: The focus of this review is to perform the new comparison of the proteomic profiles of the solid component and the intracystic fluid of adamantinomatous craniopharyngioma based on our previous results, obtained by both the top-down and the bottom-up proteomic approaches, to disclose differences and similarities, and to discuss the results in the context of the most recent literature. RESULTS AND CONCLUSIONS: Proteins and peptides identified in the cyst fluid and in the solid component of adamantinomatous craniopharyngioma (AC) include beyond markers of inflammation (i.e., alpha-defensins), proteins involved in cell migration and protein degradation (i.e., beta-thymosin and ubiquitin peptides), whose main role might be in tumor growth and infiltration of the surrounding neural structures. These last appeared different in the solid components compared with the cyst fluid, missing their terminal part in the solid tissue, a feature generally associated to malignancies, which might represent a distinct molecular site for an aggressive behavior of AC.

2.
J Proteomics ; : 103890, 2020 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-32629195

RESUMO

Gingival Crevicular Fluid (GCF), a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process. The results obtained confirmed the presence in GCF of α-Defensins 1-4, Thymosin ß4 and Thymosin ß10, as described in previous works and revealed the presence of other interesting peptides never described before in GCF such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin; fragments of Thymosin ß4 and Thymosin ß10; Fibrinopeptide A and its fragments and Fibrinopeptide B; S100A8 and S100A9, LVV Hemorphin-7 (hemoglobin chain ß fragment), as well as some other peptides deriving from α and ß subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups. Our study demonstrate that an in-depth analysis of a biological fluid like GCF, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption. Data are available via ProteomeXchange with identifier PXD016010 and PXD016049. SIGNIFICANCE: GCF due to his site-specific nature has a great potential in containing factors that are specific for action at a given site and might have diagnostic value to detect qualitative and quantitative variations of proteins/peptides composition linked to physiological or pathological conditions.

3.
J Clin Immunol ; 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32519288

RESUMO

We analyzed by bidimensional electrophoresis the acid-insoluble fraction of saliva from three classes of angioedema patients and a healthy control group, highlighting significant variations of several normalized spot volumes. Characterization of the corresponding proteins was performed by in-gel tryptic digestion of the spots, followed by high-resolution HPLC-ESI-MS/MS analysis of tryptic mixtures. By this strategy, 16 differentially-expressed proteins among two or more groups were identified. We found higher concentration of proteins involved in immune response (interleukin-1 receptor antagonist and annexin A1), and of moonlighting proteins acting as plasminogen receptors (glyceraldehyde-3-phosphate dehydrogenase, α-enolase, and annexin A2) in patients affected by the idiopathic non-histaminergic or hereditary angioedema with unknown origin with respect to healthy controls. These data provide new information on the molecular basis of these less characterized types of angioedema. Graphical Abstract Graphical Abstract.

4.
J Proteome Res ; 2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32575983

RESUMO

Mastocytosis is a myeloproliferative neoplasm causing abnormal clonal mast cell accumulation in different tissues, such as skin and bone marrow. A cutaneous subtype (CM) is distinguished from a systemic one (SM); SM patients can be grouped into SM with (SM+C) or without (SM-C) additional cutaneous lesions, and their classification is often challenging. This study was purposed to highlight variations in the salivary proteome of patients with different mastocytosis subtypes and compared to healthy controls. A top-down proteomics approach coupled to a label-free quantitation revealed salivary profiles in patients different from those of controls and a down-regulation of peptides/proteins involved in the mouth homeostasis and defense, such as statherin, histatins, and acidic proline-rich proteins (aPRPs), and in innate immunity and inflammation, such as the cathepsin inhibitors, suggesting a systemic condition associated with an exacerbated inflammatory state. The up-regulation of antileukoproteinase and S100A8 suggested a protective role against the disease status. The two SM forms were distinguished by the lower levels of truncated forms of aPRPs, statherin, P-B peptide, and cystatin D and the higher levels of thymosin ß4 and α-defensins 1 and 4 in SM-C patients with respect to SM+C. Data are available via ProteomeXchange with identifier PXD017759.

5.
Sci Rep ; 10(1): 8943, 2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488029

RESUMO

Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.

6.
J Clin Immunol ; 40(2): 329-339, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916122

RESUMO

PURPOSE: Present study is designed to discover potential salivary biomarkers associated with predominantly antibody deficiencies, which include a large spectrum of disorders sharing failure of antibody production, and B cell defects resulting in recurrent infections, autoimmune and inflammatory manifestations, and tumor susceptibility. Understanding and clinical classification of these syndromes is still challenging. METHODS: We carried out a study of human saliva based on liquid chromatography-mass spectrometry measurements of intact protein mass values. Salivary protein profiles of patients (n = 23) and healthy controls (n = 30) were compared. RESULTS: Patients exhibited lower abundance of α-defensins 1-4, cystatins S1 and S2, and higher abundance of glutathionylated cystatin B and cystatin SN than controls. Patients could be clustered in two groups on the basis of different levels of cystatin SN, S1 and S2, suggesting that these proteins may play different roles in the disease. CONCLUSIONS: Quantitative variations of these pro-inflammatory and antimicrobial peptides/proteins may be related to immunodeficiency and infectious condition of the patients. The high incidence of tumors in the group with the highest level of cystatin SN, which is recognized as tumoral marker, appeared an intriguing result deserving of future investigations. Data are available via ProteomeXchange with identifier PXD012688.

7.
Hum Genet ; 139(2): 227-245, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31919630

RESUMO

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.


Assuntos
Ataxia/patologia , Metilação de DNA , Proteína do X Frágil de Retardo Mental/metabolismo , Síndrome do Cromossomo X Frágil/patologia , Mitocôndrias/patologia , Proteínas Mitocondriais/metabolismo , Mutação , Proteoma/análise , Tremor/patologia , Ataxia/genética , Ataxia/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína do X Frágil de Retardo Mental/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , RNA Interferente Pequeno/genética , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Tremor/genética , Tremor/metabolismo
8.
J Proteome Res ; 19(1): 300-313, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31638822

RESUMO

Because of the distinctive features of the oral cavity, the determination of the proteins involved in the formation of the "oral protein pellicle" is demanding. The present study investigated the susceptibility of several human basic proline-rich peptides, named P-H, P-D, P-F, P-J, and II-2, as substrates of transglutaminase-2. The reactivity of the P-C peptide and statherin was also investigated. Peptides purified from human whole saliva were incubated with the enzyme in the presence or in the absence of monodansyl-cadaverine. Mass spectrometry analyses of the reaction products highlighted that P-H and P-D (P32 and A32 variants) were active substrates, II-2 was less reactive, and P-F and P-J showed very low reactivity. P-C and statherin were highly reactive. All of the peptides formed cyclo derivatives, and only specific glutamine residues were involved in the cycle formation and reacted with monodansyl-cadaverine: Q29 of P-H, Q37 of P-D, Q21 of II-2, Q41 of P-C, and Q37 of statherin were the principal reactive residues. One or two secondary glutamine residues of only P-H, P-D P32, P-C, and statherin were hierarchically susceptible to the reaction with monodansyl-cadaverine. MS and MS/MS data were deposited to the ProteomeXchange Consortium ( http://www.ebi.ac.uk/pride ) via the PRIDE partner repository with the data set identifier PXD014658.

9.
J Sep Sci ; 43(1): 313-336, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31631532

RESUMO

More than 300 different protein post-translational modifications are currently known, but only a few have been extensively investigated because modified proteoforms are commonly present in sub-stoichiometry amount. For this reason, improvement of specific enrichment techniques is particularly useful for the proteomic characterization of post-translationally modified proteins. Enrichment proteomic strategies could help the researcher in the challenging issue to decipher the complex molecular cross-talk existing between the different factors influencing the cellular pathways. In this review the state of art of the platforms applied for the enrichment of specific and most common post-translational modifications, such as glycosylation and glycation, phosphorylation, sulfation, redox modifications (i.e. sulfydration and nitrosylation), methylation, acetylation, and ubiquitinylation, are described. Enrichments strategies applied to characterize less studied post-translational modifications are also briefly discussed.

10.
J Clin Med ; 8(12)2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31816910

RESUMO

In the grey zone of testosterone levels between 8 and 12 nmol/L, the usefulness of therapy is controversial; as such, markers of tissue action of androgens may be helpful in adjusting clinical decisions. To better understand the effect of the hypothalamic-pituitary-testicular axis on male accessory secretion, we performed a proteomic quantitative analysis of seminal plasma in patients with secondary hypogonadism, before and after testosterone replacement therapy (TRT). Ten male patients with postsurgical hypogonadotrophic hypogonadism were enrolled in this study, and five of these patients were evaluated after testosterone treatment. Ten men with proven fertility were selected as a control group. An aliquot of seminal plasma from each individual was subjected to an in-solution digestion protocol and analyzed using an Ultimate 3000 RSLC-nano HPLC apparatus coupled to a LTQ Orbitrap Elite mass spectrometer. The label-free quantitative analysis was performed via Precursor Ions Area Detector Node. Eleven proteins were identified as decreased in hypogonadic patients versus controls, which are primarily included in hydrolase activity and protein binding activity. The comparison of the proteome before and after TRT comes about within the discovery of six increased proteins. This is the primary application of quantitative proteomics pointed to uncover a cluster of proteins reflecting an impairment not only of spermatogenesis but of the epididymal and prostate epithelial cell secretory function in male hypogonadism. The identified proteins might represent putative clinical markers valuable within the follow-up of patients with distinctive grades of male hypogonadism.

11.
Artigo em Inglês | MEDLINE | ID: mdl-31202182

RESUMO

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.


Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Hiper-Homocisteinemia/metabolismo , Proteoma/metabolismo , Complexo Vitamínico B/análise , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Química Encefálica , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hiper-Homocisteinemia/etiologia , Hiper-Homocisteinemia/genética , Masculino , Espectrometria de Massas , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteoma/química , Proteoma/genética , Complexo Vitamínico B/metabolismo
12.
Dis Markers ; 2019: 3609789, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191748

RESUMO

Although histologically benign, adamantinomatous craniopharyngioma (AC) pediatric brain tumor is a locally aggressive disease that frequently determines symptoms and hormonal dysfunctions related to the mass effect on the surrounding structures. Another typical feature of this benign neoplasm is the presence of voluminous liquid cysts frequently associated with the solid component. Even if studies have been devoted to the proteomic characterization of the tumor intracystic fluid, poor explorations have been performed on its solid part, principally investigated by transcriptomics technologies. In the present study, seven specimens of AC whole tumor tissue have been analyzed by LC-MS for a preliminary assessment of the proteomic profile by a top-down/bottom-up integrated approach. Thymosin beta 4, ubiquitin, calmodulin, S100 proteins, prothymosin α isoform 2, alpha-defensins 1-4, and fragments largely belonging to vimentin, hemoglobin, and glial fibrillary acidic protein characterized the intact proteome. The identification of alpha-defensins, formerly characterized in AC intracystic fluid, reinforces the hypothesis of a role for inflammation in tumor pathogenesis. A total number of 1798 unique elements were identified by a bottom-up approach with a special focus on the 433 proteins commonly characterized in the 85.7% of the samples analyzed. Their gene ontology classification evidenced the involvement of the adherence system, intermediate filaments, and actin cytoskeleton in tumor pathogenesis and of elements part of the Wnt, FGF, and EGFR signaling pathways. In addition, proteins involved in calcium modulation, innate immunity, inflammation, CCKR and integrin signaling, and gonadotropin-releasing hormone receptor pathways were also outlined. Further than confirming proteomic data previously obtained on AC intracystic fluid, these results offer a preliminary overview of the AC whole tissue protein phenotype, adding new hints towards the comprehension of this still obscure pediatric brain tumor.


Assuntos
Neoplasias Encefálicas/metabolismo , Craniofaringioma/metabolismo , Proteoma/metabolismo , Adolescente , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Craniofaringioma/genética , Craniofaringioma/patologia , Feminino , Humanos , Masculino , Proteoma/genética
13.
Redox Biol ; 23: 101162, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30876754

RESUMO

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.


Assuntos
Síndrome de Down/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Administração Intranasal , Animais , Autofagia , Biomarcadores , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma , Proteômica/métodos
14.
Arch Oral Biol ; 98: 148-155, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30496935

RESUMO

OBJECTIVE: In the present study the salivary proteome of burning mouth syndrome patients and healthy subjects was characterized by a top-down proteomic approach and compared to highlight possible qualitative and quantitative differences that may give suggestions about the causes of this pathology which are still unknown. MATERIALS AND METHODS: Resting and stimulated whole saliva, stimulated parotid and submandibular/sublingual saliva samples were collected from burning mouth syndrome patients (n = 16) and age- and gender-matched healthy subjects (n = 14). An equal volume of 0.2% trifluoroacetic acid was added to each sample immediately after collection and the supernatants were analysed by liquid chromatography coupled to electrospray-ionisation mass spectrometry. Proteins and peptides were quantified using a label-free approach measuring the extracted ion current peak areas of the main salivary proteins and peptides. RESULTS: The quantitation of the main salivary proteins and peptides revealed a higher concentration of cystatin SN in resting saliva of burning mouth syndrome patients with respect to healthy controls and no other conspicuous changes. CONCLUSIONS: The reported data showed that the salivary protein profile was not affected, in composition and relative abundance, by the burning mouth syndrome, except for the cystatin SN, a protein up-regulated in several pathological conditions, that might be considered potentially indicative of the disease.


Assuntos
Síndrome da Ardência Bucal/complicações , Síndrome da Ardência Bucal/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Saliva/química , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/análise , Idoso , Cromatografia Líquida , Feminino , Humanos , Pessoa de Meia-Idade , Glândula Parótida/metabolismo , Salivação , Espectrometria de Massas por Ionização por Electrospray , Xerostomia/complicações
15.
Free Radic Biol Med ; 129: 430-439, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30321702

RESUMO

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-ß plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3+ lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3+ T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.


Assuntos
Doença de Alzheimer/diagnóstico , Linfócitos/química , Nitrocompostos/imunologia , Proteoma/imunologia , Tirosina/análogos & derivados , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Complexo CD3/genética , Complexo CD3/imunologia , Estudos de Casos e Controles , Separação Celular , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/imunologia , Metabolismo Energético/genética , Feminino , Expressão Gênica , Humanos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Nitrocompostos/química , Nitrocompostos/isolamento & purificação , Estresse Nitrosativo , Estresse Oxidativo , Cultura Primária de Células , Proteoma/genética , Proteoma/metabolismo , Transdução de Sinais , Tirosina/metabolismo
16.
Sci Rep ; 8(1): 16050, 2018 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-30375487

RESUMO

Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (KD = 0.3 mM), causes a fast glutathionylation of this residue (t1/2 = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.


Assuntos
Cisteína/química , Dissulfetos/química , Muramidase/química , Agregados Proteicos , Dissulfeto de Glutationa/química , Isoenzimas/química , Isoenzimas/metabolismo , Muramidase/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
17.
J Proteomics ; 187: 212-222, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30086402

RESUMO

Multiple sclerosis is a chronic disease of the central nervous system characterized by inflammation, demyelination and neurodegeneration which is of undetermined origin. To date a single diagnostic test of multiple sclerosis does not exists and novel biomarkers are demanded for a more accurate and early diagnosis. In this study, we performed the quantitative analysis of 119 salivary peptides/proteins from 49 multiple sclerosis patients and 54 healthy controls by a mass spectrometry-based top-down proteomic approach. Statistical analysis evidenced different levels on 23 proteins: 8 proteins showed lower levels in multiple sclerosis patients with respect to controls and they were mono- and di-oxidized cystatin SN, mono- and di-oxidized cystatin S1, mono-oxidized cystatin SA and mono-phosphorylated statherin. 15 proteins showed higher levels in multiple sclerosis patients with respect to controls and they were antileukoproteinase, two proteoforms of Prolactin-Inducible Protein, P-C peptide (Fr.1-14, Fr. 26-44, and Fr. 36-44), SV1 fragment of statherin, cystatin SN Des1-4, cystatin SN P11 → L variant, and cystatin A T96 → M variant. The differences observed between the salivary proteomic profile of patients suffering from multiple sclerosis and healthy subjects is consistent with the inflammatory condition and altered immune response typical of the pathology. Data are available via ProteomeXchange with identifier PXD009440. SIGNIFICANCE: To date a single diagnostic test of multiple sclerosis does not exist, and diagnosis is based on multiple tests which mainly include the analysis of cerebrospinal fluid. However, the need for lumbar puncture makes the analysis of cerebrospinal fluid impractical for monitoring disease activity and response to treatment. The possible use of saliva as a diagnostic fluid for oral and systemic diseases has been largely investigated, but only marginally in multiple sclerosis compared to other body fluids. Our study demonstrates that the salivary proteome of multiple sclerosis patients differs considerably compared to that of sex and age matched healthy individuals and suggests that some differences might be associated with the different disease-modifying therapy used to treat multiple sclerosis patients.


Assuntos
Esclerose Múltipla/metabolismo , Proteoma/análise , Proteômica/métodos , Proteínas e Peptídeos Salivares/análise , Adulto , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Proteoma/metabolismo , Saliva/química , Saliva/metabolismo , Proteínas e Peptídeos Salivares/metabolismo
18.
Expert Opin Biol Ther ; 18(sup1): 199-203, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-30063862

RESUMO

OBJECTIVES: We reevaluated a lyophilized sample of thymosin fraction 5, stored for 37 years at room temperature, by high-resolution mass spectrometry in terms of stability and yet uncharacterized polypeptides that could be biological important substances. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to molecular characterization of polypeptides present in thymosin fraction 5. RESULTS: We detected more than 100 monoisotopic masses corresponding to thymosin ß4 and truncated forms of ubiquitin, prothymosin α, thymosin ß4, and thymosin ß9. Additionally, we discovered a new polypeptide present in thymosin fraction 5 and identified it as intact SH3 domain-binding glutamic acid-rich-like protein 3. CONCLUSION: In spite of the well-known proteolytic processes inherent to the preparation of thymosin fraction 5, still uncharacterized polypeptides as well as truncated forms of already well-known thymosins are present in fraction 5 after long-term storage. Therefore, continuing characterization of thymosin fraction 5 is even nowadays highly promising.


Assuntos
Espectrometria de Massas/métodos , Timosina/análogos & derivados , Animais , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Humanos , Precursores de Proteínas/análise , Precursores de Proteínas/isolamento & purificação , Proteômica/métodos , Timosina/análise , Timosina/química , Timosina/isolamento & purificação , Fatores de Tempo , Ubiquitina/análise , Ubiquitina/isolamento & purificação
19.
J Proteome Res ; 17(9): 3292-3307, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30064219

RESUMO

Human basic proline-rich proteins and basic glycosylated proline-rich proteins, encoded by the polymorphic PRB1-4 genes and expressed only in parotid glands, are the most complex family of adult salivary proteins. The family includes 11 parent peptides/proteins and more than 6 parent glycosylated proteins, but a high number of proteoforms with rather similar structures derive from polymorphisms and post-translational modifications. 55 new components of the family were characterized by top-down liquid chromatography-mass spectrometry and tandem-mass platforms, bringing the total number of proteoforms to 109. The new components comprise the three variants P-H S1 → A, P-Ko P36 → S, and P-Ko A41 → S and several of their naturally occurring proteolytic fragments. The paper represents an updated reference for the peptides included in the heterogeneous family of proteins encoded by PRB1/PRB4. MS data are available via ProteomeXchange with the identifier PXD009813.


Assuntos
Processamento de Proteína Pós-Traducional , Saliva/química , Proteínas Salivares Ricas em Prolina/metabolismo , Adulto , Sequência de Aminoácidos , Cromatografia Líquida , Feminino , Glicosilação , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Glândula Parótida/química , Glândula Parótida/metabolismo , Peptídeos/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteólise , Proteômica/métodos , Proteínas Salivares Ricas em Prolina/química , Proteínas Salivares Ricas em Prolina/genética , Proteínas Salivares Ricas em Prolina/isolamento & purificação , Espectrometria de Massas em Tandem
20.
Infect Drug Resist ; 11: 969-979, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30046246

RESUMO

Background: A peptide of 2,733 Da named SP-E, previously isolated from pig saliva and already described for its antifungal activity and absence of toxicity against mammalian cells, is characterized by a high content of proline residues (70% of entire sequence), that confer structural features probably related to peptide activity. Purpose: The aim of this study was to evaluate the activity of SP-E against Gram-negative bacteria, including drug-resistant clinical isolates. Methods: SP-E and shorter fragments of the same peptide were tested in vitro against the selected bacteria by colony forming unit assays. Scanning electron microscopy and confocal microscopy were also applied. SP-E potential therapeutic activity was evaluated in vivo in a Galleria mellonella model of bacterial infection. Results: SP-E proved to be active against the tested bacteria with EC50 values in the micro-molar range. Though maintaining antibacterial properties, the shorter peptides showed lower activity in respect to the parental molecule. Kinetics of killing action and nonmembranolytic internalization within Escherichia coli and Pseudomonas aeruginosa cells strongly suggested a cytosolic mechanism of action involving one or more intracellular molecular targets. A single injection of SP-E exerted a therapeutic effect in G. mellonella larvae infected with P. aeruginosa. Conclusion: The biological properties of SP-E strongly back this peptide as a new promising multitasking antimicrobial molecule.

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