The long-term objective of this project is to gain insight into the structure and function of the transporters associated with antigen processing (TAP molecules). TAP transporters play a key role in the MHC class I antigen presentation pathway, which in turn, is fundamental to the generation of an anti-tumor immune response. Peptide epitopes produced in the cytosol are transported by TAP across the endoplasmic reticulum membrane for assembly with class I molecules. The subsequent expression of the peptide epitopes on the cell surface allows for the recognition of the cell by tumor-specific effector cells of the immune system leading to destruction of the tumor cell.
The specific aims of the proposal are (1) to reconstitute TAP function from purified components, (2) to identify the peptide binding regions of TAP, and (3) to generate TAP/MDR chimeric molecules as a way to analyze TAP function.
The first aim will provide definitive evidence that TAP transports peptides by assaying the function of TAP in isolation, i.e., purified TAP incorporated into defined lipid membranes. In addition, the functional reconstitution of TAP will provide the basis for further biochemical and biophysical analysis of TAP function.
The second aim attempts to identify those regions of the TAP molecule that are involved in peptide recognition, leading to a structural and mechanistic understanding of the peptide translocation process. This will be carried out by the covalent modification of the TAP peptide binding site through the use of photoreactive peptide analogues, followed by proteolytic digestion, and identification of the TAP peptide fragments which contain the modification.
The third aim i s designed to compare and contrast the mechanistic features of TAP and the multi-drug resistance transporter (MDR). TAP and MDR are highly homologous members of the ATP- binding cassette (ABC) family of transporters. Through the generation of TAP/MDR chimeric proteins, and TAP/MDR cross-complementation assays, we hope to gain insight into the mechanism of action of TAP, as well as MDR. Overall, this proposal will provide fundamental knowledge of the structure and function of TAP, which could eventually play a role in the treatment or cure of cancer.
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