Stress response, p97, and Nrf2 in arsenic-mediated toxicity

描述（由申请人提供）：砷及其砷衍生物估计影响全世界超过2亿人。接触砷有多种来源，如受污染的饮用水、土壤或空气污染物。各种流行病学研究已将慢性砷暴露与多种疾病状态联系起来，包括肺癌、膀胱癌或皮肤癌；代谢性疾病，如糖尿病；心血管和其他血管疾病；还有皮肤问题，比如“黑足病”。除了流行病学研究之外，人们还付出了巨大的努力来理解病理机制，但迄今为止，沿着这些路线的许多问题仍然模糊不清。理解砷毒性的部分问题在于砷改变了细胞系统的数量。例如，砷会导致氧化应激、蛋白质质量控制、热休克反应和细胞周期改变等。我们实验室的工作已经确定了砷对细胞影响的一个关键环节。长期使用低水平的亚砷酸盐（砷的一种氧化物）治疗会导致自噬受损，自噬是一种主要的蛋白质质量控制途径。这种破坏发生在自噬体/溶酶体融合的步骤中，导致自噬体的积累和高水平的自噬特异性因子p62。关键的是，p62含有Keap1的识别元件，Keap1是Cul3-Keap1-Rbx1 E3泛素连接酶复合物中的一个底物识别因子。这种E3复合物通常维持低水平的氧化应激反应转录因子Nrf2。在过量p62存在的情况下，Keap1被占用，允许Nrf2的组成性、高水平表达和随后的抗氧化反应元件调控基因的激活。这种高水平的表达赋予细胞生长优势，并可能导致癌症等疾病。尽管有这些机制上的飞跃，但亚砷酸盐干扰自噬的机制仍是未知的。在目前的研究计划中，我们提出砷干扰AAA+蛋白质量控制机器的假设，p97。这提供了砷与自噬以及其他蛋白质质量控制机制之间的关键联系。为了探索这种砷介导的破坏的详细机制基础，我们将使用一系列详细的机制酶学研究，结合细胞生物化学和体内研究。这些努力将得到我们组建的多项目负责人团队的极大帮助。

DESCRIPTION (provided by applicant): Arsenic and its arsenical derivatives are estimated to effect greater than 200 million people worldwide. Exposure to arsenicals comes from a number of sources such as contaminated drinking water, soil or as an airborne pollutant. Various epidemiological studies have linked chronic arsenic exposure to a number of disease states including cancer of the lungs, bladder, or skin; metabolic diseases such as diabetes; cardiovascular and other vascular diseases; and skin problems such as 'black foot disease'. In addition to the epidemiological studies there has been a great deal of effort to understand the mechanisms of pathology, but to date many questions along these lines remain obfuscated. Part of the problem with understanding arsenic toxicity is the sheer number of cellular systems that arsenic alters. For instance arsenic leads to oxidative stress, compromise of protein quality control, heat-shock response, and cell-cycle alterations to name a few. Work from our lab has identified a crucial link in the effects of arsenic on cells. Chronic treatment with low levels of arsenite (one of the oxides of arsenic) leads to a compromise of autophagy, a major protein quality control pathway. This breach comes at the step of autophagosome/lysosome fusion, leading to a build-up of autophagosomes and high levels of the autophagy specificity factor, p62. Critically, p62 contains a recognition element for Keap1, which is a substrate recognition factor in the Cul3-Keap1-Rbx1 E3 ubiquitin ligase complex. This E3 complex normally maintains a low level of the oxidative stress responsive transcription factor, Nrf2. In the presence of excess p62, Keap1 is occupied, allowing for constitutive, high level expression of Nrf2 and subsequent activation of antioxidant response element regulated genes. This high-level expression confers a growth advantage on the cells and can lead to diseases such as cancer. Despite these mechanistic leaps, it remains the mechanism by which arsenite interferes with autophagy is unknown. In the present research program we propose the hypothesis that arsenicals interfere with the AAA+ protein quality control machine, p97. This provides a critical link between arsenic and autophagy as well as other protein quality control mechanisms. To probe the detailed mechanistic underpinnings of this arsenic-mediated breach we will use an array of detailed mechanistic enzymology studies, coupled with cellular biochemistry, and in vivo studies. These efforts will be greatly aided by the multi-PI team we have assembled.