Modification of PDI by 4-HNE and 4-ONE and its Role in Ethanol-Induced ER Stress

描述（由申请人提供）：内质网（ER）中错误折叠和/或未折叠蛋白质的积累被描述为未折叠蛋白质反应（UPR）。这种反应的病理结果被称为内质网（ER）应激，最近被认为是许多疾病状态（包括酒精性肝）的开始和进展的可能机制。蛋白二硫异构酶（PDI）是内质网分子伴侣，是氧化折叠和异构化反应的关键酶。已发表的数据表明，活性醛- 4-羟基壬烯醛（4-HNE）修饰了PDI的活性位点。4-HNE和4-氧壬烯醛（4-ONE）是脂质过氧化的产物，在酒精性肝的进展中起作用。通过活性部位半胱氨酸的内聚，这些醛已被证明能引起许多蛋白质酶活性的改变。本实验旨在验证4-HNE和4-ONE对PDI的共价内聚增加内质网中的错误蛋白负荷并随后诱导内质网应激反应的一般工作假设。本提案的第一阶段旨在阐明PDI在体外内质网应激反应中的作用，以及在慢性乙醇啮齿动物喂养模型中的作用。其次，在长期乙醇喂养后，PDI氧化还原状态的变化将被检查。最后，PDI酶活性的变化将在乙醇饲养后进行体内检测，以及在体外用上述醛处理后进行检测。完成后，本提案提供的数据将进一步阐明PDI在乙醇诱导的内质网应激反应中的作用。仅在美国，酒精性肝病就影响了近200万人，其发展背后的机制仍远未阐明。终末期肝病的四年生存率仍然低得惊人，只有35%，这就强调了揭示其发病机制的紧迫性。酒精性肝的进展是多因素的，这是最有可能的；然而，目前的研究表明内质网应激反应和脂质过氧化产物都有作用。

DESCRIPTION (provided by applicant): The build-up of misfolded and/or unfolded proteins in the endoplasmic reticulum (ER) is characterized as the unfolded protein response (UPR). The pathological consequence of this response is known as endoplasmic reticulum (ER) stress and has recently emerged as a possible mechanism for the initiation and progression of numerous disease states, including the alcoholic liver. The ER-resident molecular chaperone, protein disulfide isomerase (PDI), is a key enzyme in both oxidative folding and isomerization reactions. Published data has shown modification of the active-site of PDI by the reactive aldehyde 4-hydroxynonenal (4-HNE). 4-HNE, as well as 4-oxononenal (4-ONE), are products of lipid peroxidation and have been implicated to have a role in the progression of the alcoholic liver. Through the adduction of active site cysteines, these aldehydes have been shown to cause alterations in the enzymatic activity of numerous proteins. The experiments outlined in this proposal are designed to test the general working hypothesis that covalent adduction of PDI by 4-HNE and 4-ONE increase the erred protein burden in the ER and subsequently induces the ER stress response. The first phase of this proposal is designed to elucidate the role of PDI in the ER stress response in vitro, as well as in a chronic ethanol rodent feeding model. Secondly, the alterations in the redox status of PDI will be examined following chronic ethanol feeding. Finally, alterations in the enzymatic activities of PDI will be examined in vivo following ethanol feeding, as well as in vitro, following treatment with the aforementioned aldehydes. Upon completion, the data provided by this proposal will further elucidate the role of PDI in an ethanol-induced ER stress response. With alcoholic liver disease affecting nearly 2 million people in the United States alone, the mechanisms behind its progression remain far from elucidated. The four-year survival rate for end-stage liver disease remains a staggeringly low 35%, stressing an urgency for unraveling the mechanisms behind its pathogenesis. It is most conceivable that the progression of the alcoholic liver is multi-factorial; however, current research suggests a role for both the ER stress response and lipid peroxidation products.