CONFORMATIONAL CHANGES IN AN IMPORTANT E3 UBIQUITIN LIGASE COMPLEX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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，重点关注来自自然来源的小分子以及它们如何以有益的和细胞保护的方式影响我们的身体。其中几种化学预防化合物，如来自西兰花的萝卜硫素和来自啤酒花的黄腐酚，共价修饰细胞中一种特殊的大分子复合体，称为基于CUL3的E3泛素连接酶。这种蛋白质复合体泛素化底物蛋白Nrf2，Nrf2是调节细胞保护酶水平的主要转录因子。当这些化合物中的一种被引入细胞时，泛素化通过一种未知的信号机制被关闭。这导致Nrf2的核积累，允许参与氧化还原动态平衡、谷胱甘肽生物合成、炎症抑制和DNA修复的酶的转录增强。用质谱仪观察到这些化合物与Keap1分子中活性半胱氨酸的共价加合作用。据推测，这种共价修饰通过改变络合物的四元结构而导致泛素化关闭。这一假设可以很容易地通过小角度散射实验来探索，其中，在用一种化学预防化合物攻击之前和之后测量复合体的旋转半径(Rg)和最大线性尺寸(Dmax)。如果修饰导致络合物解离成更小的粒子，则Rg和Dmax将相应改变。了解这些有益的小分子如何影响基于CUL3的E3泛素连接酶复合体的结构，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.