Regulation of Protein Translocation into the ER

该项目旨在了解新合成的分泌蛋白和膜蛋白是如何在细胞中合成、成熟、分类和代谢的。这类蛋白质对所有细胞间和细胞内通讯都是必不可少的，它们的精确位置和丰度受到严格调控，以维持正常的细胞和生物生理学。事实上，目前大多数药物靶点影响分泌蛋白和膜蛋白，强调了它们在人类生物学中的核心作用。我们的目标是在分子水平上了解分泌和膜蛋白的生物合成和代谢途径。我们不仅对这些正常的细胞事件感兴趣，而且还想发现它们在某些疾病状态下受到干扰的方式。 以哺乳动物朊蛋白（PrP）的生物合成为模型系统，我们发现PrP最初分离到内质网（ER）的过程中，最重要和最受严格调控的步骤是PrP信号序列与蛋白质转位子之间的相互作用。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。在其他蛋白质（钙网蛋白和p58-IPK）的情况下，我们发现，信号translocon相互作用是至关重要的，允许这些蛋白质存在于两个隔室（ER腔和胞质溶胶），在那里他们可能提供独立的功能。 因此，在过去几年中的进展开始阐明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。最近，这些见解已被应用于揭示蛋白质进入内质网的方式是由细胞在应激条件下有效地调节。这种分析导致了一种新的降解途径的发现，我们称之为先发制人的质量控制（或pQC）。促进pQC和降解机制的机制，目前正在研究的生化系统，重演这些事件在体外。 除了机械功能的研究，我们正在从事合作研究，以深入了解哺乳动物ER易位的结构特征。冷冻电子显微镜方法正在与生化重建相结合，以确定含有不同组分或在不同功能状态下被捕的translocon复合物的结构。 最后，平行的合作研究正在使用体内系统来了解调节特定蛋白质易位到哺乳动物ER的生理重要性。在一个项目中，我们正在研究小鼠模型中易位调节产生的胞浆钙网蛋白的作用。在另一个项目中，我们正在研究PrP的蛋白质易位在与该蛋白质相关的各种疾病中所起的作用。

This project seeks to understand how newly synthesized secretory and membrane proteins are made, matured, sorted, and metabolized in cells. This class of proteins is essential to all intercellular and intracellular communication, and their precise locations and abundances are tightly regulated to maintain normal cellular and organismal physiology. Indeed, the majority of current drug targets affect secreted and membrane proteins, underscoring their central role in human biology. Our goals are to develop a molecular level understanding of the pathways of secretory and membrane protein biosynthesis and metabolism. Not only are we interested in these normal cellular events, but also in discovering the ways they are perturbed in certain disease states. Using the biosynthesis of mammalian Prion protein (PrP) as a model system, we have discovered that the most important and tightly regulated step in its initial segregatin to the endoplasmic reticulum (ER) 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 other proteins (Calreticulin and p58-IPK), we find that signal-translocon interactions are critical in allowing these proteins to exist in two compartments (the ER lumen and the cytosol), where they could potentially serve independent functions. Thus, advances during the past few years 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. This analysis has led to the discovery of a new degradation pathway we have termed pre-emptive quality control (or pQC). The mechanisms that facilitate pQC and the degradative machinery are currently being studied in biochemical systems that recapitulate these events in vitro. In addition to mechanistic functional studies, we are engaged in collaborative studies to gain insight into the structural features of the mammalian ER translocon. Cryo-electron microscopy approaches are being combined with biochemical reconstitution to determine the structures of translocon complexes containing different components or arrested in different functional states. And finally, parallel collaborative studies are using in vivo systems to understand the physiologic importance of regulating translocation of specific proteins into the mammalian ER. In one project, we are examining the role of cytosolic calreticulin generated by translocation regulation in mice models. In another project, we are investigating the role that protein translocation of PrP plays in the various diseases associated with this protein.