Biogenesis Of Secretory And Membrane Proteins

分泌蛋白和膜蛋白的生物发生

• 批准号: 7210515
• 负责人: Ramanujan S Hegde
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7210515
• 关键词: calreticulin; cell component structure /function; chemical models; cryoelectron microscopy; endoplasmic reticulum; genetically modified animals; intracellular transport; laboratory mouse; membrane proteins; molecular assembly /self assembly; prions; protein biosynthesis; protein localization; protein protein interaction; protein signal sequence; protein transport; ribosomes; secretory protein; transport inhibitor

Our previous studies have demonstrated that alterations in the initial translocation of the Prion protein (PrP) into the endoplasmic reticulum (ER) can lead to the development of neurodegenerative disease. During the past year, my group has made significant progress towards not only providing a molecular description of PrP translocation, but demonstrating how key steps during this process can be modulated to influence the generation of potentially neurotoxic forms of PrP. In particular, we have discovered that the most important and tightly regulated step in PrP biogenesis is the interaction between its signal sequence and the protein translocon. This step was found to be critically dependent on a four protein complex of previously unknown function (termed the TRAP complex), in the absence of which PrP does not enter the ER. Our finding that not all signal sequences require TRAP suggests that different substrates are recognized differently by the translocon, an idea further supported by recent studies analyzing crosslinking between signal sequences and translocon components. More significantly, we have now shown that alterations in the nature of this signal-translocon interaction have substantial consequences for protein localization and function. In the case of PrP, the cellular burden of potentially cytotoxic forms can be reduced (or enhanced) to change the susceptibility of cells to otherwise harmful insults. In the case of another protein, Calreticulin, we find that signal-translocon interactions are critical in allowing this protein to exist in two compartments (the ER lumen and the cytosol), where it serves independent functions. Thus, advances during the past year are beginning to illuminate a novel site of potential cellular regulation, the entry of secretory and membrane protein substrates into the mammalian secretory pathway, that impacts both normal physiology and disease progression. Most recently, these insights have been applied to uncover the ways in which protein entry into the ER is modulated productively by the cell under conditions of stress. In parallel collaborative studies, we are using both physiological, structural, and pharmacological approaches to understand components of the protein translocation machinery at the mammalian ER. In the physiological approach, we are using transgenic mice to investigate the consequences for neurodegenerative disease progression of modulating translocation of PrP. Parallel studies are examining the role of cytosolic calreticulin in mice models. In the structural approach, we are applying cryo-electron microscopy to visualize intact ribosome-translocon complexes. By preparing and analyzing translocon complexes lacking or containing specific components such as the TRAP complex, we are able to determine the relative positions of the various proteins comprising the translocon. In the pharmacologic approach, we are utilizing novel assays for translocation to identify, characterize, and study small molecule inhibitors of protein translocation. The goal of these studies is to develop probes that facilitate the modulation of protein translocation in vivo to understand the role of this process in normal and pathological cellular physiology.

我们以前的研究表明，prion蛋白（PRP）最初转运为内质网（ER）的改变会导致神经退行性疾病的发展。在过去的一年中，我的小组不仅在提供PRP易位的分子描述方面取得了重大进展，而且还证明了如何调节此过程中的关键步骤以影响潜在的神经毒性形式的PRP。特别是，我们发现PRP生物发生中最重要，最严格的步骤是其信号序列与蛋白质转运之间的相互作用。在没有PRP不进入ER的情况下，发现此步骤严重取决于先前未知功能的四蛋白复合物（称为陷阱复合物）。我们的发现，并非所有信号序列都需要陷阱都表明易位对不同的底物识别不同，这一想法是通过最近的研究进一步支持的想法，该研究分析了信号序列和转运成分之间的交联。更重要的是，我们现在已经表明，这种信号跨环节相互作用的性质改变对蛋白质定位和功能具有实质性后果。在PRP的情况下，可以减少（或增强）潜在的细胞毒性形式的细胞负担，以改变细胞对其他有害损伤的敏感性。对于另一种蛋白质，钙网蛋白，我们发现信号 - 跨洛肯相互作用对于允许该蛋白质存在于两个隔室（ER Lumen和cytosol）中至关重要，它具有独立的功能。因此，过去一年的进步开始阐明一个潜在的细胞调节的新部位，分泌蛋白和膜蛋白底物进入哺乳动物分泌途径，这会影响正常的生理和疾病进展。最近，这些见解已应用于揭示蛋白质进入ER的方式在压力条件下通过细胞有效地调节蛋白质的方式。 在平行的协作研究中，我们同时使用生理，结构和药理方法来了解哺乳动物ER蛋白质易位机械的成分。在生理方法中，我们正在使用转基因小鼠研究PRP调节易位的神经退行性疾病进展的后果。平行研究正在研究胞质钙网蛋白在小鼠模型中的作用。在结构方法中，我们正在应用冷冻电子显微镜来可视化完整的核糖体 - 透射配合物。通过准备和分析缺少或包含特定成分（例如陷阱复合物）的转运络合物，我们能够确定包含转运的各种蛋白质的相对位置。在药理学方法中，我们正在利用新颖的测定法进行易位来识别，表征和研究蛋白质易位的小分子抑制剂。这些研究的目的是开发探针，以促进体内蛋白质易位的调节，以了解该过程在正常和病理细胞生理学中的作用。