RESUMEN
The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Complejo Mayor de Histocompatibilidad , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Miembro 3 de la Subfamilia B de Transportadores de Casetes de Unión a ATP , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacocinética , Sustitución de Aminoácidos , Animales , Azidas/farmacocinética , Sitios de Unión , Línea Celular , Células HeLa , Humanos , Lisina , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Etiquetas de Fotoafinidad , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The 70 kDa mycobacterial heat shock protein (Mtb HSP70) stimulates mononuclear cells to release CC-chemokines. We now show that this function of Mtb HSP70, but not human HSP70, is dependent on the cell surface expression of CD40. Deletion of the CD40 cytoplasmic tail abolished, and CD40 antibody inhibited, Mtb HSP70 stimulation of CC-chemokine release. Mtb HSP70 stimulated THP1, KG1 cells, and monocyte-derived dendritic cells to produce RANTES. Specific binding of CD40-transfected HEK 293 cells to Mtb HSP70 was demonstrated by surface plasmon resonance. Coimmunoprecipitation of Mtb HSP70 with CD40 indicates a physical association between these molecules. The results suggest that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production.
Asunto(s)
Antígenos CD40/fisiología , Quimiocina CCL5/biosíntesis , Células Dendríticas/efectos de los fármacos , Ácido Egtácico/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas Inflamatorias de Macrófagos/biosíntesis , Monocitos/efectos de los fármacos , Mycobacterium tuberculosis/fisiología , Proteínas Bacterianas , Antígenos CD40/química , Antígenos CD40/genética , Calcio/fisiología , Señalización del Calcio/efectos de los fármacos , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Membrana Celular/metabolismo , Quelantes/farmacología , Quimiocina CCL4 , Quimiocina CCL5/genética , Células Dendríticas/metabolismo , Ácido Egtácico/farmacología , Proteínas de Escherichia coli/farmacología , Proteínas HSP70 de Choque Térmico/farmacología , Humanos , Riñón , Lipopolisacáridos/farmacología , Sustancias Macromoleculares , Proteínas Inflamatorias de Macrófagos/genética , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Unión Proteica , Proteínas Recombinantes de Fusión/fisiología , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismoRESUMEN
Assembly of antigen-presenting complexes between class I MHC molecules and peptide requires formation of a complex between the 'ABC' peptide transporter, TAP, and newly synthesized class I molecules. Recent studies have provided new insights into the role of ATP in peptide binding, transport and release.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Presentación de Antígeno , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/inmunología , Regulación Alostérica , Transporte Biológico , Metabolismo Energético , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Linfocitos/inmunología , Linfocitos/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Conformación ProteicaRESUMEN
In its attempt to evade cytotoxic T cell recognition, human cytomegalovirus encodes several genes that target MHC class I molecules at different points in their assembly pathway. We show here that the human cytomegalovirus US6 gene encodes a 22-kDa glycoprotein that binds the transporter-associated with antigen processing (TAP)/class I complex and inhibits translocation of peptide from the cytosol to the endoplasmic reticulum. Major histocompatibility complex class I molecules are therefore unable to load TAP-dependent peptides, resulting in the retention of MHC class I molecules in the endoplasmic reticulum, with a consequent reduction in class I at the cell surface. Interferon-gamma treatment of US6 transfected cells overcomes this inhibition of peptide translocation and restores class I at the cell surface to wild type levels. The functional consequence of TAP inhibition is that US6 transfected cells are unable to present endogenous antigen to cytotoxic T lymphocytes and are therefore resistant to cytotoxic T lymphocyte lysis.
Asunto(s)
Presentación de Antígeno/inmunología , Citomegalovirus/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Proteínas del Envoltorio Viral/inmunología , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/genética , Línea Celular Transformada , Técnicas de Transferencia de Gen , Células HeLa , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/farmacología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Flow cytometric analyses of the surface immunoglobulins of murine memory B cells revealed the existence of populations expressing multiple isotypes, including an IgM+/IgG+ population that could be stimulated in vitro with antigen to secrete both IgM and IgG. Female BALB/c mice were immunized with R-phycoerythrin (RPE), a fluorescent photosynthetic accessory protein from red algae. Pooled splenocytes from these mice at different stages of immunization were stained with RPE as well as with allophycocyanin- and fluorescein-conjugated anti-isotype antibodies and analysed on a two-laser FACS. RPE-binding cell sub-populations were defined and selectively sorted to verify their phenotype and to demonstrate that the various subpopulations (IgM+/IgG+, IgM+/IgG-, IgM-/IgG+) had different isotype-secretion patterns when challenged with RPE in vitro. These results re-affirm the notion that a transcriptional processing mechanism may be responsible for the simultaneous expression of multiple isotypes in memory cells.