Pathophysiology of environmentally-induced protein malfolding

描述(申请人提供)：蛋白质折叠起重要作用的神经退行性疾病，如帕金森氏病，阿尔茨海默病和运动神经元疾病。越来越多的证据表明，环境因素可能通过直接或间接影响细胞代谢来干扰蛋白质折叠，从而促进这些常见疾病的病理生理学。然而，关于细胞如何适应环境诱导的蛋白毒性的威胁，人们知之甚少。这项研究将利用砷作为一种环境毒素的模型，这种毒素会对蛋白质折叠产生不利影响，并代表着一种影响多器官系统的重要公共健康危害。最近确定的两种对砷暴露的适应将作为本研究的出发点：(1)新蛋白质合成的调节减弱。(2)修改细胞的蛋白质降解装置，以更好地适应砷诱导的蛋白质毒性。应激诱导的翻译起始因子2a(ElF2a)的磷酸化减弱了蛋白质的合成，并激活了一个被称为整合应激反应(ISR)的有益基因表达程序，它减少了由砷诱导的蛋白质错折叠造成的压力。因此，elF2a的磷酸化已成为细胞未折叠蛋白反应(UPR)的重要组成部分。将对使elF2a去磷酸化的磷酸酶进行表征，以努力确定其抑制激活ISR的特定生化步骤。抑制elF2a磷酸酶的生理意义将在神经退行性疾病的小鼠模型中进行测试。这些研究将揭示通过抑制elF2a磷酸酶来保护蛋白毒性的治疗策略的前景和潜在的局限性。AIRAP是一种新的亚砷酸盐诱导蛋白，它使蛋白酶体的调节帽适应亚砷酸盐诱导的细胞蛋白毒性的条件，从而提高细胞处理错误折叠蛋白的能力。为了了解细胞内蛋白质降解机制如何适应蛋白毒性，将用蛋白质组学方法表征亚砷酸盐诱导的和AIRAP依赖的蛋白酶体组成的变化。小鼠和蠕虫的基因敲除实验将被用来建立缺乏AIRAP的实验系统，这些实验系统将被用作工具来鉴定亚砷酸盐修饰的蛋白质，其降解依赖于AIRAP的诱导和AIRAP与19S蛋白酶体调节颗粒的整合。含有AIRAP的纯化蛋白酶体的体外生化分析将被用来表征蛋白酶体对环境诱导的蛋白质麦芽折叠的功能性适应。这项研究的目标是减少环境毒素对其分子组成的蛋白质折叠诱导的细胞适应。这将为确定暴露的相关生物标记物以及未来针对神经变性的预防和治疗干预措施奠定基础。

DESCRIPTION (provided by applicant): Protein malfolding plays an important role neurodegenerative conditions, such as Parkinson's Disease, Alzheimer's Disease and Motor Neuron Disease. Accumulating evidence suggests that environmental agents may contribute to the pathophysiology of these common disorders by perturbing protein folding, either directly or indirectly through their effects on cell metabolism. However, little is known about how cells adapt to the threat of environmentally-induced proteotoxicity. This study will exploit arsenic as a model for an environmental toxin that adversely affects protein folding and one that represents an important public health hazard affecting multiple organ systems. Two recently-identified adaptations to arsenic exposure will serve as this study's point of departure: (1) Regulated attenuation of new protein synthesis. (2) Modification of the cell's protein degradation apparatus to better accommodate it to arsenic-induced proteotoxicity. Stress-induced phosphorylation of translation initiation factor 2a (elF2a) attenuates protein synthesis and activates a salubrious gene expression program known as the Integrated Stress Response (ISR), which reduces the stress caused by arsenic-induced protein malfolding. Therefore, elF2a phosphorylation has emerged as an important component of cellular unfolded protein responses (UPR). Phosphatases that dephosphorylate elF2a will be characterized in an effort to identify specific biochemical steps whose inhibition activates the ISR. The physiological significance of inhibiting elF2a phosphatases will be tested in mouse models of neurodegenerative diseases. These studies will uncover the promise and potential limitations of therapeutic strategies to protect against proteotoxicity by inhibiting elF2a phosphatases. AIRAP, a novel arsenite induced protein, adapts the proteasome's regulatory cap to the conditions in cells experiencing arsenite-induced proteotoxicity and thereby promotes the cell's ability to deal with malfolded proteins. In an effort to understand how the intracellular protein degradation machinery adapts to proteotoxicity, arsenite-induced and AIRAP-dependent changes in the composition of the proteasome will be characterized by proteomic approaches. Gene knock out experiments in mouse and worms will be used to create experimental systems lacking AIRAP, and these will be applied as tools to identify arsenite-modified proteins whose degradation depends on AIRAP induction and AIRAP integration into the 19S proteasome regulatory particle. In vitro biochemical assays of purified proteasomes containing AIRAP will be used to characterize functionally proteasomal adaptation to environmentally-induced protein malfolding. The goal of this research program is to reduce the cellular adaptations to protein malfolding induced by environmental toxins to their molecular constituents. This will lay the groundwork for identifying relevant bio- markers of exposure and for future preventive and therapeutic interventions against neurodegeneration.