RESUMO
Dendritic cells are unique in their capacity to process antigens and prime naive CD8(+) T cells. Contrary to most cells, which express the standard proteasomes, dendritic cells express immunoproteasomes constitutively. The melanoma-associated protein Melan-A(MART1) contains an HLA-A2-restricted peptide that is poorly processed by melanoma cells expressing immunoproteasomes in vitro. Here, we show that the expression of Melan-A in dendritic cells fails to elicit T-cell responses in vitro and in vivo because it is not processed by the proteasomes of dendritic cells. In contrast, dendritic cells lacking immunoproteasomes induce strong anti-Melan-A T-cell responses in vitro and in vivo. These results suggest that the inefficient processing of self-antigens, such as Melan-A, by the immunoproteasomes of professional antigen-presenting cells prevents the induction of antitumor T-cell responses in vivo.
Assuntos
Células Dendríticas/enzimologia , Proteínas de Neoplasias/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/imunologia , Células Dendríticas/imunologia , Antígeno HLA-A2/imunologia , Humanos , Imunoterapia Adotiva/métodos , Ativação Linfocitária , Antígeno MART-1 , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitin-acceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells.
Assuntos
Lisossomos/metabolismo , Melanossomas/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Antígenos de Neoplasias , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Antígeno MART-1 , Melanossomas/química , Ubiquitina-Proteína Ligases Nedd4 , Proteínas de Neoplasias/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Repressoras/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The proteasome produces MHC class I-restricted antigenic peptides carrying N-terminal extensions, which are trimmed by other peptidases in the cytosol or within the endoplasmic reticulum. In this study, we show that the N-terminal editing of an antigenic peptide with a predicted low TAP affinity can occur in the cytosol. Using proteomics, we identified two cytosolic peptidases, tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, that trimmed the N-terminal extensions of the precursors produced by the proteasome, and led to a transient enrichment of the final antigenic peptide. These peptidases acted either sequentially or redundantly, depending on the extension remaining at the N terminus of the peptides released from the proteasome. Inhibition of these peptidases abolished the CTL-mediated recognition of Ag-expressing cells. Although we observed some proteolytic activity in fractions enriched in endoplasmic reticulum, it could not compensate for the loss of tripeptidyl peptidase II/puromycin-sensitive aminopeptidase activities.