Mechanistic analysis of post-translation membrane protein insertion into the ER.

翻译后膜蛋白插入内质网的机制分析。

• 批准号: 8450736
• 负责人: Vladimir Denic
• 金额: $ 30.33万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8450736
• 关键词: ATP phosphohydrolase; Address; Affinity; Animal Model; Apoptosis; Binding; Biogenesis; Cell physiology; Cells; Cisplatin; Complement; Complex; Data; Drug resistance; Drug usage; Endoplasmic Reticulum; Fluorescence Microscopy; Genetic; Goals; Homologous Gene; Human; Image; In Vitro; Light; Lipid Bilayers; Location; Malignant Neoplasms; Membrane; Membrane Proteins; Mitochondria; Molecular Chaperones; Molecular Conformation; Molecular Machines; Monitor; Mutation; Nucleotides; Outer Mitochondrial Membrane; Pathway interactions; Pharmaceutical Preparations; Play; Point Mutation; Protein Binding; Protein translocation; Proteins; Recruitment Activity; Regulation; Resistance; Role; Saccharomycetales; Signal Transduction; Sorting - Cell Movement; Specific qualifier value; Structure; System; Tail; Testing; Translations; Transmembrane Domain; Work; chemotherapy; dimer; improved; in vivo; insight; interest; mutant; novel; protein complex; proteoliposomes; reconstitution; research study; tool

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the mechanism of the Guided Entry of Tail-anchored proteins (GET) pathway. This pathway targets tail-anchored (TA) proteins destined for the secretory pathway for post-translational insertion into the endoplasmic reticulum (ER) membrane. The central component of this pathway is the chaperone Get3. We know that once Get3-TA protein complexes are formed they are recruited to the ER membrane for insertion. Beyond this conceptual framework, we still know very little about how Get3 finds its TA protein substrates and by what mechanism they are inserted into the ER membrane. To gain a deeper mechanistic understanding of the GET pathway, this project will determine (1) how an upstream chaperone complex that we identified delivers TA proteins with ER-targeting signals to Get3, and (2) how TA proteins associated with Get3 are inserted by two membrane components of the pathway that we have reconstituted into proteoliposomes. These studies will be performed in budding yeast because this model organism has facile genetics, is biochemically tractable, and cell biologically accessible to imaging using fluorescence microscopy. The proposed studies will reveal the mechanism of a conserved targeting pathway that enables the biogenesis of hundreds of TA proteins in human cells. From an academic standpoint, the GET pathway is interesting because it accurately distinguishes the membrane targeting signals of TA proteins destined for the secretory pathway form those of mitochondrial TA proteins. Furthermore, this pathway uses a novel membrane insertion machinery, distinct from the canonical Sec61 protein translocation channel, to integrate transmembrane domains into the ER lipid bilayer. From a practical standpoint, these studies will shed light on how tail-anchored Bcl2 proteins regulate apoptosis by partitioning between the ER and the mitochondrial outer membrane. Lastly, Asna1, the metazoan Get3 homolog, has emerged as a critical factor for cellular sensitivity to cisplatin. Thus, the proposed studies will inform pharmacological approaches for manipulating Asna1 activity to minimize drug resistance and enhance existing cisplatin chemotherapies.

描述（由申请人提供）：该项目的长期目标是阐明尾锚定蛋白引导进入（GET）途径的机制。该通路靶向尾锚定 (TA) 蛋白，该蛋白将进入分泌通路，用于翻译后插入内质网 (ER) 膜。该通路的核心成分是伴侣 Get3。我们知道，一旦 Get3-TA 蛋白复合物形成，它们就会被招募到内质网膜上进行插入。除了这个概念框架之外，我们对 Get3 如何找到其 TA 蛋白底物以及它们通过什么机制插入 ER 膜仍然知之甚少。为了更深入地了解 GET 通路的机制，该项目将确定 (1) 我们确定的上游伴侣复合物如何将具有 ER 靶向信号的 TA 蛋白递送至 Get3，以及 (2) 与 Get3 相关的 TA 蛋白如何被我们重构到脂蛋白体中的通路的两个膜成分插入。这些研究将在芽殖酵母中进行，因为这种模型生物具有简单的遗传学，在生化上易于处理，并且细胞在生物学上易于使用荧光显微镜成像。拟议的研究将揭示保守靶向途径的机制，该途径能够实现人体细胞中数百种 TA 蛋白的生物合成。从学术角度来看，GET 途径很有趣，因为它准确地区分了前往分泌途径的 TA 蛋白的膜靶向信号和线粒体 TA 蛋白的膜靶向信号。此外，该途径使用一种不同于典型 Sec61 蛋白易位通道的新型膜插入机制，将跨膜结构域整合到 ER 脂质双层中。从实践的角度来看，这些研究将揭示尾部锚定的 Bcl2 蛋白如何通过 ER 和线粒体外膜之间的分隔来调节细胞凋亡。最后，后生动物 Get3 同源物 Asna1 已成为细胞对顺铂敏感性的关键因素。因此，拟议的研究将为操纵 Asna1 活性的药理学方法提供信息，以最大限度地减少耐药性并增强现有的顺铂化疗。