The Energetic Cost of Protein Retrotranslocation during ER-associated Degradation

内质网相关降解过程中蛋白质逆转位的能量消耗

• 批准号: 7801761
• 负责人: Christopher James Guerriero
• 金额: $ 4.76万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7801761
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Address; Biological Models; C-terminal; Cell Cycle; Cells; Characteristics; Chimera organism; Chimeric Proteins; Complex; Cues; Cystic Fibrosis; Cytoplasmic Tail; Data; Defect; Disease; Drug Delivery Systems; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Gene Mutation; Goals; Heat-Shock Proteins 70; Homologous Gene; Human; Hydrophobicity; Hypertension; In Vitro; Inherited; Integral Membrane Protein; Knowledge; Length; Lesion; Link; Location; Mediating; Membrane; Modeling; Molecular Chaperones; Molecular Conformation; Mutation; Nature; Pathway interactions; Pheromone; Play; Protein Biosynthesis; Proteins; Quality Control; Research; Role; Saccharomyces cerevisiae; Signal Transduction; Structure; System; Techniques; Transmembrane Domain; Ubiquitination; Variant; Yeasts; a-mating factor; base; cell type; cohort; cost; human disease; in vivo; lipid metabolism; luminal membrane; membrane model; multicatalytic endopeptidase complex; protein degradation; protein misfolding; salt balance

DESCRIPTION (provided by applicant): Approximately one-third of all newly synthesized proteins pass through the secretory pathway. These proteins use cues within their primary structure as well as a conserved chaperone system in order to fold into their native, functional conformations. When folding is disrupted, i.e. by an inherited genetic mutation, misfolded proteins are "sensed" by the cellular proteostasis machinery and may then be targeted for ER-associated degradation (ERAD). ERAD substrates have been classified based on the location of the folding lesion within their ER lumenal, membrane, or cytoplasmic domains. Specifically, it appears that an ERAD substrate can be recognized at different points during its synthesis depending on the location of the "folding lesion". Ste6p is an ABC transporter found in the yeast S. cerevisiae, and is required for export of the a-factor mating pheromone. Truncation of Ste6p's large C-terminal cytoplasmic domain (Ste6p*) converts the protein into a substrate for the ERAD-C (cytoplasmic) pathway. Because the mutation that renders Ste6p* into an ERAD substrate resides at the extreme C-terminus, the "decision" for degradation must occur post-translationally. Recent evidence from the Brodsky lab indicates that when the truncated C-terminus of Ste6p* is transferred to other proteins it is sufficient to induce the degradation of the chimera, raising the possibility that the sequence can act as a "degron" to target proteins for degradation. I propose to dissect the ERAD pathway taken by chimeric proteins containing the Ste6p* degron. The first goal of this research is to determine the chaperones, ubiquitination machinery, and requirements for retrotranslocation for an ERAD substrate with a post-translational ERAD-C-type lesion. Second, since little is known about how integral membrane hydrophobicity influences ERAD efficiency, I propose to generate chimeric constructs including the Ste6p* degron with varied transmembrane domains. The goal of these studies is to determine if the difference in transmembrane hydrophobicity of an ERAD substrate correlates with degradation and/or membrane extraction efficiency. These studies will help to extend our knowledge of the complex mechanisms used by cells to degrade misfolded proteins, identify new factors that catalyze ERAD, and identify potential drug targets for disease-associated ERAD substrates.

描述（申请人提供）：大约所有新合成蛋白的三分之一通过分泌途径。这些蛋白质在其主要结构和保守的伴侣系统中使用提示，以折叠成本机的功能构象。当折叠被破坏时，即通过遗传基因突变，细胞蛋白抑制剂机制将“检测到”错误折叠的蛋白质，然后可以针对ER相关的降解（ERAD）。 ERAD底物已根据其ER腔，膜或细胞质结构域内的折叠病变的位置进行了分类。具体而言，似乎可以根据“折叠病变”的位置在其合成过程中在其合成过程中在不同点上识别出ERAD底物。 Ste6p是在酵母菌酿酒酵母中发现的ABC转运蛋白，是出口A因子交配信息素所必需的。 Ste6p的大C末端细胞质结构域（Ste6p*）的截断将蛋白质转化为ERAD-C（细胞质）途径的底物。由于将Ste6p*变成ERAD底物的突变存在于极端的C端，因此降解的“决定”必须在后翻译后进行。 Brodsky实验室的最新证据表明，当将STE6P*的截短的C末端转移到其他蛋白质上时，足以诱导嵌合体的降解，从而提高了该序列可以作为靶向蛋白靶向蛋白质降解的可能性。我建议剖析包含ste6p* degron的嵌合蛋白所采取的ERAD途径。这项研究的第一个目的是确定伴侣，泛素化机制以及对具有翻译后ERAD-C-TYPE病变的ERAD基板的转化转换要求。其次，由于对整体膜疏水性如何影响ERAD效率的鲜明了解知之甚少，因此我建议生成嵌合构建体，包括具有变化的跨膜结构域的Ste6p* Degron。这些研究的目的是确定ERAD底物的跨膜疏水性差异是否与降解和/或膜提取效率相关。这些研究将有助于扩展我们对细胞降解错误折叠蛋白质的复杂机制的了解，确定催化ERAD的新因素，并确定与疾病相关的ERAD底物的潜在药物靶标。