Role of a Novel E3-Ubiquitin Ligase in Chemoprevention

这项提案中的工作重点是研究一种主要的癌症预防信号转导途径，它可以触发 保护细胞免受包括致癌物在内的活性化学物质影响的酶的转录诱导 和氧化应激。这一途径被两种自然产生的化学预防药物激活，这些药物可以在 种类繁多的水果和蔬菜以及合成分子。我们认为，一个根本的 了解这一信号转导途径将有助于识别食物、饮食 将显著降低人类患癌症风险的补充剂和药物。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，拟议的研究将对 人类健康。 该途径的关键靶标转录因子Nrf2通常被BTB-Kelch抑制 蛋白质，Keapl。化学预防药物使Nrf2逃脱Keapl介导的抑制并激活 转录其目标基因，以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CUL3-的底物适配蛋白发挥作用的假设 依赖的E3泛素连接酶复合体。这一假说代表了一种新的范式，可以理解 Keap1能够抑制Nrf2依赖的转录，并提供了一个有效的框架来定义 化学预防药物使Nrf2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GAN1和ENC1中与疾病相关的突变，这些突变与巨细胞 轴突神经病变和导致脑癌。 拟议的实验将(1)定义Nrf2如何通过一种 Keapl：CUL3：Rbx1复合体，(2)定义Nrf2如何逃避Keapl介导的抑制，(3)将Keapl用作 定义疾病相关突变如何扰乱BTB底物适配器功能的模型系统- Kelch蛋白，以及(4)定义了Keapl识别底物的结构基础。

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.