Sunday, July 24, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
The ubiquitin-proteasome pathway is the primary route of regulated proteolysis in eukaryotes. Disruption of this key cellular process with small molecule inhibitors has implications in a number of human diseases such as cancer and inflammation and has exposed the proteasome as an important therapeutic target. The marine actinomycete Salinispora tropica produces salinosporamide A, a potent 20S proteasome inhibitor which has emerged as a promising clinical agent due to its distinct mechanism of action and unique chemical structure. Intriguingly, actinobacteria such as S. tropica also possess 20S proteasome machinery, raising questions of self-resistance. A gene encoding a redundant 20S proteasome β-subunit, SalI, has been identified within the salinosporamide biosynthetic cluster. We have heterologously expressed and biochemically characterized 20S proteasome complexes containing either the housekeeping β-subunit or the SalI-subunit. The SalI proteasome complex had an altered substrate specificity profile and displayed 30-fold resistance to salinosporamide A relative to the housekeeping β-subunit. Cross-resistance to other peptidyl proteasome inhibitors such as the FDA-approved bortezomib was also observed. We identified two residues, position 45 and 49, in the substrate binding pocket of SalI that differ from previously characterized eubacterial β-subunits and influence salinosporamide affinity. A similar mutation at position 49 in the human 20S proteasome β5-subunit has recently been observed that confers resistance to bortezomib. SalI represents the first proteasome subunit resistant to an endogenously produced proteasome inhibitor and suggests that intrinsic resistance to natural product proteasome inhibitors may predict clinical outcomes.