Redox modulation of the immunoproteasome in dendritic cells under oxidative challenge: implications upon generation of peptides for antigenic presentation by MHC I

Abstract

Dendritic cells are crucial to initiate immune response. Peptides generated to antigenic presentation by MHC I complex are derived from immunoproteasomal cleavage. The difference between the standard and imunoproteasome consists on the increased generation of peptides for antigenic presentation by a kinetic effect attributed to the substitution of three catalytic subunits: beta1,beta2 and beta3 by their counterparts: LMP2, MECL1 and LMP7, respectively. Differently from other nucleate cells, the immunoproteasome is constitutive in dendritic cells. Chymotrypsin- as well as trypsin-like activities are increased in the immunoproteasome when compared to those activities in the standard protease. Increased generation of peptides with hydrophobic residues (chymotrypsin-like activity) increases peptide binding to MHC I molecules and consequently, increases antigenic presentation. The laboratory has accumulated thus far data showing that the proteasome is redox modulated by its Cys residues through S-glutathionylation. That process has been described for other proteins and it occurs when alterations on the intracellular redox state takes place. One of the mechanisms proposed is the oxidation of Cys-SH to Cys-SOH followed by S-glutathionylation that implies on the formation of a mixed disulfide between protein Cys residues and glutathione: Cys-SG. S-glutathionylation of the proteasome decreases its chymotrypsin- and caspase-like activities without any alteration on the trypsin-like activity. The goal of the present work is to investigate the role of imunoproteasome S-thionylation upon the generation of peptides for antigenic presentation. For that, dendritic cells will be submitted to moderate oxidative challenge in order to promote S-glutathionylation of the immunoproteasome. Next, we will examine antigenic presentation by MHC I, monitored by flow cytometry with anti-MHC I antibody. Immunoproteasome S-glutathionylation will be evaluated by western blot analysis with the anti-GSH antibody. Our hypothesis is that redox modifications on the immunoproteasome alters the pattern of peptides generated during proteolysis modifying antigenic presentation. Ovalbumin will be utilized as the standard protein for those studies. We believe that such studies might contribute to better understanding MHC I antigen presentation by dendritic cells. (AU)