CONFORMATIONAL CHANGES IN AN IMPORTANT E3 UBIQUITIN LIGASE COMPLEX

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Cancer chemoprevention is an exciting field of research focusing on small molecules from natural sources and how they affect our body in beneficial and cytoprotective ways. Several of these chemopreventive compounds, such as sulforaphane from broccoli and xanthohumol from hops, covalently modify a specific complex of macromolecules in the cell called the Cul3- based E3 ubiquitin ligase. This complex of proteins ubiquitinate a substrate protein, Nrf2, which is a major transcription factor regulating the levels of cytoprotective enzymes. When one of these compounds is introduced to cells, ubiquitination is shutdown through an unknown signaling mechanism. This leads to the nuclear accumulation of Nrf2, allowing for enhanced transcription of enzymes involved in redox homeostasis, glutathione biosynthesis, inflammation suppression, and DNA repair. Covalent adduction of the compounds to reactive cysteines within the Keap1 molecule has been observed by mass spectrometry. It is hypothesized that this covalent modification leads to the ubiquitination shutdown through an alteration in the complex's quaternary structure. This hypothesis can be readily explored using small angle scattering experiments, where the radius of gyration (Rg) and maximal linear dimension (Dmax) of the complex is measured before and after challenge with one of the chemopreventive compounds. If modification causes the complex to dissociate into smaller particles, then the Rg and Dmax will change accordingly. Understanding how these beneficial small molecules affect the structure of the Cul3-based E3 ubiquitin ligase complex will aid in the development of better, more potent chemopreventive regimens in the future.