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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Kisselev AF, Akopian TN, Woo KM, Goldberg AL (1999) The sizes of peptides generated from protein by mammalian 26 and 20S proteasomes. Implications for understanding the degradative mechanism and antigen presentation. J Biol Chem 274:3363–3371
Yewdell JW, Reits E, Neefjes J (2003) Making sense of mass destruction: quantitating MHC class I antigen presentation. Nat Rev Immunol 3:952–961
Fruci D, Lauvau G, Saveanu L, Amicosante M, Butler RH, Polack A, Ginhoux F, Lemonnier F, Firat H, van Endert PM (2003) Quantifying recruitment of cytosolic peptides for HLA class I presentation: impact of TAP transport. J Immunol 170:2977–2984
Reits E, Griekspoor A, Neijssen J, Groothuis T, Jalink K, van Veelen P, Janssen H, Calafat J, Drijfhout JW, Neefjes J (2003) Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I. Immunity 18:97–108
Rammensee HG, Falk K, Rotzschke O (1993) Peptides naturally presented by MHC class I molecules. Annu Rev Immunol 11:213–244
Beninga J, Rock KL, Goldberg AL (1998) Interferon-gamma can stimulate post-proteasomal trimming of the N terminus of an antigenic peptide by inducing leucine aminopeptidase. J Biol Chem 273:18734–18742
Geier E, Pfeifer G, Wilm M, Lucchiari-Hartz M, Baumeister W, Eichmann K, Niedermann G (1999) A giant protease with potential to substitute for some functions of the proteasome. Science 283:978–981
Stoltze L, Schirle M, Schwarz G, Schroter C, Thompson MW, Hersh LB, Kalbacher H, Stevanovic S, Rammensee HG, Schild H (2000) Two new proteases in the MHC class I processing pathway. Nat Immunol 1:413–418
Mo XY, Cascio P, Lemerise K, Goldberg AL, Rock K (1999) Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides. J Immunol 163:5851–5859
Reits E, Neijssen J, Herberts C, Benckhuijsen W, Janssen L, Drijfhout JW, Neefjes J (2004) A major role for TPPII in trimming proteasomal degradation products for MHC class I antigen presentation. Immunity 20:495–506
Acknowledgment
This work was supported by NWO-ZonMW VIDI grant.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stargardt, A., Reits, E. (2013). Kinetic Studies of Cytoplasmic Antigen Processing and Production of MHC Class I Ligands. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology™, vol 960. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-218-6_4
Download citation
DOI: https://doi.org/10.1007/978-1-62703-218-6_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-217-9
Online ISBN: 978-1-62703-218-6
eBook Packages: Springer Protocols