Biogenesis Of Secretory And Membrane Proteins

分泌蛋白和膜蛋白的生物发生

• 批准号: 6993728
• 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/6993728
• 关键词: calreticulin; cell component structure /function; cryoelectron microscopy; endoplasmic reticulum; 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. In parallel collaborative studies, we are using both structural and pharmacological approaches to understand components of the protein translocation machinery at the mammalian ER. 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.

我们之前的研究表明，朊病毒蛋白（PrP）进入内质网（ER）的初始易位的改变可能导致神经退行性疾病的发生。在过去的一年里，我的团队取得了重大进展，不仅提供了 PrP 易位的分子描述，而且展示了如何调节该过程中的关键步骤以影响潜在神经毒性形式 PrP 的生成。特别是，我们发现 PrP 生物发生中最重要且受严格调控的步骤是其信号序列与蛋白质易位子之间的相互作用。发现这一步骤严重依赖于先前未知功能的四蛋白复合物（称为 TRAP 复合物），如果没有该复合物，PrP 就不会进入 ER。我们发现并非所有信号序列都需要 TRAP，这表明易位子对不同底物的识别方式不同，最近分析信号序列和易位子成分之间交联的研究进一步支持了这一观点。更重要的是，我们现在已经证明，这种信号-易位子相互作用性质的改变对蛋白质定位和功能具有重大影响。就 PrP 而言，可以减少（或增强）潜在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。就另一种蛋白质钙网蛋白而言，我们发现信号转位子相互作用对于允许该蛋白质存在于两个区室（内质网腔和胞质溶胶）中至关重要，在两个区室中它发挥独立的功能。因此，过去一年的进展开始阐明潜在细胞调节的新位点，即分泌和膜蛋白底物进入哺乳动物分泌途径，影响正常生理和疾病进展。 在并行合作研究中，我们正在使用结构和药理学方法来了解哺乳动物内质网蛋白质易位机制的组成部分。在结构方法中，我们应用冷冻电子显微镜来可视化完整的核糖体-易位子复合物。通过制备和分析缺乏或含有特定成分（例如TRAP复合物）的易位子复合物，我们能够确定构成易位子的各种蛋白质的相对位置。在药理学方法中，我们正在利用新的易位测定法来识别、表征和研究蛋白质易位的小分子抑制剂。这些研究的目标是开发促进体内蛋白质易位调节的探针，以了解该过程在正常和病理细胞生理学中的作用。