Role of a Novel E3-Ubiquitin Ligase in Chemoprevention

该提案中的工作重点是触发癌症的主要预防信号传导途径 转录诱导酶，保护细胞免受反应性化学物质，包括致癌物质的影响 和氧化应激。这一途径被两种天然存在的化学预防剂激活， 各种各样的水果和蔬菜以及合成分子。我们认为， 了解这种信号转导途径将有助于识别食物，饮食， 补充剂和药物，将显着降低人类患癌症的风险。此外如 氧化应激是许多病理生理状况，包括神经变性， 心血管疾病和骨骼肌萎缩，拟议的研究将产生广泛的影响， 人体健康 该途径的关键靶标，转录因子Nrf 2，通常被BTB-Kelch抑制。 蛋白质，Keapl.化学预防剂使Nrf 2能够逃避Keapl介导的抑制并激活 其靶基因的转录，消除反应性物种和恢复细胞氧化还原稳态。 我们的初步数据表明，Keapl作为Cul 3的底物衔接蛋白发挥作用的假设。 依赖性E3泛素连接酶复合物。这一假设代表了一种新的范式， Keapl能够抑制Nrf 2依赖性转录，并提供了一个有效的框架来定义如何 化学预防剂使Nrf 2能够逃脱Keapl介导的抑制。这一假设还提供了 对所有BTB-Kelch蛋白的生物学功能的新见解。我们建议检验这一假设 进一步通过表征GAN 1和ENC 1中的疾病相关突变， 轴突神经病变和脑癌的原因。 拟议的实验将（1）定义Nrf 2如何被泛素依赖性降解靶向， Keapl：Cul 3：Rbxl复合物，（2）定义Nrf 2如何逃脱Keapl介导的阻遏，（3）使用Keapl作为Nrf 2的抑制剂。 模型系统来定义疾病相关突变如何干扰BTB的底物衔接子功能， Kelch蛋白，和（4）定义的底物识别Keapl的结构基础。

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 Keapl-mediated repression andactivate transcription of its target genes that eliminate reactive species and restore cellular redox homeostasis. Our preliminary data suggest the hypothesis that Keapl 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 Keapl-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 Keapl-mediated repression, (3) use Keapl 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 Keapl.