The proteasome is a multicatalytic enzyme complex expressed in eukaryotic cells (present in the nucleus and cytoplasm). The main function of the proteasome is to degrade unneeded or damaged proteins by proteolysis. Thus, it plays a key role in the cell cycle regulation, apoptosis, angiogenesis and expression of adhesion molecules. Disturbances in the functioning of this enzyme system are important in tumor progression, drug resistance, and a number of nervous system diseases such as Alzheimer’s disease or Parkinson’s. The 20S proteolytic core consists of four heptameric rings forming a cylindrical particle. Each outer ring consists of seven α subunits and each inner ring consists of seven β subunits. The proteolytic activity of the proteasome is connected with the β-subunits, each of which has a different specificity. The β1-subunit (caspase-like) is associated with hydrolysis of the peptide bond after acidic amino acids and the β2-subunit (trypsin-like) is associated with cleavage after basic amino acids, and the β5-subunit (chymotrypsin-like) is associated with cleavage after hydrophobic amino acids [Adams J., Cancer Treat. Rev.,2003].
So far, the substrate cleavage preference of the 20S proteasome in the presence or absence of PA28 activator subunit was determined using fluorogenic peptide libraries containing only natural amino acids [Harris J.L., et al., Chem. Biol.,2001]. Knowledge of 20S proteasome substrate specificity can be greatly extended through the use of unnatural amino acids in peptide substrates. Therefore, we determined the substrate specificity profile for each activity of 20S proteasome using HyCoSuL (Hybrid Combinatorial Substrate Library) approach. Based on the obtained substrate specificity profile for each activity new, specific fluorogenic tetrapeptide substrate were synthesized and their kinetics parameters were determined. Profiling human 20S proteasome by HyCoSuL approach is the first step to obtain a specific Activity Based Probe for each catalytic β subunit, that could be used for proteasome detection and monitoring its activity.
This work was supported by the National Science Centre grant 2014/13/B/ST5/00240 in Poland