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: 10152599
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10152599
• 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; 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.

项目总结 内质网负责所有蛋白质中超过三分之一的折叠和翻译后修饰 真核生物。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 疾病。在这里，我们研究了内质网相关蛋白降解(ERAD)途径，它调控着 从内质网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体 它们被运输到细胞质，在那里它们被蛋白酶体降解。ERAD的靶向是癌症 由于癌细胞需要更大的蛋白质折叠和降解能力，因此需要进行更多的治疗。 属于硒蛋白家族的两种完整的膜蛋白参与了ERAD 机制：硒蛋白S(SELS)和硒蛋白K(SELK)。因为所有的硒蛋白都是酶，Sels和 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能够裂解自己的肽键，释放一种含硒半胱氨酸 多肽，从而终止酶的活性。我们认为这种自身蛋白分解是一种调节性的 SELK与不同膜复合体结合的机理。我们将描述 裂解机制、裂解位点和硒半胱氨酸对多肽的前所未有的贡献 键断裂。然后我们将研究SELK蛋白伙伴是否影响卵裂率或位点和 截短形式的SELK是否能够与选定的蛋白质伙伴结合。 在一个相关的重点中，我们将研究Selk的蛋白质合作伙伴SELS如何协调AAA的招募 含Valosin蛋白(VCP)的ATPase p97转位错折叠蛋白的膜通道 (Dislocon)。细胞质p97提供了将错误折叠的蛋白质从错位中拉出所需的能量。 因此，它是ERAD进程的核心。因为硒蛋白经常被发现可以解毒或调节 我们假设SELS不仅招募p97，而且还调节其ATPase活性 以及对氧化修饰的敏感性。我们将映射SEL与p97和Derlin-1的相互作用，a 错位蛋白的跨膜贡献者。此外，SELS与额外蛋白质底物相互作用的能力 而结合p97或DERLIN-1将被评估。 这项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， 从而阐明了错位粒子及其能源P97的复杂组装所需的步骤。此外, 我们将阐明SELS在多大程度上以氧化还原状态依赖的方式作为氧化剂的传感器和保护作用。 P97免受伤害。我们的研究将极大地促进我们对赛尔斯和赛尔克的理解 对蛋白质降解的贡献以及它们的硒半胱氨酸在络合物形成和 酶促反应。由于与硒半胱氨酸、SELS和SELK有关的特殊化学物质 将自己作为独特的药物靶点，其基于硒的反应性可以被靶向。