The long term goal of this proposal is to understand the molecular antigen processing mechanism by which endogenously synthesized proteins yield precisely cleaved peptide/MHC class I complexes on the cell surface. The expression of these peptide/MHC complexes is essential for CD8+ cytotoxic T-cell immunity specific for tumors, intracellular pathogens, allogeneic tissue grafts and for autoimmunity. Failure to express peptide/MHC complexes is a major mechanism by which tumor cells and viruses such as herpes simplex virus, escape immune surveillance. Elucidating the molecular mechanism that allows endogenously synthesized proteins to be fragmented and displayed as peptide/MHC will also lead to understanding a key factor in determining epitope dominance, and thus improve design of vaccines for eliciting immunity against established tumors and intracellular pathogens. Novel T-cell based """"""""lacZ"""""""" assays have been developed that permit quantitation and identification of processed peptides in the attomole range. Together with methods to express DNA constructs encoding defined antigenic precursors and for characterizing the processed peptide precursors and products in model cell lines the following problems will be addressed. (1) Whether naturally processed peptides arise as a consequence of cellular protein turn-over and/or from nascent proteins will be established by selective alteration of stability of antigenic precursor proteins via the N-end rule. (2) The influence of flanking residues on the generation of processed peptides will be determined by introducing specific mutations in model precursors and full-length proteins. The immunological consequences of altering the quantitative or qualitative outcome of antigen processing will be evaluated by measurement of T-cell responses to different dominant and sub-dominant peptides in these precursors. (3) The identity of processed peptide intermediates during the pre- and post-TAP step and their chaperones, that eventually yield the final precisely cleaved peptide products will be determined by analysis of HPLC fractionated cell extracts and novel assays. Finally, (4) we will generate and analyze new mutations in the antigen processing pathway. These genetic mutants are likely to reveal steps that occur during processing of endogenously synthesized precursors in living cells but may be inaccessible to conventional biochemical analysis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Allergy and Immunology Study Section (ALY)
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Prasad, Shiv A
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University of California Berkeley
Schools of Arts and Sciences
United States
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Prasad, Sharanya; Starck, Shelley R; Shastri, Nilabh (2016) Presentation of Cryptic Peptides by MHC Class I Is Enhanced by Inflammatory Stimuli. J Immunol 197:2981-2991
Shastri, Nilabh; Nagarajan, Niranjana; Lind, Kristin C et al. (2014) Monitoring peptide processing for MHC class I molecules in the endoplasmic reticulum. Curr Opin Immunol 26:123-7
Nagarajan, Niranjana A; Shastri, Nilabh (2013) Immune surveillance for ERAAP dysfunction. Mol Immunol 55:120-2
Kanaseki, Takayuki; Lind, Kristin Camfield; Escobar, Hernando et al. (2013) ERAAP and tapasin independently edit the amino and carboxyl termini of MHC class I peptides. J Immunol 191:1547-55
Nagarajan, Niranjana A; Gonzalez, Federico; Shastri, Nilabh (2012) Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum. Nat Immunol 13:579-86
Cardinaud, Sylvain; Starck, Shelley R; Chandra, Piyanka et al. (2010) The synthesis of truncated polypeptides for immune surveillance and viral evasion. PLoS One 5:e8692
Blanchard, Nicolas; Kanaseki, Takayuki; Escobar, Hernando et al. (2010) Endoplasmic reticulum aminopeptidase associated with antigen processing defines the composition and structure of MHC class I peptide repertoire in normal and virus-infected cells. J Immunol 184:3033-42
Blanchard, Nicolas; Shastri, Nilabh (2010) Topological journey of parasite-derived antigens for presentation by MHC class I molecules. Trends Immunol 31:414-21
Blanchard, Nicolas; Shastri, Nilabh (2010) Cross-presentation of peptides from intracellular pathogens by MHC class I molecules. Ann N Y Acad Sci 1183:237-50
Blanchard, Nicolas; Shastri, Nilabh (2008) Coping with loss of perfection in the MHC class I peptide repertoire. Curr Opin Immunol 20:82-8

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