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1.
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.

2.
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.

3.
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.

4.
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.

5.
Biomedicines ; 7(3)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261938

RESUMO

BACKGROUND: Zimmermann-Laband-1 syndrome (ZLS-1; OMIM# 135500) is a rare genetic disorder whose oral pathognomonic sign is the development of progressive, diffuse, and severe gingival hypertrophy. Most children with abnormally gingival hyperplasia may also present multiple unerupted teeth and skeletal deformities of maxillary arches (i.e., skeletal anterior open bite). Despite phenotypic variability of the clinical spectrum, gingival fibromatosis is the hallmark of ZLS-1. METHOD: In this study, we report a 3-year-old male patient with a ZLS-1-related gingival overgrowth and failure of eruption of the deciduous teeth in the molar area. Surgical excision was performed under general anesthesia. RESULTS: At three weeks follow-up, esthetics was significantly improved in terms of gingival appearance, and teeth eruption allowed an adequate masticatory function. CONCLUSION: In severe cases, surgical removal of the hyperplasic fibrous tissue may be required to expose unerupted teeth and establish a proper gingival contour. Surgical excision under general anesthesia is an elective procedure for patients with special needs, mental disability, as well as young and adult patients with dental anxiety type II and IV associated with poor oral health.

6.
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
7.
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
8.
Front Pediatr ; 7: 30, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30815432

RESUMO

Bronchopulmonary dysplasia is a major issue affecting morbidity and mortality of surviving premature babies. Preterm newborns are particularly susceptible to oxidative stress and infants with bronchopulmonary dysplasia have a typical oxidation pattern in the early stages of this disease, suggesting the important role of oxidative stress in its pathogenesis. Bronchopulmonary dysplasia is a complex disease where knowledge advances as new investigative tools become available. The explosion of the "omics" disciplines has recently affected BPD research. This review focuses on the new evidence coming from microbiomics, metabolomics and proteomics in relation to oxidative stress and pathogenesis of bronchopulmonary dysplasia. Since the pathogenesis is not yet completely understood, information gained in this regard would be important for planning an efficacious prevention and treatment strategy for the future.

9.
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
10.
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
11.
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
12.
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
13.
Biochim Biophys Acta Mol Basis Dis ; 1864(10): 3309-3321, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30031227

RESUMO

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.


Assuntos
Acetilglucosamina/metabolismo , Doença de Alzheimer/genética , Proteínas/metabolismo , Proteômica/métodos , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , N-Acetilglucosaminiltransferases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
14.
Crit Rev Biochem Mol Biol ; 53(3): 246-263, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29564928

RESUMO

Proteomic surveys with top-down platforms are today revealing thousands of naturally occurring fragments of bigger proteins. Some of them have not functional meaning because they derive from pathways responsible for protein degradation, but many have specific functions, often completely different from that one of the parent proteins. These peptides encrypted in the protein sequence are nowadays called cryptides. They are frequent in the animal and plant kingdoms and represent a new interesting -omic field of investigation. To point out how much widespread is their presence, we describe here the most studied cryptides from very common sources such as serum albumin, immunoglobulins, hemoglobin, and from saliva and milk proteins. Given its vastness, it is unfeasible to cover the topic exhaustively, therefore only several selected examples of cryptides from other sources are thereafter reported. Demanding is the development of new -omic platforms for the functional screening of new cryptides, which could provide suggestion for peptides and peptido-mimetics with variegate fields of application.


Assuntos
Peptídeos , Proteínas de Plantas , Plantas , Animais , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/química , Plantas/genética , Plantas/metabolismo
15.
J Proteome Res ; 17(1): 455-469, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29083190

RESUMO

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.


Assuntos
Consumo de Bebidas Alcoólicas/genética , Proteoma/análise , Proteômica/métodos , Saliva/química , Animais , Itália , Ratos , Glândula Submandibular
16.
J Proteome Res ; 16(11): 4196-4207, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29019242

RESUMO

Cystatins are a complex family of cysteine peptidase inhibitors. In the present study, various proteoforms of cystatin A, cystatin B, cystatin S, cystatin SN, and cystatin SA were detected in the acid-soluble fraction of human saliva and characterized by a top-down HPLC-ESI-MS approach. Proteoforms of cystatin D were also detected and characterized by an integrated top-down and bottom-up strategy. The proteoforms derive from coding sequence polymorphisms and post-translational modifications, in particular, phosphorylation, N-terminal processing, and oxidation. This study increases the current knowledge of salivary cystatin proteoforms and provides the basis to evaluate possible qualitative/quantitative variations of these proteoforms in different pathological states and reveal new potential salivary biomarkers of disease. Data are available via ProteomeXchange with identifier PXD007170.


Assuntos
Polimorfismo Genético , Processamento de Proteína Pós-Traducional , Cistatinas Salivares/análise , Sequência de Aminoácidos , Inibidores de Cisteína Proteinase , Humanos , Espectrometria de Massas , Fosforilação , Saliva/química , Cistatinas Salivares/metabolismo
17.
Arch Oral Biol ; 77: 68-74, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28178587

RESUMO

OBJECTIVE: This study aims to analyze the salivary peptidome/proteome of edentulous subject with respect to dentate control subjects. DESIGN: Unstimulated whole saliva, collected from 11 edentulous subjects (age 60-76 years) and 11 dentate age-matched control subjects, was immediately treated with 0.2% aqueous trifluoroacetic acid and the acidic soluble fraction analyzed by High Performace Liquid Chromatography-Mass Spectrometry. The relative abundance of the salivary peptides/proteins was determined by measuring the area of the High Performace Liquid Chromatography-Mass Spectrometry eXtracted Ion Current peaks which is linearly proportional to peptide/protein concentration under identical experimental conditions. Levels of salivary peptides/proteins in the two groups were compared by the nonparametric Mann-Whitney test to evidence statistically significant differences. RESULTS: Levels of cystatin A, S-glutathionylated, S-cystenylated, S-S dimer derivatives of cystatin B and S-glutathionylated derivative of SPRR3, were found significantly higher in edentulous subjects with respect to dentate controls. The major peptides and proteins typically deriving from salivary glands did not show any statistically significant differences. CONCLUSIONS: Cystatin A, S-glutathionylated, S-cystenylated, S-S dimer derivatives of cystatin B and S-glutathionylated derivative of SPRR3, which are mainly of intracellular origin and represent the major constituents of the cornified cell envelope are a clue of inflammation of mucosal epithelia.


Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Cistatina A/metabolismo , Cistatina B/metabolismo , Proteômica/métodos , Saliva/química , Idoso , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Boca Edêntula , Espectrometria de Massas por Ionização por Electrospray
18.
J Chemother ; 29(3): 164-172, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27439026

RESUMO

In the present study, three strains of Candida glabrata have been investigated to shed light on the mechanisms involved in azole resistance during adherence and biofilm formation. In particular, a clinical isolate, susceptible to azole-based drugs, DSY562 and two different resistant mutagenic strains deriving from DSY562, SFY114 and SFY115, have been analysed with different approaches for their cell wall composition and properties. A proteomic analysis revealed that the expression of six cell wall-related proteins and biofilm formation varied between the strains. The SFY114 and SFY115 strains resulted to be less hydrophobic than the susceptible parental counterpart DSY562, on the other hand they showed a higher amount in total cell wall polysaccharides fraction in the total cell wall. Accordingly to the results obtained from the hydrophobicity and adherence assays, in the resistant strain SFY115 the biofilm formation decreased compared to the parental strain DSY562. Finally, the total glucose amount in resistant SFY115 was about halved in comparison to other strains. Taken together all these data suggest that azole drugs may affect the cell wall composition of C. glabrata, in relation to the different pathogenic behaviours.


Assuntos
Antifúngicos/farmacologia , Biofilmes , Candida glabrata/fisiologia , Parede Celular/metabolismo , Farmacorresistência Fúngica Múltipla , Proteínas Fúngicas/biossíntese , Triazóis/farmacologia , Adesividade , Azóis/farmacologia , Biofilmes/efeitos dos fármacos , Candida glabrata/efeitos dos fármacos , Candida glabrata/isolamento & purificação , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Polissacarídeos Fúngicos/biossíntese , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Mutação , Proteômica/métodos , Especificidade da Espécie , Propriedades de Superfície , Eletroforese em Gel Diferencial Bidimensional
19.
Arthritis Res Ther ; 18(1): 229, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27716395

RESUMO

BACKGROUND: In the present study, we investigated whether thymosin ß (Tß) in saliva and in minor salivary glands is differentially expressed in patients with primary Sjögren's syndrome (pSS) and patients with autoimmune diseases (systemic sclerosis [SSc], systemic lupus erythematosus [SLE], and rheumatoid arthritis [RA], with and without sicca syndrome [ss]). METHODS: Saliva specimens of nine patients with pSS, seven with ss/SSc, seven with ss/SLE, seven with ss/RA, seven with SSc, seven with SLE, and seven with RA, as well as ten healthy subjects, were analyzed using a high-performance liquid chromatograph coupled with a mass spectrometer equipped with an electrospray ionization source to investigate the presence and levels of Tß4, Tß4 sulfoxide, and Tß10. Immunostaining for Tß4 and Tß10 was performed on minor salivary glands of patients with pSS and ss. RESULTS: Tß4 levels were statistically higher in patients with pSS with respect to the other subgroups. Tß10 was detectable in 66.7 % of patients with pSS and in 42.8 % of those with ss/SSc, while Tß4 sulfoxide was detectable in 44.4 % of patients with pSS and in 42.9 % of those with ss/SSc. Tß10 and Tß4 sulfoxide were not detectable in patients without associated ss and in healthy control subjects. Regarding thymosin immunostaining, all patients had immunoreactivity for Tß10, and a comparable distribution pattern in the four different subgroups of patients was observed. Tß4 immunoreactivity was present in patients with ss/SSc and those with ss/SLE, while it was completely absent in patients with pSS and those with ss/RA. CONCLUSIONS: Our data show that higher salivary Tß expression characterizes patients with pSS, while Tß4 sulfoxide and Tß10 salivary expression was selectively present in patients with sicca symptoms. Moreover, at the immunohistochemical level in patients with pSS, minor salivary glands showed a peculiar pattern characterized by immunostaining for Tß10 in acinar cells in the absence of any reactivity for Tß4. These findings, taken together, suggest a different role for Tß4 and Tß10 in patients with pSS who have ss and other autoimmune disease.


Assuntos
Saliva/metabolismo , Glândulas Salivares Menores/metabolismo , Síndrome de Sjogren/metabolismo , Timosina/biossíntese , Idoso , Doenças Autoimunes/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Proteômica , Saliva/química , Espectrometria de Massas por Ionização por Electrospray
20.
FEBS J ; 283(22): 4113-4127, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27685835

RESUMO

After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.


Assuntos
Cisteína/química , Dissulfetos/química , Albumina Sérica/química , Reagentes de Sulfidrila/química , Animais , Sítios de Ligação , Bovinos , Cisteína/metabolismo , Dissulfetos/metabolismo , Cães , Glutationa/química , Glutationa/metabolismo , Cabras , Cavalos , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Dobramento de Proteína , Albumina Sérica/metabolismo , Ovinos , Especificidade da Espécie , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismo
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