טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMilner Elena
SubjectModulating the HLA Peptidomes by Cytokines and by Regulated
Proteolysis
DepartmentDepartment of Biology
Supervisor Professor Arie Admon
Full Thesis textFull thesis text - English Version


Abstract

Peptides displayed by HLA (Human Leukocyte Antigens) molecules are the degradation products of cellular proteins. The HLA class I peptides originate from newly synthesized defective proteins, and from long-lived proteins, which completed their functional role and are degraded within the proteasome. Autophagy is another cellular mechanism for degradation of cytoplasmic proteins and organelles during the maintenance of cellular homeostasis.

Upon virus infection, interferons (IFNs) are induced in mammalian cells and thus mediate cellular responses. IFN-γ (gamma) is also involved in cell growth, proliferation and differentiation, cell death, immune response and other cellular defense mechanisms.

The peptides of HLA class I and II can be identified by a direct biochemical approach, based on the immunoaffinity isolation of HLA molecules with their bound peptides, followed by identification of the peptides by capillary chromatography coupled with tandem mass spectrometry.

The methodology was based on dynamic-SILAC (stable isotopes labeling in tissue culture) using in-vivo incorporation of heavy isotope amino acids into the cellular proteins. The ratio between the heavy and the light isotopes incorporated into the proteins served as indication for their turnover rates.

IFN-γ and proteasome inhibitors are among the major modulators of processing and presentation of HLA antigens. In addition to the identification of altered repertoires of HLA class I and class II peptides, a large scale proteomic analysis of the cellular responses to IFN-γ was performed in this research. The proteome of treated cells was compared to the HLA peptidome, presented by the same cells. The data presented here both confirm earlier works and identify novel IFN-γ-regulated proteins and pathways. We have noticed changes in a number of proteins already known to be responsive to IFN-γ, and in other novel proteins, not yet known to be involved in this pathway.

The quantitative data we provide in this study suggests that there is a large involvement of a non-proteasomal pathway, presumably involving the lysosome, to the production of the HLA class I peptidome. Moreover, we propose, that while the proteasome is inhibited, the lysosomal proteolysis becomes a major supplier of HLA class I peptides. Therefore, the level of HLA molecules on the cell surface is maintained, but the repertoire is biased toward peptides derived mostly from stable and not nascent proteins. These results indicate that the HLA peptidome is shaped by the proteasome less significantly than previously thought.