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.

我们以前的研究已经证明，在初始易位的朊蛋白（PrP）到内质网（ER）的改变可以导致神经退行性疾病的发展。在过去的一年中，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这一过程中的关键步骤，以影响潜在神经毒性形式的PrP的产生。特别是，我们已经发现，在PrP生物合成中最重要和严格调控的步骤是其信号序列和蛋白质易位之间的相互作用。这一步骤被发现是严重依赖于一个四蛋白复合物的功能以前未知（称为TRAP复合物），在没有PrP不进入ER。我们的发现，不是所有的信号序列需要TRAP表明，不同的基板被识别不同的translocon，最近的研究分析信号序列和translocon组件之间的交联进一步支持的想法。更重要的是，我们现在已经表明，这种信号-易位子相互作用的性质的改变对蛋白质的定位和功能有重大影响。在PrP的情况下，可以降低（或增强）潜在细胞毒性形式的细胞负荷，以改变细胞对其他有害损伤的易感性。在另一种蛋白质，钙网蛋白的情况下，我们发现，信号translocon的相互作用是至关重要的，允许这种蛋白质存在于两个隔室（ER腔和胞质溶胶），在那里它提供独立的功能。因此，在过去的一年中的进展开始照亮一个新的网站的潜在的细胞调节，进入哺乳动物分泌途径的分泌和膜蛋白底物，影响正常的生理和疾病的进展。 在平行的合作研究中，我们正在使用结构和药理学方法来了解哺乳动物ER的蛋白质易位机制。在结构方法中，我们应用冷冻电子显微镜来观察完整的核糖体-转位子复合物。通过制备和分析缺乏或含有特定组分的易位子复合物，例如TRAP复合物，我们能够确定包含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检测方法来鉴定、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探针，促进体内蛋白质易位的调节，以了解这一过程在正常和病理细胞生理学中的作用。