alpha-chymotrypsin and dihydroeponemycin

The immunoproteasome, having been linked to neurodegenerative diseases and hematological cancers, has been shown to play an important role in MHC class I antigen presentation. However, its other pathophysiological functions are still not very well understood. This can be attributed mainly to a lack of appropriate molecular probes that can selectively modulate the immunoproteasome catalytic subunits. Herein, we report the development of molecular probes that selectively inhibit the major catalytic subunit, LMP2, of the immunoproteasome. We show that these compounds irreversibly modify the LMP2 subunit with high specificity. Importantly, LMP2-rich cancer cells compared to LMP2-deficient cancer cells are more sensitive to growth inhibition by the LMP2-specific inhibitor, implicating an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2.

Topics: Adenocarcinoma; Animals; Catalytic Domain; Cell Line, Tumor; Chymotrypsin; Cysteine Endopeptidases; Humans; Male; Mice; Molecular Probes; Neovascularization, Pathologic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Serine

The chymotrypsin-like (CT-L) activity of the proteasome is downregulated by substrates of the peptidyl-glutamyl peptide hydrolyzing (PGPH) activity. To investigate the nature of such interactions, we synthesized selective alpha',beta'-epoxyketone inhibitors of the PGPH activity. In cellular proliferation and protein degradation assays, these inhibitors revealed that selective PGPH inhibition was insufficient to inhibit protein degradation, indicating that the CT-L and PGPH sites function independently. We also demonstrated that CT-L inhibition by a PGPH substrate does not require the occupancy of the PGPH site or hydrolysis of the PGPH substrate. Thus, these results support a model in which a substrate of one subunit regulates the activity of another via binding to a noncatalytic site(s) rather than through binding to an active site.

Topics: Allosteric Regulation; Animals; Binding Sites; Cattle; Cell Division; Cells, Cultured; Chymotrypsin; Cysteine Endopeptidases; Endopeptidases; Epoxy Compounds; Humans; Hydrolysis; Ketones; Kinetics; Models, Biological; Multienzyme Complexes; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Subunits; Recombinant Fusion Proteins; Serine; Substrate Specificity; Transfection