INVESTIGATION OF IMMUNOPROTEASOME DYNAMICS IN CANCER BIOLOGY AND IMMUNOSENESCENCE REVEALS COMMON TRAITS OF APPARENTLY DIVERGENT PROCESSES

The proteasome is a multi-subunit proteolytic complex that plays a pivotal role in maintaining protein homeostasis as it degrades unfolded and misfolded proteins. It is also responsible for the degradation of key regulatory proteins involved in processes such as proliferation, apoptosis, trafficking and signalling. The proteasome consists of two rings of seven α subunits and two rings of seven β subunits forming a barrel-like structure (α,β,β,α) called 20S core in which β1, β2, and β5 subunits show chymotrypsin-like, trypsin-like, and caspase-like activity, respectively. The immunoproteasome (IP) is a specialized variant in which the three catalytic subunits are replaced by their IP counterparts β1i, β2i and β5i (i20S). High expression of IP was found in immune cells where it is involved in several aspects of the immune response. The IP is also constitutively expressed in many types of cancers or induced in other cell types after cytokine stimulation. Different regulatory complexes such as the 19S and PA28αβ regulatory particles (RPs) can be assembled to the 20S catalytic core to modulate its activity and substrate specificity. Proteasome complexes are highly plastic; mixed complexes containing both constitutive and IP subunits and/or hybrid proteasomes with different RP combinations can be present in cells. Proteasome content is tightly regulated to maintain cellular homeostasis and its dysregulation has been reported in many diseases. The qualitative and quantitative expression of proteasome complexes vary between cell types, reflecting the different requirements of individual cells and representing a possible target of interventions when dysregulated. However, our knowledge of proteasome complex dynamics and their role/evolution in many biological processes is very limited. AIM OF THE THESIS: This PhD thesis focuses on the study of the dynamic regulation of the proteasome/immunoproteasome in different physio-pathological conditions. METHODS: Proteasome subunit expression was analysed by Real Time PCR and western immunoblotting, proteasome activity was assessed using fluorogenic substrates both in cell-free extracts and in “in gel activity” assays, proteasome complex composition was determined by native PAGE analysis followed by western immunoblotting. RESULTS AND CONCLUSIONS: In gastric cancer (GC) cells, IP complexes were found more expressed and preferentially capped with the 19S regulator in diffuse type metastatic cells compared to epithelial types, playing a role in GC cell migration and possibly in the epithelial/mesenchymal transition (published). Furthermore, we found that a small synthetic redox-modulating molecule (I-152) could induce early IP expression in the lymph nodes of mice infected with a leukaemia virus, thus demonstrating the feasibility to modulate proteasome expression in vivo and contributing to explain the antiviral activity of this compound (published). Finally, loss of the PA28αβ regulator, coupled with the interchange between constitutive and IP catalytic core subunits, was found as a specific trait of immunosenescence in both an in vitro model of senescent macrophages and in vivo in the lymph nodes of two distinct mouse strains. These findings provide a molecular rationale for IP dysfunction with remodelling of the proteasome, pointing to the loss of the PA28αβ regulator as the most critical event and a hallmark of immunosenescence (manuscript draft).