The Turnover Kinetics of Major Histocompatibility Complex Peptides of Human Cancer Cells

Eilon Barnea,Ilan Beer,Elena Milner,Arie Admon

doi:10.1074/mcp.m500241-mcp200

Abstract

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins. Thus, the repertoire of these peptides (the MHC peptidome) should correlate better with the cellular protein degradation scheme (the degradome) than with the cellular proteome. To test the validity of this statement and to determine whether the majority of MHC peptides are derived from short lived proteins, from defective ribosome products, or from regular long lived cellular proteins we analyzed in parallel the turnover kinetics of both MHC peptides and cellular proteins in the same cancer cells. The analysis was performed by pulse-chase experiments based on stable isotope labeling in tissue culture followed by capillary chromatography and tandem mass spectrometry. Indeed only a limited correlation was observed between the proteome and the MHC peptidome observed in the same cells. Moreover a detailed analysis of the turnover kinetics of the MHC peptides helped to assign their origin to normal, to short lived or long lived proteins, or to the defective ribosome products. Furthermore the analysis of the MHC peptides turnover kinetics helped to direct attention to abnormalities in the degradation schemes of their source proteins. These observations can be extended to search for cancer-related abnormalities in protein degradation, including those that lead to loss of tumor suppressors and cell cycle regulatory proteins.

Highlights

Peptides presented by the major histocompatibility complex (MHC) are derived from the degradation of cellular proteins
MHC peptides are presented only following the degradation of their proteins of origin and because proteins degrade at vastly differing rates, it can be assumed that the MHC peptidome of the cells correlates better with the pattern of protein degradation rather than with the proteome of the cells
The stable isotope pulse-chase method described by Pratt et al [25], modified for this research, enabled the independent determination of the turnover kinetics of many of these MHC peptides and of the cellular proteins

Summary

The MHC Peptidome and the Transient Proteome

Identifying individual DRiPs or SLiPs, both of which are rapidly degrading proteins, and assigning them to one of these categories is technically challenging due to the scarcity of these protein molecules and their peptide degradation products. DRiPs and SLiPs are degraded rapidly in the cytoplasm all the way to free amino acids and may not accumulate to detectable levels even by the modern high sensitivity analytical tools Some of their peptide degradation products are stabilized for relatively long periods of time while bound to the MHC molecules and accumulate and become amenable to analysis. A recent modification to this method involves the recombinant expression of soluble, secreted MHC (sMHC) molecules by the cells These are collected with their bound peptides from the growth medium of the cells by immunoaffinity isolation resulting in large amounts of peptides free of contaminating cellular debris or detergents (16 –19). Rapid turnover of some of these MHC peptides may help to direct attention to the rapid degradation of their source proteins, which are possibly of significance to the carcinogenic transformation of the cells

EXPERIMENTAL PROCEDURES

RESULTS

Protein similar to heterogeneous nuclear ribonucleoprotein K

Analysis of Turnover Kinetics Based on SILAC with Heavy Leucine

Distinguishing between DRiPs and Degradation Products of Normal Protein

Bioinformatic Analysis of the Kinetic SILAC Data

Limitation of Analysis of Very Rapid Turnover Kinetics

Abnormally Rapid Degradation

Examples for Rapidly Turning Over Peptides of Possible Interest