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In cells, proteins are continuously synthesized and degraded to control protein levels and thereby regulate a wide variety of biochemical processes. The proteasome is the main cellular degradation machinery, responsible for the degradation of key proteins involved in the regulation of a wide range of cellular processes, including quality control, cell cycle progression, cell differentiation, signal transduction and apoptosis. Inhibition of the proteasome causes disruption of many regulatory processes, eventually leading to cell death. The observation that many cancer cells are more sensitive to proteasome inhibition than normal cells has led to the development of several proteasome inhibitors for the treatment of cancer. Proteasome activity in its broadest sense is the central theme of this thesis. The development and characterization of different chemical proteasome activity probes that can be used to study proteasome activity in a wide range of tissue types using a variety of assays are described. Subsequently, these probes are used to assess the effects of different proteasome inhibitors in preclinical and clinical studies and to purify proteasome and characterize its activity and composition. Finally, the molecular mechanism of proteasomal antigen splicing is studied.

Proteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP) and one or two 19S regulatory particles (RP). The constitutive proteasome core particle (cCP), which is expressed in all mammalian tissues, contains three catalytically active subunits, namely β1c, β2c and β5c. Lymphoid cells express another proteasome core particle known as the immunoproteasome (iCP). In iCPs, β1c, β2c and β5c are replaced by β1i, β2i and β5i. The research described in this thesis reports on the development of new subunit-selective inhibitors and activity-based probes, on the development of an assay to simultaneously monitor all cCP and iCP catalytic activities and on the development of a method that reports on CP catalytic active subunit composition. The tools that stem from the work described in this thesis can now be used to unravel the role of each individual catalytic subunit in a chemical genetics setting (selective and (near) complete inhibition of each subunit), and to clarify the role of mCPs, in, for instance, antigen presentation and cancer. Furthermore, these tools could possibly serve as leads in the discovery of agents for future treatment of cancer and autoimmune diseases.