RESUMEN
BACKGROUND: Bioinformatics methods are helpful to identify new molecules for diagnostic or therapeutic applications. For example, the use of peptides capable of mimicking binding sites has several benefits in replacing a protein which is difficult to produce, or toxic. Using peptides is less expensive. Peptides are easier to manipulate, and can be used as drugs. Continuous epitopes predicted by bioinformatics tools are commonly used and these sequential epitopes are used as is in further experiments. Numerous discontinuous epitope predictors have been developed but only two bioinformatics tools have been proposed so far to predict peptide sequences: Superficial and PEPOP 2.0. PEPOP 2.0 can generate series of peptide sequences that can replace continuous or discontinuous epitopes in their interaction with their cognate antibody. RESULTS: We have developed an improved version of PEPOP (PEPOP 2.0) dedicated to answer to experimentalists' need for a tool able to handle proteins and to turn them into peptides. The PEPOP 2.0 web site has been reorganized by peptide prediction category and is therefore better formulated to experimental designs. Since the first version of PEPOP, 32 new methods of peptide design were developed. In total, PEPOP 2.0 proposes 35 methods in which 34 deal specifically with discontinuous epitopes, the most represented epitope type in nature. CONCLUSION: Through the presentation of its user-friendly, well-structured new web site conceived in close proximity to experimentalists, we report original methods that show how PEPOP 2.0 can assist biologists in dealing with discontinuous epitopes.
Asunto(s)
Biología Computacional/métodos , Epítopos/metabolismo , Programas Informáticos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Epítopos/química , Sueros Inmunes , Internet , Ratones , Péptidos/sangre , Péptidos/química , Péptidos/inmunología , Dominios Proteicos , Proteínas/químicaRESUMEN
We show here high levels of expression and secretion of the chemokine CXC ligand 5 (CXCL5) in the macrophage fraction of white adipose tissue (WAT). Moreover, we find that CXCL5 is dramatically increased in serum of human obese compared to lean subjects. Conversely, CXCL5 concentration is decreased in obese subjects after a weight reduction program, or in obese non-insulin-resistant, compared to insulin-resistant, subjects. Most importantly we demonstrate that treatment with recombinant CXCL5 blocks insulin-stimulated glucose uptake in muscle in mice. CXCL5 blocks insulin signaling by activating the Jak2/STAT5/SOCS2 pathway. Finally, by treating obese, insulin-resistant mice with either anti-CXCL5 neutralizing antibodies or antagonists of CXCR2, which is the CXCL5 receptor, we demonstrate that CXCL5 mediates insulin resistance. Furthermore CXCR2-/- mice are protected against obesity-induced insulin resistance. Taken together, these results show that secretion of CXCL5 by WAT resident macrophages represents a link between obesity, inflammation, and insulin resistance.
Asunto(s)
Tejido Adiposo/metabolismo , Quimiocina CXCL5/metabolismo , Resistencia a la Insulina , Obesidad/metabolismo , Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Animales , Quimiocina CXCL5/deficiencia , Quimiocina CXCL5/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Rosiglitazona , Transducción de Señal/efectos de los fármacos , Tiazolidinedionas/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaAsunto(s)
Biomarcadores/orina , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/orina , Adiponectina/orina , Albuminuria/orina , Colágeno Tipo IV/orina , Proteínas de la Matriz Extracelular/orina , Ligamiento Genético , Predisposición Genética a la Enfermedad , Productos Finales de Glicación Avanzada/orina , Humanos , Factor de Crecimiento Transformador beta/orinaRESUMEN
IA-2 and IA-2beta are highly related proteins that are autoantigens in type 1 diabetes, and provide a model for developing reagents and assays that distinguish similar proteins with unique autoantibody epitopes. Monoclonal antibodies (mAb) to IA-2 and IA-2beta were prepared and tested for their ability to bind to the related proteins and their ability to compete for specific autoantibody epitope binding by sera from patients with type 1 diabetes. Monoclonal antibodies that specifically bound IA-2 (76F) or bound both IA-2 and IA-2beta (A9) were isolated and characterized. 76F mAb recognized IA-2 of human, rat and mouse origin in native and denatured forms and had an epitope specificity for residues 626-630 (FEYQD) which are found in the juxtamembrane (JM) region of human and mouse IA-2, but not IA-2beta. This region overlaps with the autoantibody epitope JM2. Binding to the 76F monoclonal antibody was specifically inhibited by sera with antibodies to the JM2 epitope but not with antibodies to the adjacent JM1 epitope, indicating that unique epitopes can be distinguished by this approach. 76F mAb has the unique property to distinguish between the two closely related autoantigens IA-2 and IA-2beta by targeting an IA-2 specific epitope of the juxtamembrane region. The findings define an approach to develop assays for specific antibody epitope measurements which may be relevant for disease prognosis and monitoring intervention therapies.
