Abstract
Proteasome inhibitors have been described as an important target for cancer therapy due to their potential to regulate the ubiquitin-proteasome system in the degradation pathway of cellular proteins. Here, we reported the effects of a Bowman-Birk-type protease inhibitor, the Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI), on proteasome 20S in MCF-7 breast cancer cells and on catalytic activity of the purified 20S proteasome from horse erythrocytes, as well as the structural analysis of the BTCI-20S proteasome complex. In vitro experiments and confocal microscopy showed that BTCI readily crosses the membrane of the breast cancer cells and co-localizes with the proteasome in cytoplasm and mainly in nucleus. Indeed, as indicated by dynamic light scattering, BTCI and 20S proteasome form a stable complex at temperatures up to 55°C and at neutral and alkaline pHs. In complexed form, BTCI strongly inhibits the proteolytic chymotrypsin-, trypsin- and caspase-like activities of 20S proteasome, indicated by inhibition constants of 10−7 M magnitude order. Besides other mechanisms, this feature can be associated with previously reported cytostatic and cytotoxic effects of BTCI in MCF-7 breast cancer cells by means of apoptosis.
Highlights
Proteases are involved in many biological processes such as the hydrolysis of intracellular proteins, transcription, cell cycle, cell invasion and apoptosis [1]
Black-eyed pea Trypsin/Chymotrypsin Inhibitor (BTCI) and 20S Proteasome Association Evaluated by Dynamic Light Scattering (DLS)
The parameters obtained from DLS measurements indicate that the 20S proteasome appears as a monomer at 21.4 nM and pH 7.5 with hydrodynamic diameter of 15 nm (Fig. 1a), in agreement with previously reported data [71,72]
Summary
Proteases are involved in many biological processes such as the hydrolysis of intracellular proteins, transcription, cell cycle, cell invasion and apoptosis [1]. As reported in 2005 [55], BBI from soybean inhibits the chymotrypsin activity of the proteasome in breast cancer cells, but the molecular mechanism involved in this process is not fully understood. We report the effects of BTCI on the 20S proteasome of MCF-7 breast cancer cells and on the catalytic activity of the 20S proteasome purified from horse erythrocytes, as well as the molecular analysis of the BTCI-proteasome complex.
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