Selenoproteins in the ER-associated protein degradation pathway

ER 相关蛋白降解途径中的硒蛋白

• 批准号: 9690137
• 负责人: Sharon Rozovsky
• 金额: $ 24.27万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9690137
• 关键词: ATP phosphohydrolase; Active Sites; Affect; Affinity; Amino Acids; Binding; Binding Proteins; Biochemical Reaction; Cancerous; Cell physiology; Cells; Chemistry; Cleaved cell; Complex; Cytoplasm; Degradation Pathway; Disease; Disulfides; Drug Targeting; Endoplasmic Reticulum; Energy Supply; Energy-Generating Resources; Enzymes; Equilibrium; Estrogen receptor positive; Eukaryota; Exercise; Family; Homeostasis; Impairment; In Vitro; Integral Membrane Protein; Ion Channel; Length; Link; Lipids; Maintenance; Maps; Measurement; Measures; Membrane; Membrane Proteins; Modification; Molecular; Monitor; Nerve Degeneration; Oxidants; Oxidation-Reduction; Oxidoreductase; Peptides; Physiological; Post-Translational Protein Processing; Predisposition; Process; Production; Proteins; Reagent; Reducing Agents; Role; Selenium; Selenocysteine; Site; Stress; Transferase; Work; base; cancer therapy; cardiovascular health; cofactor; grasp; misfolded protein; multicatalytic endopeptidase complex; mutant; new therapeutic target; p97 ATPase; protein K; protein complex; protein degradation; protein folding; protein misfolding; proteostasis; recruit; selenoprotein; sensor; stoichiometry; targeted treatment; valosin-containing protein

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.

项目概要 ER 负责蛋白质中三分之一以上的折叠和翻译后修饰。 真核生物的蛋白质降解受损与神经退行性变和蛋白质错误折叠密切相关。 在这里，我们检查了 ER 相关蛋白降解 (ERAD) 途径，该途径控制着疾病。 从内质网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， 它们被转运到细胞质，在那里被蛋白酶体降解。ERAD 是癌症的靶标。 由于癌细胞需要增加蛋白质折叠和降解的能力，因此需要进行治疗。 属于硒蛋白家族的两种完整膜蛋白有助于 ERAD 机制：硒蛋白 S (SelS) 和硒蛋白 K (SelK) 因为所有硒蛋白都是酶 SelS 和 SelK。 SelK 很可能具有催化活性，但其对 ERAD 途径的具体贡献尚不清楚。 最近发现 SelK 能够裂解自己的肽键，释放出含有硒代半胱氨酸的物质 肽，从而终止酶活性，我们认为这种自蛋白水解是一种调节作用。 我们将描述负责 SelK 与不同膜复合物关联的机制。 裂解机制、裂解位点以及硒代半胱氨酸对肽的前所未有的贡献 然后我们将检查 SelK 蛋白伴侣是否影响裂解速率或位点以及 SelK 的截短形式是否能够结合选定的蛋白质伴侣。 在相关的主旨中，我们将研究 SelK 的蛋白质伙伴 SelS 如何协调 AAA 的招募 含 ATP 酶缬氨肽的蛋白 (VCP) p97 至膜通道，可将错误折叠的蛋白转位 (脱位子) 细胞质 p​​97 提供将错误折叠的蛋白质从脱位子中拉出所需的能量。 因此是 ERAD 过程的核心，因为硒蛋白经常被发现具有解毒或调节作用。 我们认为 SelS 不仅可以招募 p97，还可以调节其 ATP 酶活性 我们将绘制 SelS 与 p97 和 derlin-1（a）的相互作用。 SelS 还具有与其他蛋白质底物相互作用的能力。 同时将评估与 p97 或 derlin-1 的结合。 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， 从而阐明了复杂组装 dislocon 及其能源所需的步骤，p97。 将会阐明 SelS 在多大程度上以氧化还原态依赖的方式作为氧化剂传感器发挥作用并提供保护 p97 免受损害，我们的研究将极大地增进我们对 SelS 和 SelK 的理解。 对蛋白质降解的贡献及其硒代半胱氨酸在复合物形成和中的作用 由于与硒代半胱氨酸、SelS 和 SelK 相关的特殊化学反应。 将其自身作为独特的药物靶标，其基于硒的反应性可以被靶向。