Selenoproteins in the ER-associated protein degradation pathway

项目摘要 ER负责细胞中超过三分之一的蛋白质的折叠和翻译后修饰。 真核生物蛋白质降解受损与神经变性和蛋白质错误折叠密切相关 疾病在这里，我们研究了ER相关蛋白降解（ERAD）途径，该途径控制着 从ER的膜和腔中提取错误折叠的蛋白质或错误组装的蛋白质复合物， 它们运输到细胞质，在那里它们被蛋白酶体降解。ERAD靶向于癌症 因为癌细胞需要增加蛋白质折叠和降解的能力，所以癌症治疗是非常有效的。 属于硒蛋白家族的两种膜蛋白参与ERAD的形成 硒化机制：硒蛋白S（SelS）和硒蛋白K（SelK）。由于所有的硒蛋白都是酶SelS， SelK很可能具有催化活性，但它们对ERAD途径的具体贡献尚不清楚。我们 最近发现SelK能够切割其自身的肽键，释放出含有硒代半胱氨酸的 肽，从而终止酶活性。我们认为，这种蛋白质自水解是一种调节性的机制。 负责SelK与不同膜复合物结合的机制。我们将描述 切割机制、切割位点和硒代半胱氨酸对肽的前所未有的贡献 键裂然后，我们将检查SelK蛋白伴侣是否影响切割速率或位点， SelK的截短形式是否能够结合选定的蛋白质伴侣。 在相关的研究中，我们将研究SelK的蛋白质伴侣SelS如何协调AAA的募集。 ATP酶valosin-containing蛋白（VCP）p97的膜通道，易位错误折叠的蛋白质 （dislocon）.胞质p97提供将错误折叠的蛋白质拉出错位所需的能量 因此是ERAD过程的核心。因为硒蛋白通常被发现可以解毒或调节 我们推测SelS不仅募集p97，而且还调节其ATP酶活性 和对氧化修饰的敏感性。我们将绘制SelS与p97和derlin-1的相互作用， 跨膜贡献者的脱位。SelS与其他蛋白质底物相互作用的能力 而与p97或derlin-1结合的。 拟议的实验工作将揭示SelS、SelK、derlin-1和p97之间的分子相互作用， 从而阐明了位错及其能量源的复杂组装所需的步骤，P97。此外，本发明还提供了一种方法， 将阐明SelS在何种程度上以氧化还原状态依赖的方式作为氧化剂的传感器并保护 p97的伤害总之，我们的研究将极大地促进我们对SelS和SelK的理解 对蛋白质降解的贡献以及它们的硒代半胱氨酸在复合物形成和 酶促反应由于硒代半胱氨酸的特殊化学性质，SelS和SelK 它们自身表现为独特的药物靶标，其基于硒的反应性可以被靶向。

PROJECT SUMMARY The ER is responsible for the folding and posttranslational modification of over a third of all proteins in eukaryotes. Impaired degradation of proteins is strongly linked to neurodegenerative and protein misfolding diseases. Here we examine the ER-associated protein degradation (ERAD) pathway, which governs the extraction of misfolded proteins or misassembled protein complexes from the ER's membrane and lumen and their transport to the cytoplasm where they are degraded by the proteasome. The ERAD is targeted in cancer treatments since cancerous cells require an increased capacity for protein folding and degradation. Two integral membranes proteins that belong to the family of selenoproteins contribute to the ERAD machinery: selenoprotein S (SelS) and selenoprotein K (SelK). Since all selenoproteins are enzymes SelS and SelK are most likely catalytically active but their specific contribution to the ERAD pathway is yet unknown. We recently discovered that SelK is able to cleave its own peptide bond, releasing a selenocysteine–containing peptide, and thus terminating enzymatic activity. We propose that this autoproteolysis is a regulatory mechanism responsible for SelK associations with different membrane complexes. We will characterize the cleavage mechanism, cleavage sites and the unprecedented contribution of selenocysteine to the peptide bond cleavage. We will then examine whether SelK protein partners affect the cleavage rate or sites and whether truncated forms of SelK are able to bind selected protein partners. In a related thrust, we will examine how SelK's protein partner, SelS, coordinates the recruitment of the AAA ATPase valosin-containing protein (VCP) p97 to the membrane channel that translocates misfolded proteins (dislocon). The cytoplasmic p97 provides the energy necessary for pulling misfolded protein out of the dislocon and hence is central to the ERAD process. Because selenoproteins are often found to detoxify or regulate reactive oxidative species we hypothesize that SelS not only recruits p97 but also regulates its ATPase activity and sensitivity to oxidative modifications. We will map SelS interactions with p97 and derlin-1, a transmembrane contributor to the dislocon. Also SelS's ability to interact with additional protein substrates while bound to p97 or derlin-1 will be assessed. The proposed experimental work will unveil the molecular interactions between SelS, SelK, derlin-1, and p97, thus clarifying the steps required for complex assembly of the dislocon and its energy source, p97. In addition, it will be clarified to what extent SelS acts -in a redox state dependent way- as sensor of oxidants and protects p97 from damage. Together, our studies will dramatically advance our understanding of SelS's and SelK's contribution to protein degradation and of the role of their selenocysteine in complex formation and in enzymatic reactions. Because of the specialized chemistry associated with selenocysteine, SelS and SelK present themselves as unique drug targets whose selenium based reactivity can be targeted.