Project menager: professor Marcin Drąg

Research project objectives/Hypothesis

The ubiquitin-proteasome system plays a key role in maintaining the intracellular protein homeostasis. The biological role of this system in regulation of cellular processes is well established, but little is known about the pathophysiological significance of inducible form of proteasome called immunoproteasome. Upon induction by inflammatory cytokines such as interferon-?, three constitutively expressed ß-subunits are replaced by three new immuno-subunits. The first known function of the immunoproteasome is production of antigenic peptides for major histocompatibility complex class I (MHC I) presentation. Further studies revealed that this enzyme possesses broader biological significance and it is involved in the regulation of both immune and nonimmune responses. Its function makes it an important therapeutic target for the treatment of neurodegenerative and autoimmune diseases as well as cancer. Chemical tools such as substrates, inhibitors or activity based probes can be very useful for understanding enzyme functions. The main requirement for the use of aforementioned tools in biological research, is their selectivity toward tested enzymes. In the case of immunoproteasome, which possesses three catalytic subunits in its structure, it is necessary to design tools that would enable selective examination of each subunit. Currently, the same fluorogenic substrates are utilized in immunoproteasome and proteasome 20S studies due to similar substrate specificity of these enzymes. Thus, it is impossible to determine the activity of one subunit or enzyme. The main objective of this project is to develop a screening platform by which it will be possible to design and synthesize specific substrates for monitoring activity of each human immunoproteasome and 20S proteasome catalytic subunit. In the first step, the substrate specificity profile will be determined in P6-P2' pockets. The obtained substrate specificity matrix of the immunoproteasome will be compared with 20S proteasome specificity in order to design selective peptide sequence for each catalytic site of immunoproteasome. In the final stage of research, the selectivity of designed substrates toward both enzymes will be determined. We hope that this approach will be of more general use and could be applied for other members of proteases family.