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

描述(申请人提供)：错误折叠和/或未折叠蛋白质在内质网(ER)中的积聚被描述为未折叠蛋白质反应(UPR)。这种反应的病理后果被称为内质网(ER)应激，最近被认为是包括酒精性肝脏在内的许多疾病状态的启动和发展的可能机制。内质网驻留分子伴侣蛋白二硫键异构酶(PDI)是氧化折叠和异构化反应中的关键酶。已发表的数据表明，反应醛4-羟基壬烯醛(4-HNE)修饰了PDI的活性部位。4-HNE和4-羟甲基烯醛(4-酮)是脂质过氧化的产物，已被认为在酒精性肝脏的进展中起作用。通过活性部位半胱氨酸的加成，这些醛已被证明能引起许多蛋白质的酶活性的改变。本提案中概述的实验旨在检验一般的工作假说，即4-HNE和4-ONE共价加成PDI增加了内质网中错误的蛋白质负担，并随后诱导内质网应激反应。该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，将检查慢性酒精摄入后PDI氧化还原状态的变化。最后，将在体内检测乙醇喂养后PDI的酶活性的变化，以及在用上述醛处理后的体外检测。完成后，这项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。由于酒精性肝病仅在美国就影响了近200万人，其进展背后的机制仍远未阐明。终末期肝病的四年存活率仍然低到惊人的35%，强调了揭开其发病机制的紧迫性。最有可能的是，酒精性肝脏的进展是多因素的；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.