Redox and Protein Homeostasis in Arsenic Tumorigenicity

项目总结 砷被归类为一种人类致癌物质，与增加患癌症的风险有关 肺、皮肤和膀胱。然而，砷致癌的机制仍不完全清楚。 描述。砷已被证明上调NRF2，NRF2是细胞应激反应的主要调节因子 体外和体内。在非应激条件下，Keap1-CUL3-RBX1-1使NRF2的基础水平保持在较低水平。 介导泛素化和随后的26S蛋白酶体降解。在规范的NRF2激活期间 Keap1-Cys151被修饰，阻止了NRF2泛素化，增加了NRF2及其下游的水平 保护细胞免受环境侮辱的基因。一旦细胞动态平衡恢复，NRF2就会回到低水平 基本水平，确保瞬时NRF2上调。然而，我们发现砷通过一种非 正则机制。与环境相关的剂量的砷阻止自噬体成熟，从而导致 P62介导的Keap1滞留到自噬小体中，阻断NRF2泛素化和降解。 这导致NRF2及其抗氧化反应元件(ARE)的持续高表达 基因，赋予细胞生存优势。有趣的是，我们的小说未发表的结果表明 与多种细胞活动(AAA)相关的ATPase伴侣p97在其 推动者。在功能上，p97利用atp的结合和水解来产生一种分离泛素化的力量。 来自其他生物分子的蛋白质，通常用于蛋白酶体的后续降解。我们已经证明了 P97通过从Keap1-CUL3-RBX1 E3泛素中提取泛素化的NRF2来负性调节NRF2 促进蛋白酶体降解NRF2的连接酶复合体。P97的这种分离酶活性发挥了 在蛋白质动态平衡中的突出作用--蛋白质平衡。P97作为一个新的NRF2靶基因的发现表明 NRF2不仅控制氧化应激，还控制蛋白毒性应激。与NRF2一样，p97在 人类肿瘤已有报道，p97被认为是治疗癌症的靶点。我们还观察到， 在砷转化的细胞系和人肺肿瘤中，NRF2和p97的蛋白水平都增加 组织(初步数据)。基于这些发现，我们提出了如下模型：在基本条件下， 通过NRF2-p97-NRF2负反馈环维持NRF2和p97的低水平表达。 然而，在慢性砷暴露期间，这种NRF2-p97-NRF2负馈送浸泡循环自NRF2以来被禁用 不再泛素化，导致NRF2和p97持续上调。因此，砷会增加 氧化应激反应和蛋白毒性应激反应。我们将检验我们的假设 环境砷暴露对NRF2和NRF2介导的p97持续上调的反应 提供了一种生存优势，使暴露在砷中的细胞能够在氧化和 蛋白毒性应激，并积累足够的分子事件，推动恶性转化。

PROJECT SUMMARY Arsenic is classified as a human carcinogen that has been associated with increased risk of developing cancers of the lung, skin, and bladder. However, the mechanism of arsenic tumorgenicity remains incompletely described. Arsenic has been shown to upregulate NRF2, the master regulator of the cellular stress response in vitro and in vivo. Under non-stress conditions, the basal level of NRF2 is kept low by KEAP1-CUL3-RBX1- mediated ubiquitylation and subsequent 26S proteasomal degradation. During canonical NRF2 activation KEAP1-Cys151 is modified, preventing NRF2 ubiquitylation and increasing the level of NRF2 and its downstream genes to protect cells from environmental insults. Once cellular homeostasis is restored, NRF2 returns to low basal level, ensuring transient NRF2 upregulation. However, we discovered arsenic activates NRF2 by a non- canonical mechanism. Environmentally relevant doses of arsenic block autophagosome maturation, which leads to p62-mediated sequestration of KEAP1 into autophagosomes, blocking NRF2 ubiquitylation and degradation. This leads to sustained hyperexpression of NRF2 and its antioxidant response element (ARE) containing target genes, conferring a cellular survival advantage. Interestingly, our novel unpublished results demonstrate that the ATPase associated with various cellular activities (AAA+) chaperone, p97, contains a functional ARE in its promoter. Functionally, p97 uses the binding and hydrolysis of ATP to generate a force to segregate ubiquitylated proteins from other biomolecules, often for subsequent degradation by the proteasome. We have demonstrated that p97 negatively regulates NRF2 by extracting ubiquitinated NRF2 from the KEAP1-CUL3-RBX1 E3 ubiquitin ligase complex that facilitates NRF2 degradation by proteasome. This segregase activity of p97 plays a prominent role in protein homeostasis – proteostasis. Our finding of p97 as a novel NRF2 target gene indicates that NRF2 controls not only oxidative stress but also proteotoxic stress. Like NRF2, hyperexpression of p97 in human tumors has been reported and p97 is considered a target for treating cancer. We also observed that the protein levels of both NRF2 and p97 were increased in arsenic transformed cell lines and in human lung tumor tissues (preliminary data). Based on these findings, we propose the following model: Under basal conditions, low level expression of NRF2 and p97 is maintained through the NRF2-p97-NRF2 negative feedback loop. However, during chronic arsenic exposure, this NRF2-p97-NRF2 negative feed bask loop is disabled since NRF2 is no longer ubiquitylated, resulting in sustained upregulation of both NRF2 and p97. Thus, arsenic increases both the oxidative stress response and the proteotoxic stress response. We will test our hypothesis that sustained NRF2 and NRF2-mediated p97 upregulation in response to environmental arsenic exposure provides a survival advantage that enables arsenic-exposed cells to survive both oxidative and proteotoxic stress and to accumulate sufficient molecular events that drive malignant transformation.