RESUMO
Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.
Assuntos
Diabetes Mellitus/sangue , Hemoglobinas Glicadas/metabolismo , Glicoproteínas/sangue , Albumina Sérica/metabolismo , Animais , Glicemia/metabolismo , Western Blotting , Análise por Conglomerados , Diabetes Mellitus Experimental/sangue , Eletroforese em Gel Bidimensional , Hemoglobinas Glicadas/química , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Glicoproteínas/química , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Proteômica , Albumina Sérica/químicaRESUMO
Cancer is associated with increased glycolysis and carbonyl stress. In view of this, AGE modified proteins were identified from clinical breast cancer tissue using 2DE-immunoblot and mass-spectrometry. These proteins were identified to be serotransferrin, fibrinogen gamma chain, glycerol-3-phosphate dehydrogenase, lactate dehydrogenase, annexin II, prohibitin and peroxiredoxin 6, which have established role in cancer. Further, RAGE expression and its downstream signaling proteins NADPH oxidase and NF-kB were studied. Role of these AGE modified proteins and RAGE signaling in breast cancer is discussed.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Produtos Finais de Glicação Avançada/metabolismo , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas de Neoplasias/metabolismo , Receptor para Produtos Finais de Glicação Avançada/biossíntese , Sequência de Aminoácidos , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Feminino , Humanos , Dados de Sequência Molecular , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Invasividade Neoplásica , Processamento de Proteína Pós-Traducional , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismoRESUMO
Post-translational modifications (PTMs) are very important to biological function, however their identification and characterization is technically challenging. In this study, we have identified glycation modifications by nano LC-MSE, a data independent acquisition work flow, followed by database search using the Protein Lynx Global Server (PLGSJ). PLGS search with a complete human protein database hardly identified glycation modifications in a glycated human serum albumin (HSA), which was detected to be glycated by western blotting with advanced glycation end products (AGE) antibody and fluorescence spectroscopy. To overcome this difficulty, "Zoom-In" approach, a targeted database search was used to identify glycation modifications in a glycated HSA, which were further manually validated. This approach was useful for identification of glycation modifications from untargeted tandem mass spectrometryworkflow such as MSE, but may require the development of a new algorithm or an upgrade of the existing software.
Assuntos
Bases de Dados de Proteínas , Produtos Finais de Glicação Avançada/análise , Proteínas/química , Albumina Sérica/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Western Blotting , Glicopeptídeos/química , Glicopeptídeos/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Albumina Sérica/metabolismo , Espectrometria de FluorescênciaRESUMO
Glycation, a non-enzymatic reaction between glucose and protein is the primary cause of diabetic complications. Albumin, the most abundant plasma protein undergoes glycation both in vivo and in vitro. The influence of albumin on glycation of less abundant proteins has not been addressed. For the first time, we show that albumin competitively inhibits the glycation of less abundant proteins. This study suggests that at least in the initial stages of diabetes, albumin may protect other proteins from glycation.
Assuntos
Proteínas/química , Proteínas/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Ligação Competitiva , Anidrases Carbônicas/metabolismo , Complicações do Diabetes/metabolismo , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação , Humanos , Técnicas In Vitro , Insulina/química , Insulina/metabolismo , Cinética , Mioglobina/química , Mioglobina/metabolismo , Papaína/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Adhesive interactions between molecules on tumor cells and those on target organs play a key role in organ specific metastasis. Poly-N-acetyl-lactosamine (polyLacNAc) substituted N-oligosaccharides on melanoma cell surface glycoproteins promote lung specific metastasis via galectin-3 by facilitating their arrest and extravasation. This study reports the identification and characterization of galectin-3 interacting proteins using a combination of galectin-3 sepharose affinity and leucoagglutinating phytohemagglutinin (L-PHA) columns. A total of 83 proteins were identified as galectin-3 interacting glycoproteins, of which 35 were constituents of the L-PHA bound fraction, suggesting that these proteins carry polyLacNAc substituted ß1,6 branched N-glycans. The identities of some of these proteins, like LAMP-1, LAMP-3, basigin, embigin, and α5 and ß1 Integrin, have been confirmed by western blotting, and functional relevance with respect to metastatic properties has been established.
Assuntos
Proteínas de Transporte/metabolismo , Galectina 3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Espectrometria de Massas , Melanoma/patologia , Mapeamento de Interação de Proteínas/métodos , Animais , Cromatografia de Afinidade , Cromatografia Líquida , Espectrometria de Massas/métodos , Melanoma Experimental , Camundongos , Ligação Proteica , Reprodutibilidade dos Testes , Fluxo de TrabalhoRESUMO
Glycation, a nonenzymatic reaction between reducing sugars and proteins, is a proteome wide phenomenon, predominantly observed in diabetes due to hyperglycemia. Glycated proteome of plasma, kidney, lens, and brain are implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative diseases, cancer, and aging. This review discusses the strategies to characterize protein glycation, its functional implications in different diseases, and intervention strategies to protect the deleterious effects of protein glycation.
Assuntos
Glicosilação , Proteoma/metabolismo , Proteômica , Animais , Produtos Finais de Glicação Avançada/metabolismo , Glicosilação/efeitos dos fármacos , Humanos , Proteoma/químicaRESUMO
Proteomic approaches aid in gaining a better understanding of the pathophysiology of diabetic complications. In view of this, differential protein expression in diabetic plasma samples was studied by a combination of proteomic and western blot analyses. Diabetic plasma samples were categorized based on glycated haemoglobin levels as controlled diabetes (CD; 7-8%), poorly controlled diabetes (PCD; >8%) and non-diabetic control (ND;<6.4%). Two-dimensional electrophoresis and liquid chromatographymass spectrometry revealed differential expression of proteins including upregulation of fibrinogen and haptoglobin and downregulation of vitamin D binding protein, α-1-antitrypsin, transthyretin and apolipoprotein A1 (Apo A1) in diabetic compared with non-diabetic plasma samples. Amongst these proteins, Apo A1 downregulation was prominent in PCD. Downregulation of Apo A1 may serve as an early predictive marker of diabetic complications.
Assuntos
Apolipoproteína A-I/sangue , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus/metabolismo , Espectrometria de Massas , Proteoma/análise , Diabetes Mellitus/patologia , Regulação para Baixo , Eletroforese em Gel Bidimensional , Hemoglobinas Glicadas/análise , Humanos , Projetos PilotoRESUMO
Breast cancer, especially ER positive/HER2/neu negative IDC, is the predominant subtype of invasive ductal carcinoma. Although proteomic approaches have been used towards biomarker discovery in clinical breast cancer, ER positive/HER2/neu negative IDC is the least studied subtype. To discover biomarkers, as well as to understand the molecular events associated with disease progression of estrogen receptor positive/HER2/neu negative subtype of invasive ductal carcinoma, differential protein expression profiling was performed by using LC-MS(E) (MS at elevated energy). A total of 118 proteins were identified, of which 26 were differentially expressed. These identified proteins were functionally classified and their interactions and coexpression were analyzed by using bioinformatic tools PANTHER (Protein Analysis THrough Evolutionary Relationships) and STRING (Search Tool for the Retrieval of Interacting Genes). These proteins were found to be upregulated and were involved in cytoskeletal organization, calcium binding, and stress response. Interactions of annexin A5, actin, S100 A10, glyceraldehyde 3 phosphate dehydrogenase, superoxide dismutase 1, apolipoprotein, fibrinogen, and heat shock proteins were prominent. Differential expression of these proteins was validated by two-dimensional gel electrophoresis and Western blot analysis. The cluster of these proteins may serve as a signature profile for estrogen receptor positive/ HER2/neu negative subtype.