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2.
Nature ; 404(6779): 774-8, 2000 Apr 13.
Artículo en Inglés | MEDLINE | ID: mdl-10783892

RESUMEN

The transporter associated with antigen processing (TAP) is a member of the family of ABC transporters that translocate a large variety of substrates across membranes. TAP transports peptides from the cytosol into the endoplasmic reticulum for binding to MHC class I molecules and for subsequent presentation to the immune system. Here we follow the lateral mobility of TAP in living cells. TAP's mobility increases when it is inactive and decreases when it translocates peptides. Because TAP activity is dependent on substrate, the mobility of TAP is used to monitor the intracellular peptide content in vivo. Comparison of the diffusion rates in peptide-free and peptide-saturated cells indicates that normally about one-third of all TAP molecules actively translocate peptides. However, during an acute influenza infection TAP becomes fully employed owing to the production and degradation of viral proteins. Furthermore, TAP activity depends on continuing protein translation. This implies that MHC class I molecules mainly sample peptides that originate from newly synthesized proteins, to ensure rapid presentation to the immune system.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas/metabolismo , 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/genética , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Transporte Biológico , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Fluorescentes Verdes , Antígeno HLA-A2/metabolismo , Humanos , Proteínas Luminiscentes , Conformación Proteica , Transfección , Células Tumorales Cultivadas , Proteínas Virales/metabolismo
3.
Proc Natl Acad Sci U S A ; 96(18): 10326-31, 1999 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-10468607

RESUMEN

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH(4)Cl-sensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.


Asunto(s)
Linfocitos B/inmunología , Retículo Endoplásmico/fisiología , Endosomas/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Línea Celular Transformada , Membrana Celular/fisiología , Proteínas Fluorescentes Verdes , Antígenos HLA-D/fisiología , Herpesvirus Humano 4/genética , Humanos , Concentración de Iones de Hidrógeno , Cinética , Proteínas Luminiscentes/metabolismo , Virus del Sarampión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales de Fusión/metabolismo
4.
J Immunol ; 161(1): 83-9, 1998 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-9647210

RESUMEN

MHC class I molecules are cell surface glycoproteins that play a pivotal role in the response to intracellular pathogens. The loading of MHC class I molecules with antigenic substrates takes place in the endoplasmic reticulum. This requires a functional TAP transporter, which translocates peptides into the endoplasmic reticulum from the cytosol. The generation of antigenic peptides from polypeptide precursors is thought to be mediated in the cytosol by the proteasome. Previously, we have demonstrated that inhibiting the proteasome with the specific covalent inhibitor lactacystin results in a direct reduction of peptide-loaded MHC class I molecules. This indicates that the proteasome is the limiting step in the MHC class I pathway. In this study we use isoelectric focusing to demonstrate that two related MHC class I alleles, HLA-A3 and HLA-A11, as well as HLA-B35 do not follow this behavior. In contrast to other class I alleles expressed by the same cells, these alleles are loaded with peptides and mature normally when proteasome activity is severely inhibited. Our observations highlight a new level of diversity in the MHC class I system and indicate that there are allele-specific differences in the linkage between proteasome activity and MHC class I peptide loading.


Asunto(s)
Alelos , Cisteína Endopeptidasas/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Complejos Multienzimáticos/metabolismo , Péptidos/metabolismo , Línea Celular , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/inmunología , Inhibidores de Cisteína Proteinasa/farmacología , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Antígeno HLA-A11 , Antígeno HLA-A3/genética , Antígeno HLA-A3/inmunología , Células HeLa , Humanos , Focalización Isoeléctrica , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/inmunología , Péptidos/inmunología , Complejo de la Endopetidasa Proteasomal
5.
Eur J Immunol ; 27(4): 898-904, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9130642

RESUMEN

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of approximately 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Diseño de Fármacos , Péptidos/química , Péptidos/inmunología , 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 , Transportadoras de Casetes de Unión a ATP/inmunología , Secuencia de Aminoácidos , Unión Competitiva/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Péptidos/síntesis química , Unión Proteica/inmunología , Estereoisomerismo , Especificidad por Sustrato/inmunología
6.
Eur J Immunol ; 25(4): 1133-6, 1995 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7737286

RESUMEN

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico , Línea Celular , Humanos , Ratones , Microsomas/metabolismo , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/metabolismo , Prolina/metabolismo , Ratas
7.
J Exp Med ; 180(5): 1591-7, 1994 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-7964447

RESUMEN

Cytosolic peptides are translocated to the endoplasmic reticulum (ER) lumen by the transporters associated with antigen processing (TAP), where major histocompatibility complex (MHC) class I molecules associate with peptides of about 8-10 amino acids. TAP translocates peptides of 9-13 amino acids with the highest relative affinity but also longer and shorter peptides. The fate of the peptides that fail to associate with class I molecules because of incorrect sequence or length, is unknown. Here we show that the bulk of the translocated peptides are rapidly released from the ER by a mechanism that requires adenosine triphosphate (ATP) and that could not be inhibited by GTP gamma S. TAP does not appear to be involved in this process. Whereas free peptides are slowly trimmed in the ER lumen, they are rapidly degraded in the cytosol. A fraction of the peptides released from the ER escapes complete degradation in the cytosol and recycles back to the ER in a TAP-dependent fashion. These results suggest that peptides that are too long for binding to class I molecules in the ER can be trimmed further in the ER lumen or, alternatively, can be transported back to the cytosol where a fraction of the peptides is trimmed to a size suitable for association to MHC class I molecules and recycles back to the ER.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Glicosilación , Antígenos de Histocompatibilidad Clase I/metabolismo , Datos de Secuencia Molecular
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