Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit

Area of Science:

• Biochemistry
• Molecular Biology
• Proteasome Biology

Background:

• The ubiquitin/proteasome system (UPS) is crucial for protein homeostasis in eukaryotes.
• The β5 subunit is a key catalytic core within the proteasome, targeted by inhibitors for cancer therapy.

Purpose of the Study:

• To investigate the impact of P1 residue size on the conformational changes and molecular recognition of β5-selective proteasome inhibitors.
• To re-evaluate the classification of the β5 subunit's catalytic activity and the suitability of standard substrates.

Main Methods:

• Synthesis and application of modified proteasome inhibitors with varied P1 residue sizes.
• Analysis of proteasome inhibition and substrate conversion kinetics.
• Assessment of substrate hydrolysis following β5 subunit activity silencing.

Main Results:

• Ligand P1 residue size dictates a mouse-trap-like conformational change influencing proteasome recognition.
• Increasing P1 size enhances β5 subunit inhibition, while decreasing size accelerates substrate conversion.
• Significant residual substrate hydrolysis for large residues persists even after β5 silencing, suggesting alternative catalytic activity.

Conclusions:

• The β5 subunit's catalytic profile is more complex than the 'chymotrypsin-like' classification suggests.
• Standard tyrosine-containing substrates may not fully represent the β5 subunit's activity, especially with larger substrates.
• Ligand design can modulate proteasome inhibition versus substrate conversion, offering new therapeutic strategies.