Role of a Novel E3-Ubiquitin Ligase in Chemoprevention

描述（由申请人提供）：本提案的工作重点是主要的癌症预防信号转导途径，该途径触发酶的转录诱导，保护细胞免受活性化学物质（包括致癌物和氧化应激）的侵害。这条途径可以被多种水果和蔬菜中天然存在的化学预防剂和合成分子激活。我们相信，对这一信号转导途径的基本理解将有助于识别能够显著降低人类癌症风险的食品、膳食补充剂和药物。此外，氧化应激是许多病理生理状况的驱动力，包括神经退行性疾病、心血管疾病和骨骼肌萎缩，因此拟议的研究将对人类健康产生广泛的影响。该途径的关键靶点转录因子Nrf2通常被BTB-Kelch蛋白Keapl抑制。化学预防药物使Nrf2能够摆脱keap1介导的抑制，并激活其靶基因的转录，从而消除活性物质并恢复细胞氧化还原稳态。我们的初步数据支持Keap1作为Cul3依赖性E3泛素连接酶复合物的底物衔接蛋白的假设。这一假设代表了理解Keapl如何能够抑制Nrf2依赖转录的新范式，并为定义化学预防剂如何使Nrf2逃避keap1介导的抑制提供了一个有效的框架。这一假设也为所有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中的疾病相关突变来进一步检验这一假设，这两种突变分别导致巨大轴突神经病变和脑癌。拟进行的实验将(1)确定Nrf2如何被Keap1:Cul3:Rbx1复合物降解为泛素依赖的靶标，(2)确定Nrf2如何逃脱Keap1介导的抑制，(3)使用Keap1作为模型系统来确定疾病相关突变如何干扰BTB- Kelch蛋白的底物接子功能，以及(4)确定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.