Structure-Function Studies of the Cul3/Rbx1:Keap1 E3 Ubiquitin Ligase Complex

Eukaryotic cells have adapted unique mechanisms to respond to environmental stressors. The Nrf2-ARE system is one of these mechanisms and is used to combat chemical stressors such as environmental toxins or endogenous metabolites. Chemopreventive compounds are chemical agents that in low doses cause Nrf2 nuclear accumulation and the subsequent up-regulation of a battery of cytoprotective enzymes which decrease carcinogenesis. These same compounds have been shown to react with and covalently modify components of the ubiquitin E3 ligase complex that directly affects the stability of Nrf2. Determining how this complex’s quaternary structure is altered upon reaction with chemopreventive compounds will provide a greater understanding of how the complex functions and is regulated. It is hypothesized that chemopreventive compounds, most of which are electrophilic in nature, react with specific cysteines within the complex to bring about a structural change thereby affecting the ability of the complex to ubiquitinate Nrf2. To test this hypothesis, ultracentrifugation and small angle X-ray scattering experiments were performed to measure the size and shape of the E3 ligase bound to its substrate Nrf2. In addition, the alteration of the size and shape of the Keap1-Nrf2 system upon site-directed mutation or modification by chemopreventive compounds was also performed. Results suggest that upon modification, conformational changes occur within the E3 ligase complex, which alter the ubiquitination of Nrf2. This research was supported by a grant from NCI of the NIH (5PO1 CA48112).