RESUMO
The majority of MHC class I epitopes is generated through the ubiquitin-proteasome system. In the present study, we have analyzed the proteasome-dependent generation of the IE pp89 MCMV-derived H-2L(d) epitope by both in vitro and in vivo experiments. As revealed by cytotoxic T-cell assays, the pp89 9mer epitope was generated with high fidelity from the recombinant IE pp89 by 20S proteasomes. In vitro processing showed that the recombinant pp89 was rapidly degraded by 20S proteasomes. Analysis of cell lysates under conditions that allowed detection of polyubiquitinated proteins provided no evidence for the presence of ubiquitin-pp89-conjugates in vivo. These findings suggest a ubiquitin-independent mechanism of proteasomal degradation for pp89.
Assuntos
Epitopos/biossíntese , Antígenos H-2/imunologia , Muromegalovirus/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Dicroísmo Circular , Antígenos H-2/química , Antígeno de Histocompatibilidade H-2D , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Especificidade por Substrato , Ubiquitina/metabolismoRESUMO
The human immunodeficiency virus-1 Tat protein inhibits the peptidase activity of the 20S proteasome and competes with the 11S regulator/PA28 for binding to the 20S proteasome. Structural comparison revealed a common site in the Tat protein and the 11S regulator alpha-subunit (REGalpha) called the REG/Tat-proteasome-binding (RTP) site. Kinetic assays found amino acid residues Lys51, Arg52 and Asp67 forming the RTP site of Tat to be responsible for the effects on proteasomes in vitro. The RTP site identified in REGalpha consists of the residues Glu235, Lys236 and Lys239. Mutation of the REGalpha amino acid residues Glu235 and Lys236 to Ala resulted in an REGalpha mutant that lost the ability to activate the 20S proteasome even though it still forms complexes with REGbeta and binds to the 20S proteasome. The REGalpha RTP site is needed to enhance the presentation of a cytomegalovirus pp89 protein-derived epitope by MHC class I molecules in mouse fibroblasts. Cell experiments demonstrate that the Tat amino acid residues 37-72 are necessary for the interaction of the viral protein with proteasomes in vivo. Full-length Tat and the Tat peptide 37-72 suppressed 11S regulator-mediated presentation of the pp89 epitope. In contrast, the Tat peptide 37-72 with mutations of amino acid residues Lys51, Arg52 and Asp67 to Ala was not able to reduce antigen presentation.
