title: Kinetic studies of cytoplasmic antigen processing and production of MHC class I ligands.
abstract: MHC class I molecules present peptides that are derived from intracellular proteins degraded by proteasomes. These peptides often require additional trimming by peptidases to fit into the peptide-binding grove of MHC class I. However, most peptides are rapidly recycled by the large heterogeneous pool of peptidases. Here, we describe a technique to quantify peptide degradation both in living cells and in cell lysates, using quenched peptides that contain a quencher and fluorophore. As degradation results in separation of the quencher and fluorophore, fluorescence will increase. This technique enables the examination of changes in peptide length and amino acid sequence on its half-life, and hence its chances to become presented by MHC class I.
authors: Department of Cell Biology & Histology, AMC/University of Amsterdam, Amsterdam, The Netherlands.
authors: Anita
authors: A
authors: Stargardt
authors: Department of Cell Biology & Histology, AMC/University of Amsterdam, Amsterdam, The Netherlands. e.a.reits@amc.uva.nl.
authors: Eric
authors: E
authors: Reits
mesh_terms: D017951:Antigen Presentation / Q000187:drug effects; D002458:Cell Fractionation; D002470:Cell Survival; D003593:Cytoplasm / Q000187:drug effects / Q000276:immunology*; D057809:HEK293 Cells; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D006801:Humans; D007267:Injections; D007700:Kinetics; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D059748:Proteolysis / Q000187:drug effects
chemical_list: D015395:Histocompatibility Antigens Class I; D008024:Ligands; D010446:Peptide Fragments; D011480:Protease Inhibitors