The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the β-chain (DR1βH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1βH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1βH81N complexes. Reintroduction of an appropriate hydrogen bond (DR1βH81N βV85H) restored DM-mediated peptide dissociation. Thus, DR1βH81N might represent a `post-DM effect' conformation. We suggest that DM may mediate peptide dissociation by a `hit-and-run' mechanism that results in conformational changes in MHC class II molecules and disruption of hydrogen bonds between βHis81 and bound peptide.Shortly after being synthesized in the antigen-presenting cell, major histocompatibility complex (MHC) class II αβ heterodimers form nonameric assemblies with invariant chain (Ii) in the endoplasmic reticulum and are then transported through the Golgi complex to the endocytic pathway 1, 2. During transport through the endocytic pathway, most Ii is removed from MHC class II molecules by low pH and acid proteases3, leaving a proteolytic fragment of Ii called `CLIP' bound to MHC class II molecule4. CLIP acts as a `placeholder' for the MHC class II groove, inhibiting conformational changes that render the groove closed5 -13 , and it must be removed to allow binding of exogenous peptides to nascent MHC class II complexes. Human HLA-DM (called `DM' here), or H2-M in mice, is a nonclassical HLA molecule that is critical in the displacement of CLIP 14-17. In addition to displacing CLIP, DM transiently interacts with empty MHC class II molecules to generate a peptide-receptive conformation and is active in the selection of specific peptide-MHC class II complexes during antigen processing18 -26. The two concurrent hypotheses for the recognition of certain peptide-MHC class II by DM relate to the intrinsic affinity between MHC class II © 2006 Nature Publishing Group Correspondence should be addressed to S.S-N (ssadegh@jhmi.edu).. 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Immunology website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Immunol. Author manuscript; available in PMC 2011 January 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and the peptide 22,27,28 or to subtle structural variations among different peptide-MHC complexes 25,[29][30][31][32] , whereby structurally flexible complexes are susceptible to DM-induced dissociation, and `rigid' complexes are resistant to DM 25 . Although those studies may have brought greater understanding of the crite...

Immunodominant epitopes are few selected epitopes from complex antigens that initiate T cell responses. Here, to provide further insights into this process, we use a reductionist cell-free antigen processing system composed of defined components. We use the system to characterize steps in antigen processing of pathogen-derived proteins or autoantigens and we find distinct paths for peptide processing and selection. Autoantigen-derived immunodominant epitopes are resistant to digestion by cathepsins, whereas pathogen-derived epitopes are sensitive. Sensitivity to cathepsins enforces capture of pathogen-derived epitopes by Major Histocompatibility Complex class II (MHC class II) prior to processing, and resistance to HLA-DM-mediated-dissociation preserves the longevity of those epitopes. We show that immunodominance is established by higher relative abundance of the selected epitopes, which survive cathepsin digestion either by binding to MHC class II and resisting DM-mediated-dissociation, or being chemically resistant to cathepsins degradation. Non-dominant epitopes are sensitive to both DM and cathepsins and are destroyed.

Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets.