Topological regulation of transmembrane proteins through Regulated Alternative Translocation

通过调节选择性易位对跨膜蛋白进行拓扑调节

• 批准号: 10396119
• 负责人: JIN YE
• 金额: $ 41万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10396119
• 关键词: Adopted; Basic Science; CCR5 gene; Ceramides; Data; Integral Membrane Protein; Mammalian Cell; Measures; Mediating; Membrane; Pharmaceutical Preparations; Physiological; Process; Protein translocation; Proteins; Proteome; Reaction; Regulation; Reporting; Ribosomes; Signal Transduction; Sphingolipids; Structure; Testing; Translations; combat; human disease; improved; insight; novel; pathogen; receptor; sensor

Summary Transmembrane proteins must adopt proper membrane topology to perform their function. In mammalian cells, the topology of transmembrane proteins is determined by the direction through which transmembrane helices are inserted into ER during their translation on ER-associated ribosomes. It has been assumed that protein translocation across the ER membranes is a constitutive process so that transmembrane proteins must adopt a fixed topology. This assumption has been challenged by our recent observation that the direction through which transmembrane helices are inserted into the ER can be reversed under certain physiological conditions. We reported that ceramide inverted the topology of two polytopic transmembrane proteins, namely TM4SF20 and CCR5. Since this regulatory mechanism does not flip transmembrane proteins that have already been synthesized but inverts the topology of newly synthesized proteins by changing the direction through which transmembrane helices are translocated across membranes, we designated this process as Regulated Alternative Translocation (RAT). This project is initiated to further characterize RAT by delineating the mechanism of this topological regulation. We will begin by testing the hypothesis that TRAM2 is the ceramide sensor that interacts with the nascent transmembrane helices subjected to RAT. The approaches developed for this study can be generalized to identify ceramide interactome, a finding that might reveal more signaling reactions mediated by the sphingolipid. These approaches may also be applied unbiasedly to identify proteins interacting with the nascent transmembrane helices subjected to RAT, thereby providing more mechanistic insights into this novel translocation regulation. This project will also identify proteins subject to RAT by a novel proteome-wide approach capable of measuring topology of transmembrane proteins globally. Achieving this part of the project will not only reveal the breadth of RAT but also provide essential experimental data for proteome- wide assembly of topology of transmembrane proteins. Considering that only 10% of mammalian transmembrane proteins have their topology defined by experimental evidence, accomplishing this project should greatly improve our understanding of transmembrane proteins.

概括 跨膜蛋白必须采用适当的膜拓扑来执行其功能。在 哺乳动物细胞，跨膜蛋白的拓扑由通过方向通过 在ER相关的翻译过程中，将哪些跨膜螺旋插入ER中 核糖体。已经假定蛋白质跨ER膜的易位是 本构过程使跨膜蛋白必须采用固定拓扑。这个假设 我们最近的观察结果受到了挑战，即跨膜的方向 在某些生理条件下，将螺旋插入ER中。我们报道 该神经酰胺倒了两种多重跨膜蛋白的拓扑，即TM4SF20和 CCR5。由于这种调节机制不会翻转已经存在的跨膜蛋白 被合成但通过改变方向来颠倒新合成蛋白的拓扑 跨膜螺旋在跨膜之间易位，我们指定了 过程作为调节的替代易位（大鼠）。 该项目旨在通过描述此机制来进一步表征大鼠 拓扑调节。我们将首先测试TRAM2是神经酰胺传感器的假设 这与受到大鼠的新生跨膜螺旋相互作用。发展的方法 因为这项研究可以概括以识别Ceramide Interactome，这一发现可能揭示了更多 由鞘脂介导的信号反应。这些方法也可以公正地应用 鉴定蛋白质与受到大鼠的新生跨膜螺旋相互作用，从而 为这一新型易位调节提供更多的机械见解。 该项目还将通过新型蛋白质组方法鉴定受大鼠约束的蛋白质 能够测量全球跨膜蛋白的拓扑结构。实现项目的这一部分 不仅会揭示大鼠的广度，而且还为蛋白质组提供必不可少的实验数据 跨膜蛋白拓扑的广泛组装。考虑到只有10％的哺乳动物 跨膜蛋白具有实验证据来定义的拓扑，实现这一目标 项目应大大提高我们对跨膜蛋白的理解。