Regulation of Proteasomal Catalytic Activity by Altering its Protein-Protein Interactions

Abstract

The proteasome is a central protease of the main intracellular pathway responsible for regulated proteolysis that uses polyubiquitinylation for substrate tagging. The basic functional unit of proteasome called 20S or catalytic particle (CP) is a tube built from four stacked heptameric rings with 3 pairs of catalytic subunits equipped with specific proteolytic activities. The catalytic sites face inside of the tube and are additionally secured by the gates on the tubes’ alpha faces and blocking the access to the CP interior. To get to the active centers, substrates have to bypass the gate, which closed state is affected by binding ligands to the alpha face or catalytic sites. To gain capability to digest polyubiquitinylated substrates, the CP binds a regulatory particle (RP) or a 19S cap to least one site of the tube. The interface between CP and RP allosterically controls dynamics of substrates uptake, digest readiness, and efficiency and specificity of the active centers. The CP also wears other types of caps that allow it to digest efficiently particular populations of substrates and generate certain types of products. Also in these cases the interface between the protein complexes plays a crucial regulatory role. Here we designed a range of peptides, peptidomimetics and small molecule compounds that affect these protein – protein interactions to study selected mechanical aspects of proteasomal regulation. Using spectroscopic and microscopic tools we found that binding of certain ligands may affect status of the proteasomal gate, alpha face conformation, stability of the complexes or/and the CP dynamics. These structural changes alter the enzyme activity and specificity, which in turn influence intracellular metabolism. We attempted to correlate particular biological effects of the ligands with their mode of molecular action and their chemical properties.