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

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7. Project Summary/Abstract Arsenic and its arsenical derivatives are estimated to effect greater then 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.