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. In addition, overexpression of recombinant soluble HLA-A*02:01, introduced to compete with the identical endogenous membrane-bound HLA-A*02:01 for peptides of the MCF-7 cells, did not alter the expression level or the presented peptidome of the membrane-bound HLA-A*02:01. Taken together, these results indicate that a surplus supply of peptides is available inside the ER for loading onto the MHC-I peptide-receptive molecules, and that cell surface MHC-I expression is likely limited by the availability of empty MHC molecules.

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 inhibitors reduced the rates of synthesis of most cellular proteins and HLA peptides, yet the synthesis rates of some of the proteins and HLA peptides was not decreased by the inhibitors and of some even increased. Therefore, we concluded that the inhibitors affected the production of the HLA peptidome in a complex manner, including modulation of the synthesis rates of the source proteins of the HLA peptides, in addition to their effect on their degradation. The collected data may suggest that the current reliance on proteasome inhibition may overestimate the centrality of the proteasome in the generation of the MHC peptidome. It is therefore suggested that the relative contribution of the proteasomal and nonproteasomal pathways to the production of the MHC peptidome should be revaluated in accordance with the inhibitors effects on the synthesis rates of the source proteins of the MHC peptides.

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. 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.

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. The resulting peptides fit to some extent, but not completely, the known consensus of the Cw4 peptide-binding motif. Among the identified peptides, there are a few that originate from proteins of possible interest for cancer immunotherapy or diagnostics, including mucin-5B, ART-1, fatty acid synthase, putative prostate cancer tumor suppressor, DNA topoisomerase-1, and Rac1. This work demonstrates that large-scale identification of HLA peptides recovered from sHLA is an advantageous approach for establishing the HLA peptide consensus of different haplotypes and the identification of useful peptides for treatment of diseases such as cancer, viral, and autoimmune diseases.