In recent years, cell-based studies have been extremely useful in providing an understanding of how key proteins of the endoplasmic reticulum (ER) cooperate together to fold and assemble class I MHC molecules. However, because these studies were carried out from different directions, it inevitably lead to a fragmented view of class I maturation. At present, the use of molecular and structural approaches to study the class I assembly pathway is not only possible but is necessary to unify and advance our knowledge of class I maturation. The long-term goal of this application is to provide an integrated understanding of functional, molecular, and structural aspects of the class I antigen presentation pathway. This project seeks to determine the mechanistic basis of the class I antigen presentation pathway with a focus on the role of tapasin (TPN).
The Specific Aims are: (1) to reconstitute interaction within the class I assembly complex in order to gain insights into the process of peptide selection by class I molecules. For this, we designed a soluble TPN/HLA-B*0801 complex tethered to a Jun/Fos linkage. The TPNjun/HLA- B*0801fos complex is stable and enables us to carry out for the first time direct peptide kinetics association/dissociation and peptide exchange experiments. The inclusion of CRT and ERp57, or their assembly intermediaries, to the TPNjun/HLA-B*0801fos complex will permit to examine how these proteins influence TPN-mediated events;(2) to understand the molecular basis of allelic differences in TPN dependencies for class I maturation;(3) to identify interaction sites between TPN and HLA-B*0801 molecules;and (4) to characterize the lectin- and polypeptide-binding sites in CRT with atomic details. These questions will be addressed using techniques such as kinetics-based assays, site-directed mutagenesis, circular dichroism, fluorescence, analytical ultracentrifugation, and x-ray crystallography. Class I MHC-restricted immune responses are essential for controlling the spread of viruses and the malignancy of carcinomas. It is therefore important to understand how events from within cells modulate the peptide repertoire presented by class I molecules since these ultimately impact on the ability of individuals to respond to challenges. Advances in understanding the class I assembly pathway will also move the field of viral immune evasion mechanisms forward given that numerous viruses have evolved strategies to interfere with class I maturation in the ER. Our studies will also provide new knowledge on the mode of action of ER- resident chaperones in preventing protein misfolding, a phenomenon linked to a growing number of diseases.
