Cell Surface MHC Class I Expression Is Limited by the Availability of Peptide-Receptive "Empty" Molecules Rather than by the Supply of Peptide Ligands.

While antigen processing and presentation (APP) by the major histocompatibility complex class I (MHC-I) molecules have been extensively studied, a question arises as to whether the level of MHC-I expression is limited by the supply of peptide-receptive (empty) MHC molecules, or by the availability of peptide ligands for loading. To this end, the effect of interferons (IFNs) on the MHC peptidomes of human breast cancer cells (MCF-7) were evaluated. Although all four HLA allotypes of the MCF-7 cells (HLA-A*02:01, B*18, B*44, and C*5) present peptides of similar lengths and C-termini, which should be processed similarly by the proteasome and by the APP chaperones, the IFNs induced differential modulation of the HLA-A, B, and C peptidomes.

The effect of proteasome inhibition on the generation of the human leukocyte antigen (HLA) peptidome.

The Major histocompatibility complex (MHC) class I peptidome is thought to be generated mostly through proteasomal degradation of cellular proteins, a notion that is based on the alterations in presentation of selected peptides following proteasome inhibition. We evaluated the effects of proteasome inhibitors, epoxomicin and bortezomib, on human cultured cancer cells. Because the inhibitors did not reduce the level of presentation of the cell surface human leukocyte antigen (HLA) molecules, we followed their effects on the rates of synthesis of both HLA peptidome and proteome of the cells, using dynamic stable isotope labeling in tissue culture (dynamic-SILAC).

The turnover kinetics of major histocompatibility complex peptides of human cancer cells.

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.

Large-scale analysis of HLA peptides presented by HLA-Cw4.

A large number of HLA-Cw4 (Cw *0402) peptides were purified, sequenced, and identified from breast and ovarian carcinoma cell lines. HLA-Cw4 molecules were expressed in these cells as soluble, secreted HLA (sHLA) and recovered from the growth medium. The peptides were separated by capillary reversed-phase HPLC and analyzed by tandem mass-spectrometry.