Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Complexo Principal de Histocompatibilidade , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/análise , Marcadores de Afinidade , Azidas , Linfócitos B , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Reagentes de Ligações Cruzadas , Dimerização , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Subunidades ProteicasRESUMO
To gain a better understanding of the intracellular sites of antigen processing we have looked at the localization of human immunodeficiency virus (HIV)-1 Nef protein by confocal microscopic and biochemical means. We found that ubiquitin (Ub)-Nef fusion proteins were localized to the centrosome in transfected COS-7 cells, and that the colocalization was inhibited by the microtubule-disrupting agent, nocodazole. Interestingly, we found that Ub-Nef trafficking to the centrosome was not dependent upon the metabolic stability of Ub-Nef nor on the inhibition of proteasome activity. We also analyzed the MHC class I antigen processing of a reporter epitope linked to the Ub-Nef fusion proteins and found that Ub-Nef was processed in COS-7 cells. In addition, we show that this processing was inhibited by nocodazole. We suggest that the centrosome may serve as a site of antigen processing in vivo.
Assuntos
Centrossomo/metabolismo , Produtos do Gene nef/metabolismo , HIV-1/metabolismo , Animais , Apresentação de Antígeno/efeitos dos fármacos , Sequência de Bases , Células COS , Centrossomo/efeitos dos fármacos , Centrossomo/imunologia , Primers do DNA/genética , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , HIV-1/genética , HIV-1/imunologia , Humanos , Nocodazol/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência HumanaAssuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Apresentação de Antígeno , Desenho de Fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Proteínas Virais , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transporte Biológico , Proteínas Imediatamente Precoces/metabolismo , Mimetismo Molecular , Proteínas do Envelope Viral/metabolismoRESUMO
Chemical cross-linking of the transporter associated with antigen processing (TAP) heterodimer was used to determine whether the herpes simplex virus inhibitor of TAP, ICP47, induces a conformational change in TAP. Cross-linking of TAP in cellular membranes produced a major species of approximately 220 kDa which was comprised solely of TAP.1 and TAP.2 and most likely represents the TAP heterodimer. Interestingly, prior treatment of TAP-containing membranes with TAP peptide substrates stimulated the formation of the cross-linked TAP heterodimer, whereas pretreatment of membranes with ICP47 completely blocked the formation of the cross-linked heterodimer. These data suggest that suitable substrates for TAP stabilize the TAP heterodimer, whereas ICP47 destabilizes the heterodimer. The results indicate that subtle conformational changes occur in the TAP heterodimer upon the binding of peptides and the inhibitor ICP47 and that ICP47 has a deleterious effect on TAP heterodimer structure, in addition to its role as a potent blocker of substrate binding to TAP.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Proteínas Virais , Sequência de Aminoácidos , Animais , Linhagem Celular , Dimerização , Radioisótopos do Iodo , SpodopteraRESUMO
To gain insight into how tumor antigens are generated and presented, a panel of peptides corresponding to melanoma-specific T cell epitopes were tested for their transport capacity by the transporter associated with antigen processing (TAP). The melanoma epitopes exhibited differential capacities to be transported by TAP in streptolysin O-permeabilized cells, as well as differential competition for peptide binding to TAP. The data indicate that some melanoma-specific epitopes are good substrates for TAP, while others are poor substrates for TAP. One of the epitopes, derived from tyrosinase, was transported into the endoplasmic reticulum (ER), in spite of being a poor competitor for reporter peptide transport and for peptide binding. These results suggest that the melanoma antigens follow distinct pathways for presentation, along the MHC class I pathway.