|M.Sc Student||Gutter-Kapon Lilach|
|Subject||The Effect of Proteasome Inhibition on the HLA Peptidome|
and Proteome of Human Cancer Cells
|Department||Department of Biology||Supervisor||Professor Emeritus Arie Admon|
|Full Thesis text|
Cells degrade their proteins and display at their surface some of the resulting peptides by the human leukocyte antigens (HLA) molecules. Presentation of peptides on HLA molecules serves the cells to alert the immune system about their health-state. It is generally assumed that the proteasome is the main protease responsible for generation of the HLA peptidome. While most studies based on inhibition of the proteasomes indicate reduction in the levels of presentation of studied peptides, others have observed only limited effects and even sporadic increases of their levels.
Previous study performed by Elena Milner noticed that proteasome inhibition did not reduce the presentation levels of the membranal HLA class I. This led us to reconsider the centrality of the proteasome in the generation of HLA class I peptidome. The proteasome inhibitors, epoxomicin and bortezomib (Velcade) were used to study their effects on the synthesis rates of HLA peptides and cellular proteins in the human breast cancer cell line MCF-7. This was done by using mass spectrometry based proteomics and peptidomics tools, combined with dynamic stable isotope labeling in tissue culture. In addition to proteasome inhibition, the inhibitors caused arrest in the synthesis rates of most proteins and of HLA peptides presentation, but increased the synthesis and presentation of others. Thus, every HLA peptide and protein could be detected with a different presentation and synthesis kinetics which were influenced in a complex ways by the proteasome inhibition. To minimize the indirect effects of the proteasome inhibition, other than protein degradation, I have resorted to short treatment periods (up to four hours).
The results suggest that the changes in the rates of protein synthesis (possibly also of HLA molecules) have major influence on the scheme of protein degradation in the cells, and therefore their processing into HLA peptides. It led us to conclude that the use of such inhibitors has an indirect effect on the production pipeline of the HLA peptidome and should be used cautiously. It is further suggested that other proteolytic pathways are likely involved in the processing of the cellular proteome into the HLA peptidome and that our new approach, based on for comparing the rates of synthesis of the cellular proteins and their derived HLA peptides, provides a powerful approach for deciphering between these pathways.