CONFORMATIONAL CHANGES IN AN IMPORTANT E3 UBIQUITIN LIGASE COMPLEX

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 癌症化学预防是一个令人兴奋的研究领域，专注于天然来源的小分子以及它们如何以有益和细胞保护的方式影响我们的身体。 这些化学预防化合物中的几种，例如来自花椰菜的萝卜硫素和来自啤酒花的黄腐酚，共价修饰细胞中称为Cul 3- based E3泛素连接酶的特定大分子复合物。 这种蛋白质复合物泛素化底物蛋白Nrf 2，Nrf 2是调节细胞保护酶水平的主要转录因子。当这些化合物之一被引入细胞时，泛素化通过一种未知的信号传导机制被关闭。这导致Nrf 2的核积累，允许参与氧化还原稳态、谷胱甘肽生物合成、炎症抑制和DNA修复的酶的转录增强。已通过质谱法观察到化合物与Keap 1分子内的反应性半胱氨酸的共价加合。据推测，这种共价修饰通过改变复合物的四级结构导致泛素化关闭。这个假设可以很容易地探索使用小角散射实验，其中的回转半径（Rg）和最大线性尺寸（Dmax）的复杂的挑战之前和之后的化学预防化合物之一进行测量。如果修饰导致复合物解离成更小的颗粒，则Rg和Dmax将相应地改变。了解这些有益的小分子如何影响基于Cul 3的E3泛素连接酶复合物的结构，将有助于未来开发更好，更有效的化学预防方案。

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.