Abstract 3236: Development of a novel cross-linking strategy to identify distinct proteasome subtypes

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Abstract Introduction: This research was conducted to develop a practical method to identify distinct proteasome subtypes. Approval of the proteasome inhibitors bortezomib and carfilzomib for clinical use has validated the proteasome as an anticancer target. These inhibitors target two well-recognized proteasome subtypes, the constitutive proteasome and the immunoproteasome, which comprise distinct sets of catalytic subunits. Additional proteasome subtypes containing mixtures of constitutive and immunoproteasome catalytic subunits have more recently been identified. These intermediate proteasome subtypes display unique proteolytic activity profiles and play important roles in the production of certain tumor antigens. However, much regarding their regulation, functions, and relevance as drug targets remains unknown, largely due to limitations in current experimental methods used to differentiate between proteasome subtypes. We have therefore developed bifunctional activity-based probes capable of cross-linking different catalytic subunit pairs within individual proteasome complexes. These probes allow a straightforward readout of proteasome catalytic subunit composition and facilitate functional studies of distinct proteasome subtypes. Experimental Procedures: Bifunctional proteasome probes were synthesized by coupling pairs of proteasome inhibitors via linkers of varying lengths and compositions. Positions for inhibitor derivatization were selected based on computational modeling, and linkers were chosen based on distances between the active sites of each targeted subunit pair. The ability of these compounds to cross-link distinct catalytic subunit pairs in both purified proteasomes and in cancer cell lysates was visualized by western blotting analysis. Competition assays with subunit-selective proteasome inhibitors were employed to confirm the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional proteasome probes were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results demonstrated the ability of these compounds to cross-link multiple pairs of proteasome catalytic subunits, allowing the composition of distinct proteasome subtypes present within cancer cells to be evaluated. Conclusions: We have successfully developed a set of bifunctional proteasome probes for analysis of proteasome catalytic subunit composition. These probes will be used to elucidate the unique functions of distinct proteasome subtypes present within cancer cells. Citation Format: Kimberly C. Carmony, Do-Min Lee, Lalit Kumar Sharma, Jieun Park, Kyung-Bo Kim, Wooin Lee. Development of a novel cross-linking strategy to identify distinct proteasome subtypes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2014-3236