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1.
Atherosclerosis ; 298: 27-35, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32169720

RESUMO

BACKGROUND AND AIMS: Reduced bioavailability of nitric oxide (NO) has been implicated in the pathogenesis of calcific aortic stenosis. Herein, we investigated the effects of l-Arginine, the main precursor of NO, on the osteogenic differentiation of aortic interstitial valve cells (VICs). METHODS: We isolated a clonal population of bovine VICs that expresses osteogenic markers and induces calcification of collagen matrix after stimulation with endotoxin (LPS 500 ng/mL). VICs were treated in vitro with different combinations of LPS ± l-Arginine (50 or 100 mM) and cell extracts were collected to perform proteomic (iTRAQ) and gene expression (RT-PCR) analysis. RESULTS: l-Arginine prevents the over-expression of alkaline phosphatase (ALP, p < 0.001) and reduces matrix calcification (p < 0.05) in VICs treated with LPS. l-Arginine also reduces the over-expression of inflammatory molecules induced by LPS (TNF-alpha, IL-6 and IL-1beta, p < 0.001). The proteomic analysis allowed to identify 49 proteins with an altered expression profile after stimulation with LPS and significantly modified by l-Arginine. These include proteins involved in the redox homeostasis of the cells (i.e. Xanthine Oxidase, Catalase, Aldehyde Oxidase), remodeling of the extracellular matrix (i.e. ADAMTSL4, Basigin, COL3A1) and cellular signaling (i.e. Fibrillin-1, Legumain, S100A13). The RT-PCR analysis confirmed the modifications of Fibrillin-1, ADAMTSL4, Basigin and Xanthine Oxidase, whose expression levels increase after stimulation with LPS and are reduced by l-Arginine (p < 0.05). CONCLUSIONS: l-Arginine prevents osteogenic differentiation of VICs and reduces matrix calcification. This effect is achieved through the modulation of proteins involved in the cellular redox system, remodeling of extracellular matrix and inflammatory activation of VICs.


Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/efeitos dos fármacos , Valva Aórtica/patologia , Arginina/metabolismo , Arginina/farmacologia , Arterite/metabolismo , Calcinose/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/metabolismo , Animais , Valva Aórtica/citologia , Valva Aórtica/metabolismo , Bovinos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Osteogênese/efeitos dos fármacos , Proteômica
2.
PLoS One ; 10(5): e0126458, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961303

RESUMO

Adenocarcinoma (AdC) is the most common lung cancer subtype and is often associated with pleural effusion (PE). Its poor prognosis is attributable to diagnostic delay and lack of effective treatments and there is a pressing need in discovering new biomarkers for early diagnosis or targeted therapies. To date, little is known about lung AdC proteome. We investigated protein expression of lung AdC in PE using the isobaric Tags for Relative and Absolute Quantification (iTRAQ) approach to identify possible novel diagnostic/prognostic biomarkers. This provided the identification of 109 of lung AdC-related proteins. We further analyzed lumican, one of the overexpressed proteins, in 88 resected lung AdCs and in 23 malignant PE cell-blocks (13 lung AdCs and 10 non-lung cancers) using immunohistochemistry. In AdC surgical samples, lumican expression was low in cancer cells, whereas it was strong and diffuse in the stroma surrounding the tumor. However, lumican expression was not associated with tumor grade, stage, and vascular/pleural invasion. None of the lung cancer cell-blocks showed lumican immunoreaction, whereas those of all the other tumors were strongly positive. Finally, immunoblotting analysis showed lumican expression in both cell lysate and conditioned medium of a fibroblast culture but not in those of A549 lung cancer cell line. PE is a valid source of information for proteomic analysis without many of the restrictions of plasma. The high lumican levels characterizing AdC PEs are probably due to its release by the fibroblasts surrounding the tumor. Despite the role of lumican in lung AdC is still elusive, it could be of diagnostic value.


Assuntos
Adenocarcinoma/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfato de Queratano/metabolismo , Neoplasias Pulmonares/metabolismo , Derrame Pleural Maligno/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Lumicana , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Proteoma , Proteômica/métodos
3.
Biochim Biophys Acta ; 1854(6): 609-23, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25278378

RESUMO

CK2 is an extremely pleiotropic Ser/Thr protein kinase, responsible for the generation of a large proportion of the human phosphoproteome and implicated in a wide variety of biological functions. CK2 plays a global role as an anti-apoptotic agent, a property which is believed to partially account for the addiction of many cancer cells to high CK2 levels. To gain information about the CK2 targets whose phosphorylation is primarily implicated in its pro-survival signaling advantage has been taken of quinalizarin (QZ) a cell permeable fairly specific CK2 inhibitor, previously shown to be able to block endogenous CK2 triggering an apoptotic response. HEK-293T cells either treated or not for 3h with 50µM QZ were exploited to perform a quantitative SILAC phosphoproteomic analysis of phosphosites readily responsive to QZ treatment. Our analysis led to the identification of 4883 phosphosites, belonging to 1693 phosphoproteins. 71 phosphosites (belonging to 47 proteins) underwent a 50% or more decreased occupancy upon QZ treatment. Almost 50% of these fulfilled the typical consensus sequence recognized by CK2 (S/T-x-x-E/D/pS) and in several cases were validated as bona fide substrates of CK2 either based on data in the literature or by performing in vitro phosphorylation experiments with purified proteins. The majority of the remaining phosphosites drastically decreased upon QZ treatment display the pS/T-P motif typical of proline directed protein kinases and a web logo extracted from them differentiates from the web logo extracted from all the proline directed phosphosites quantified during our analysis (1151 altogether). A paradoxical outcome of our study was the detection of 116 phosphosites (belonging to 92 proteins altogether) whose occupancy is substantially increased (50% or more), rather than decreased by QZ treatment: 40% of these display the typical motif recognized by proline directed kinases, while about 25% fulfill the CK2 consensus. Collectively taken our data on one side have led to the disclosure of a subset of CK2 targets which are likely to be implicated in the early steps of CK2 signaling counteracting apoptosis, on the other they provide evidence for the existence of side and off-target effects of the CK2 inhibitor quinalizarin, paving the road toward the detection of other kinases susceptible to this compound. This article is part of a Special Issue entitled: Medical Proteomics.


Assuntos
Antraquinonas/farmacologia , Caseína Quinase II/antagonistas & inibidores , Fosfoproteínas/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteoma/metabolismo , Células HEK293 , Humanos
4.
J Chromatogr A ; 1355: 278-83, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24939089

RESUMO

Shotgun proteomics of complex samples is generally coupled with at least one peptide fractionation step and, to this effect, peptide isoelectric focusing (IEF) in immobilized pH gradient (IPG) is one of the most used techniques. Fractionation with the OFFGEL 3100 Agilent Technologies apparatus allows the easy recovery of peptides that, after focusing, diffuse into the liquid phase above the gel strip. In this work we investigate the efficiency of peptide diffusion during OFFGEL fractionation and demonstrate that a recovery based only on the spontaneous diffusion process is far from being optimal. We show that a simple additional extraction step with acetonitrile increases of about 40% the amount of material that can be recovered after the focusing. Moreover, we show that the two populations of peptides obtained from the passive elution and from the extraction process are also qualitatively different and only partially overlapping.


Assuntos
Fracionamento Químico/métodos , Peptídeos/isolamento & purificação , Difusão , Força Próton-Motriz , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos
5.
Diabetologia ; 57(9): 1947-56, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24962668

RESUMO

AIMS/HYPOTHESIS: Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. METHODS: Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. RESULTS: Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with α1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. CONCLUSIONS/INTERPRETATION: We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patients.


Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores/metabolismo , Pé Diabético/metabolismo , Pé Diabético/fisiopatologia , Serpinas/metabolismo , Cicatrização/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos de Neoplasias/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Serpinas/genética , Adulto Jovem
6.
Amino Acids ; 46(5): 1415-7, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24615240

RESUMO

IEF is often used in multidimensional shotgun proteomics and the narrow range of 3.5-4.5 is the recommended pH interval for the fractionation of tryptic peptides. Usually, even if IEF is performed in IPG strip with a narrow range pH, the entire sample must be loaded onto the strip, including the "out of IPG range" peptides. We describe a simple protocol to recover at least a part of these missing peptides and show that this recovery significantly influences the overall fractionation result, increasing the number of the identified proteins and the protein coverage.


Assuntos
Focalização Isoelétrica/métodos , Peptídeos/química , Concentração de Íons de Hidrogênio , Focalização Isoelétrica/instrumentação , Proteômica/instrumentação , Proteômica/métodos
7.
Basic Res Cardiol ; 108(4): 368, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23800875

RESUMO

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.


Assuntos
Aterosclerose/fisiopatologia , Calcinose/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas dos Microfilamentos/metabolismo , Células Mieloides/fisiologia , Comunicação Parácrina/fisiologia , Regulação para Cima/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Calcinose/metabolismo , Cálcio/metabolismo , Comunicação Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Células Mieloides/patologia , Osteocalcina/metabolismo
8.
J Chromatogr A ; 1293: 1-9, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23639126

RESUMO

In shotgun proteomics, protein mixtures are proteolytically digested before tandem mass spectrometry (MS/MS) analysis. Biological samples are generally characterized by a very high complexity, therefore a step of peptides fractionation before the MS analysis is essential. This passage reduces the sample complexity and increases its compatibility with the sampling performance of the instrument. Among all the existing approaches for peptide fractionation, isoelectric focusing has several peculiarities that are theoretically known but practically rarely exploited by the proteomics community. The main aim of this review is to draw the readers' attention to these unique qualities, which are not accessible with other common approaches, and that represent important tools to increase confidence in the identification of proteins and some post-translational modifications. The general characteristics of different methods to perform peptide isoelectric focusing with natural and artificial pH gradients, the existing instrumentation, and the informatics tools available for isoelectric point calculation are also critically described. Finally, we give some general conclusions on this strategy, underlying its principal limitations.


Assuntos
Focalização Isoelétrica/métodos , Mapeamento de Peptídeos/métodos , Peptídeos/análise , Peptídeos/química , Proteômica/métodos , Humanos
9.
Amino Acids ; 43(5): 2199-202, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22434181

RESUMO

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.


Assuntos
Proteínas Sanguíneas/análise , Fracionamento Químico/métodos , Proteoma/análise , Artefatos , Cromatografia por Troca Iônica , Cromatografia de Fase Reversa , Bases de Dados de Proteínas , Proteínas do Olho/análise , Humanos , Focalização Isoelétrica , Fatores de Crescimento Neural/análise , Peptídeos/análise , Ribonuclease Pancreático/análise , Serina Endopeptidases/análise , Serpinas/análise , Software , Trombospondina 1/análise
10.
PLoS One ; 7(1): e30911, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22292075

RESUMO

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of "isobaric" spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.


Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Mapeamento de Peptídeos/métodos , Proteômica/instrumentação , Proteômica/métodos , Animais , Bovinos , Eletroforese em Gel Bidimensional/instrumentação , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/instrumentação , Eletroforese em Gel de Poliacrilamida/métodos , Desenho de Equipamento , Aumento da Imagem/instrumentação , Interpretação de Imagem Assistida por Computador/instrumentação , Modelos Teóricos , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Mapeamento de Peptídeos/instrumentação , Proteoma/análise , Proteoma/metabolismo
11.
PLoS One ; 6(5): e19603, 2011 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-21573190

RESUMO

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol.


Assuntos
Proteínas Sanguíneas/análise , Proteoma/análise , Proteômica/métodos , Fracionamento Químico , Intervalos de Confiança , Humanos , Peptídeos/sangue , Proteômica/economia
12.
J Proteome Res ; 9(11): 5913-21, 2010 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-20825172

RESUMO

Calcific degeneration represents the most frequent aortic valve disease observed in industrialized countries. Our aim is to study modifications in the cytosolic and membrane protein profile of aortic interstitial valve cells (VIC) acquiring a pro-calcific phenotype. We studied a clonal population of bovine VIC that expresses bone-related proteins (such as alkaline phosphatase [ALP]) and calcifies a collagen matrix in response to endotoxin (LPS) treatment. A proteomic analysis was performed on proteins extracted from cells treated for 12 days with LPS (100 ng/mL) versus control. We identified 34 unique cytosolic and 10 unique membrane-associated proteins showing significant changes after treatment. These proteins are involved in several cellular functions, such as chaperone-mediated protein folding, protein metabolism and transport, cell redox/nitric oxide homeostasis, and cytoskeletal organization. Reduced expression of proteins involved in NOS bioactivity (such as DDAH-1 and -2) suggested a role for the l-arginine/ADMA ratio in controlling VIC phenotypic profile. In accordance with this hypothesis, we observed that exposure of clonal cells to l-arginine prevented LPS-induced ALP expression and collagen calcification. In conclusion, we identified several proteins involved in structural, metabolic, and signaling functions that are significantly altered in aortic VIC acquiring a pro-calcific profile, thus giving new insights into the pathogenesis of aortic valve degeneration.


Assuntos
Valva Aórtica/patologia , Calcinose/metabolismo , Proteômica/métodos , Animais , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Arginina/farmacologia , Calcinose/induzido quimicamente , Calcinose/patologia , Bovinos , Células Clonais , Citosol/química , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/análise , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/fisiologia
13.
J Proteome Res ; 6(3): 976-86, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17330940

RESUMO

In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM.


Assuntos
Diabetes Mellitus Tipo 1/patologia , Nefropatias Diabéticas/patologia , Fibroblastos/química , Chaperonas Moleculares/análise , Proteínas/análise , Estudos de Casos e Controles , Células Cultivadas , Eletroforese em Gel Bidimensional , Fibroblastos/patologia , Proteínas de Choque Térmico/análise , Humanos , Isomerases de Dissulfetos de Proteínas/análise , Dobramento de Proteína , Pele/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Tumoral 1 Controlada por Tradução
14.
J Clin Endocrinol Metab ; 91(9): 3507-14, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16822825

RESUMO

CONTEXT: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. OBJECTIVE: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. DESIGN: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and alpha-actinin 4 and a decrease of tubulin-beta2 and splice isoform 2 of tropomyosin beta-chain were detected. CONCLUSIONS: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.


Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proteoma/metabolismo , Pele/metabolismo , Adulto , Biópsia , Diabetes Mellitus Tipo 1/patologia , Eletroforese em Gel Bidimensional , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Pele/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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