Biogenesis Of Secretory And Membrane Proteins

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。更值得注意的是，在小鼠中，朊病毒蛋白引起的神经变性的进展可以通过改变信号-转运子相互作用而加速或减弱。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。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）。目前正在研究促进pQC和降解机制的机制。 在平行的合作研究中，我们正在使用生理，结构和药理学的方法来了解哺乳动物ER的蛋白质易位机制的组成部分。在生理学方法中，我们使用转基因小鼠来研究调节PrP易位对神经退行性疾病进展的影响。平行研究正在检查细胞溶质钙网蛋白在小鼠模型中的作用。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。 我们还进行了系统的生物合成，贩运和疾病相关的PrP突变体的代谢分析。这些研究的目的是精确地确定PrP错误折叠启动疾病过程的细胞位置和机制。我们目前的分析已经将事件缩小到ER后的位置，这一发现是值得注意的，因为它是在分泌和膜蛋白使用的细胞质量控制的主要位点之后。在平行研究中，PrP错误折叠和聚集的下游后果正在研究中，以确定这些事件导致细胞功能障碍的机制。我们现在已经发现，这些聚集体募集各种细胞因子，从而耗尽它们的功能可用性。一个这样的因素是特别重要的，因为它在小鼠中的破坏直接导致神经退行性表型，让人想起由PrP引起的疾病。 最后，我们一直在研究膜蛋白插入ER膜的分子机制和机制。我们现在已经确定了一种新的靶向因子，该因子高度保守，广泛表达，并且在插入一大类生理上重要的膜蛋白中起关键作用。该因子似乎与胞质溶胶中的新生膜蛋白底物相互作用，并将其递送至ER膜上尚未鉴定的受体。目前正在研究该因子的作用机制以及该途径中其他成分的鉴定。

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. More remarkably, the progression of neurodegeneration caused by the prion protein in mice can be either accelerated or attenuated by alterations in the signal-translocon interaction. 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. 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 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. We are also performing a systematic analysis of the biosynthesis, trafficking, and metabolism of disease-associated PrP mutants. The aim of these studies is to identify precisely the cellular locale and mechanism of PrP misfolding that initiates the disease process. Our current analyses have narrowed the event to a post-ER location, a finding that is notable because it is after the principal site of cellular quality control used by secretory and membrane proteins. In parallel studies, the downstream consequences of PrP misfolding and aggregation are being studied to identify the mechanism by which these events lead to cellular dysfunction. We have now found that these aggregates recruit various cellular factors, therby depleting their functional availability. One such factor is of particular importance because its disruption in mice leads directlyt to a neurodegenerative phenotype reminiscent of diseases caused by PrP. And finally, we have been investigating the molecular mechanisms and machinery for the insertion of a membrane proteins into the ER membrane. We have now identified a novel targeting factor that is highly conserved, broadly expressed, and plays a key role in the insertion of a large class of physiologically important membrane proteins. This factor appears to interact with nascent membrane protein substrates in the cytosol and delivers them to a yet unidentified receptor at the ER membrane. The mechanisms by which this factor operates and the identification of additional components in this pathway are currently under investigation.