Details

Autor(en) / Beteiligte

• Serwold, Thomas Francis

Titel

Analysis of the cellular processes that generate peptides for major histocompatibility complex class I molecules

Ort / Verlag

ProQuest Dissertations Publishing

Erscheinungsjahr

2000

Link zum Volltext

• ProQuest

Quelle

ProQuest Dissertations & Theses A&I

Beschreibungen/Notizen

• All cells in the body display on their surfaces an abundance of peptides derived from the breakdown of intracellular proteins. These peptides are bound to Major Histocompatibility Complex Class I molecules, and are surveyed by CD8+ T cells of the immune system. The process that generates these peptide/MHC class I complexes is essential for immunity to intracellular pathogens as well as for the generation and maintenance of the CD8+ T cell repertoire. In these chapters, I describe the characterization of the process that generates the peptides that are displayed by MHC class I molecules. In initial studies, I characterize the proteolytic cleavages that generate two overlapping peptides from a single protein precursor. This analysis revealed that the diversity of peptides supplied to MHC class I molecules was limited by specific proteasome-mediated cleavages at the C-termini of peptides. Furthermore, generation of the N-termini of peptides was proteasome-independent, indicating that N- and C-termini of peptides for MHC class I molecules could be generated by different proteases. Next, I developed a method using a modified protein precursor and a post-extraction, protease-treatment step that enabled the detection of all proteolytic intermediates in the antigen-processing pathway. Using this method, I identified three different proteolytic steps that contributed to the generation and destruction of peptides for MHC class I molecules. Analysis of MHC class I-independent peptides in this study revealed that many peptides were generated that were longer than the optimal, antigenic peptides. To see if these longer peptides could be further trimmed in the endoplasmic reticulum (ER), we analyzed the types of antigen trimming reactions that could occur in the ER of cells. This study revealed that precursors in the ER that were longer than optimal could be cleaved to the optimal peptide only if they already had a perfectly trimmed C-terminus. This study also revealed that while generation of N-termini of peptides in the ER could occur in most cases, it did not occur when the N-terminal flanking residue was a proline. Finally, I used cellular mutagenesis to search for novel proteins that are involved in the generation of peptides for MHC class I molecules. These experiments yielded several mutants, including those that were defective in TAP1, TAP2, and tapasin, molecules already known to be important for antigen processing. (Abstract shortened by UMI.)