Abstract 2231: Development of bifunctional cross-linking agents to identify intermediate proteasomes.

Abstract

Abstract Introduction: This research was conducted to develop bifunctional cross-linking agents that can be used to identify intermediate proteasomes. Two well-characterized proteasome subtypes, the constitutive proteasome and immunoproteasome, differ with respect to their distinct sets of catalytic subunits. Additionally, intermediate proteasome subtypes comprised of non-standard mixtures of catalytic subunits have more recently been discovered. These intermediate proteasomes differ from constitutive and immunoproteasomes in their proteolytic activity profiles, suggesting that they have unique cellular functions. In agreement with this, intermediate proteasomes in cancer cell lines were shown to play important roles in the production of several tumor antigens. However, largely due to limitations of methods currently available to distinguish between subtypes, the detailed functions of intermediate proteasomes remain poorly defined. Bifunctional agents capable of cross-linking two different catalytic subunits within a single proteasome complex will facilitate a simple approach to identify distinct proteasome subtypes present within cells. These compounds will serve as important chemical probes for investigating the functions of individual proteasome subtypes in cancer cells. Experimental Procedures: Bifunctional cross-linking agents were synthesized by coupling two broad-spectrum or subunit-selective proteasome inhibitors through hydrocarbon or polyethylene glycol linkers. Positions for derivatization of these inhibitors were selected based on computational modeling, and linker lengths were varied to increase cross-linking efficiency. The ability of the resulting compounds to cross-link two proteasome catalytic subunits in cancer cell lysates was assessed via western blotting. Competition assays with subunit-selective proteasome inhibitors were conducted to verify the identities of the cross-linked subunit pairs. Data Summary: The structures of the bifunctional cross-linking agents were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Western blotting results revealed the ability of several of these bifunctional agents to cross-link two catalytic subunits within individual proteasome complexes, facilitating compositional analysis of distinct proteasome subtypes. Conclusions: We have successfully developed bifunctional agents capable of cross-linking two catalytic subunits within individual proteasome complexes. These compounds will be utilized in functional studies of intermediate proteasomes present within cancer cells. Citation Format: Kimberly C. Carmony, Lalit Kumar Sharma, Kyung-Bo Kim. Development of bifunctional cross-linking agents to identify intermediate proteasomes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2231. doi:10.1158/1538-7445.AM2013-2231