In a new line of investigation, we have recently reported that photo-activated 8-methoxypsoralen (8-MOPa) binds to a subset of cytoplasmic proteins and substantially augments the display of cell surface antigens on human lymphoblasts, in a manner dependent on the transport of additional peptide antigens and increases in expression of class I major histocompatibility complexes (MHC). This data led us to hypothesize that the increased immunogenicitv associated with 8-MOPa treatment results from the formation of 8-MOP/ protein photoadducts that fundamentally alter MHC class I processing and presentation. To understand the mechanism by which 8-MOPa leads to the observed augmentation, we proposed in the initial pilot/feasibility project to characterize cytoplasmic protein targets of 8-MOP and to link these targets to components of the antigen processing and presentation pathway. Here we report the successful completion of the first of these aims and propose an additional group of experiments to precisely define the immunological ramifications of 8-MOP binding: 1) Specific Aim 1- Sequence and characterize all intracellular 8-MOP binding proteins and verify binding in vitro utilizing specific antibodies and inhibitors. Following our initial success in identifying the first of several 8-MOP binding protein (Preliminary Results) we propose to purify and sequence three additional binding proteins observed covalently bound to 8-MOP. This selective binding will be verified by immunoprecipitation of these proteins in association with tritium labeled 8-MOP and by blocking of the binding with specific inhibitors. 2) Specific Aim 2- Determine the mechanism by which 8-MOPa augments display of class I MHC molecules. 8-MOPa -induced changes in expression of MHC class I-associated proteins (MHC class I, beta2microglobulin, TAP 1, tapasin, ubiquitin and proteosome subunits) will be quantitated. 8-MOP binding proteins will be analyzed for their interaction with components of the MHC class I processing and presentation pathways; if direct links are observed, the interaction will be interrupted with specific inhibitors.
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