Role of a Novel E3-Ubiquitin Ligase in Chemoprevention

描述(由申请人提供)：本提案中的工作重点是一种主要的癌症预防信号转导途径，该途径触发酶的转录诱导，保护细胞免受包括致癌物和氧化应激在内的活性化学物质的影响。这一途径既可由多种水果和蔬菜中的天然化学预防药物激活，也可由合成分子激活。我们相信，对这一信号转导途径的基本了解将有助于识别将显著降低人类癌症风险的食物、膳食补充剂和药物。此外，由于氧化应激是许多病理生理疾病的驱动力，包括神经退行性变、心血管疾病和骨骼肌萎缩，拟议中的研究将对人类健康产生广泛影响。该途径的关键靶点是转录因子Nrf2，通常受BTB-Kelch蛋白Keap1的抑制。化学预防药物使Nrf2能够逃脱Keap1介导的抑制并激活其靶基因的转录，从而消除活性物种并恢复细胞氧化还原动态平衡。我们的初步数据表明，Keap1作为依赖于CUL3的E3泛素连接酶复合体的底物适配器蛋白发挥作用的假设。这一假说为理解Keap1如何能够抑制Nrf2依赖的转录提供了一个新的范式，并为定义化学预防药物如何使Nrf2逃脱Keap1介导的抑制提供了一个富有成效的框架。这一假说也为所有BTB-Kelch蛋白的生物学功能提供了新的见解。我们建议通过表征GAN1和ENC1中分别导致巨大轴索神经病和脑癌的疾病相关突变来进一步检验这一假设。拟议的实验将(1)定义Nrf2如何通过Keap1：CUL3：Rbx1复合体进行泛素依赖的降解，(2)定义Nrf2如何逃避Keap1介导的抑制，(3)使用Keap1作为模型系统来定义疾病相关突变如何扰乱BTB-Kelch蛋白的底物适配器功能，以及(4)定义Keap1识别底物的结构基础。

DESCRIPTION (provided by applicant): The work in this proposal is focused on a major cancer-preventive signal transduction pathway that triggers transcriptional induction of enzymes that protect cells from reactive chemical species, including carcinogens and oxidative stress. This pathway is activated by both naturally occuring chemopreventive agents found in a wide variety of fruits and vegetables and by synthetic molecules. We believe that a fundamental understanding of this signal transduction pathway will facilitate the identification of foods, dietary supplements and drugs that will significantly decrease the risk of cancer in humans. Furthermore, as oxidative stress is a driving force of many pathophysiological conditions, including neurodegeneration, cardiovascular disease and skeletal muscle atrophy, the proposed research will have a broad impact on human health. The critical target of this pathway, the transcription factor Nrf2, is normally repressed by the BTB-Kelch protein, Keapl. Chemopreventive agents enable Nrf2 to escape Keap1-mediated repression and activate transcription of its target genes that eliminate reactive species and restore cellular redox homeostasis. Our preliminary data suggest the hypothesis that Keap1 functions as a substrate adaptor protein for a Cul3- dependent E3 ubiquitin ligase complex. This hypothesis represents a new paradigm for understanding how Keapl is able to repress Nrf2-dependent transcription and provides a productive framework for defining how chemopreventive agents enable Nrf2 to escape Keap1-mediated repression. This hypothesis also provides novel insight into the biological functions of all BTB-Kelch proteins. We propose to examine this hypothesis further by characterizing disease-associated mutations within GAN1 and ENC1 that are responsible for giant axonal neuropathy and contribute to brain cancers, respectively. The proposed experiments will (1) define how Nrf2 is targeted for ubiquitin-dependent degradation by a Keapl :Cul3:Rbx1 complex, (2) define how Nrf2 escapes Keap1-mediated repression, (3) use Keap1 as a model system to define how disease-associated mutations perturb the substrate adaptor function of BTB- Kelch proteins, and (4) define the structural basis for substrate recognition by Keap1.