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.

项目摘要 ER负责折叠和翻译后修饰超过三分之一的蛋白质的三分之一 真核生物。蛋白质的降解受损与神经退行性和蛋白质折叠不折叠密切相关 疾病。在这里，我们检查了ER相关的蛋白质降解（ERAD）途径，该途径控制 从ER的膜和腔内提取不折叠的蛋白质或毫用的蛋白质复合物 它们运输到细胞质，在那里它们被蛋白酶体降解。 ERAD针对癌症 治疗由于取消细胞需要增加蛋白质折叠和降解的能力。 属于硒蛋白家族的两个整体膜蛋白有助于Erad 机械：硒蛋白S（SELS）和硒蛋白K（SELK）。由于所有硒蛋白都是酶的， SELK很可能是催化活性的，但是它们对Erad途径的具体贡献尚不清楚。我们 最近发现SELK能够清除自己的肽键，释放硒半胱氨酸 肽，从而终止酶促活性。我们建议这种自蛋白解析是一种调节性 负责与不同膜复合物相关的机制。我们将表征 切割机制，裂解位点和硒代半胱氨酸对肽的前所未有的贡献 粘结裂解。然后，我们将检查SELK蛋白伴侣是否会影响裂解率或地点，并且 SELK的截短形式是否能够结合选定的蛋白质伴侣。 在相关的推力下，我们将研究Selk的蛋白质伴侣SELS如何协调AAA的招聘 含ATPase瓣膜蛋白（VCP）p97到膜通道的p97 p97易位的蛋白质折叠蛋白 （脱孔）。细胞质p97提供了将错误折叠蛋白从脱核中取出的必要能量 因此，是埃拉德过程的核心。因为经常发现硒蛋白来排毒或调节 反应性氧化物种类我们假设SEL不仅募集P97，还可以调节其ATPase活性 对氧化修饰的敏感性。我们将与p97和derlin-1（a 跨膜造成脱节的贡献者。同样，SELS与其他蛋白质底物相互作用的能力 虽然将绑定到P97或DERLIN-1。 提出的实验工作将揭示塞尔，selk，selk，derlin-1和p97，p97之间的分子相互作用 从而阐明了脱芯及其能源p97的复杂组装所需的步骤。此外， 它将在氧化还原依赖性的方式中阐明在多大程度上作用 - 作为氧化和保护的传感器 p97受损坏。一起，我们的研究将极大地提高我们对Sels和Selk的理解 对蛋白质降解的贡献以及其硒代半胱氨酸在复杂形成和中的作用 酶促反应。由于与硒代半胱氨酸相关的专门化学反应，SELS和SELK 将自己作为独特的药物靶标表示，其基于硒的反应性可以针对。