Identification of Immunoproteasome dependent factors involved in cytokine release and T cell differentiation

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Cells of hematopoietic origin, particularly lymphocytes and monocytes, express the catalytic subunits of the immunoproteasome low-molecular mass polypeptide (LMP)2 (beta1i), LMP7 (beta5i), and multicatalytic endopeptidase complex subunit-1 (MECL-1) (beta2i) proteins. These three subunits can also be induced in non-hematopoietic cells after exposure to inflammatory cytokines. The immunoproteasome is known for its role in antigen processing along the MHC class I pathway. Recently, novel functions of immunoproteasomes in T cell proliferation, cytokine production, T cell differentiation, and autoimmune diseases have been discovered, but the underlying molecular mechanisms have remained elusive. In order to investigate how the immunoproteasome may be mechanistically involved in the control of T cell activation and cytokine production, several experimental approaches will be pursued. Transcription factors and signalling molecules known to be involved in T cell activation and interferon-gamma stimulation will be investigated with respect to their phosphorylation status and expression level in the presence and absence of an LMP7 inhibitor. In order to identify LMP7-dependent factors involved in cytokine production we will take advantage of two newly developed methods. We will quantitatively assess the ubiquitin-modified proteome in the presence or absence of LMP7-inhbition. Additionally, we intend to identify and quantify proteolytic events by terminal amine isotopic labelling of substrates in cells subjected to LMP7-inhibition. With these experiments we intend to investigate the central hypothesis that the immunoproteasome may selectively process or degrade a factor involved in cytokine production, T cell differentiation, and T cell activation.