Biogenesis Of Secretory And Membrane Proteins

我们以前的研究已经证明，Prion蛋白(PrP)进入内质网(ER)的初始移位的改变可以导致神经退行性疾病的发展。在过去的一年里，我的团队不仅在提供PrP易位的分子描述方面取得了重大进展，而且还展示了这一过程中的关键步骤如何被调节以影响潜在的神经毒性形式PrP的产生。特别是，我们发现PrP生物发生中最重要和最严格调控的步骤是其信号序列与蛋白质转位蛋白之间的相互作用。这一步骤被发现严重依赖于一个功能未知的四蛋白复合体(称为TRAP复合体)，如果没有它，PrP就不会进入内质网。我们的发现并不是所有的信号序列都需要陷阱，这表明转运子识别不同的底物是不同的，这一观点得到了最近分析信号序列和转移子成分之间的交联性的研究的进一步支持。 更重要的是，我们现在已经证明，这种信号-转运子相互作用的性质的改变对蛋白质的定位和功能有实质性的影响。在PrP的情况下，潜在的细胞毒性形式的细胞负担可以减少(或增强)，以改变细胞对其他有害侮辱的敏感性。更值得注意的是，通过改变信号-转位蛋白的相互作用，可以加速或减弱由病毒蛋白引起的小鼠神经退行性变。在另一种蛋白质Calreticrin的情况下，我们发现信号-转位相互作用对于使该蛋白质存在于两个隔室(内质网管腔和胞浆)至关重要，在两个隔室中，它发挥着独立的功能。因此，过去一年的进展开始阐明一个潜在的细胞调节的新位置，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。最近，这些洞察力被应用于揭示细胞在应激条件下如何有效地调节蛋白质进入内质网的方式。这一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质量控制(或pQC)。促进pQC的机制和降解机制目前正在研究中。 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内质网中蛋白质转位机制的组成部分。在生理学方面，我们正在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。平行研究正在检测胞浆钙网蛋白在小鼠模型中的作用。在结构方法上，我们正在应用冷冻电子显微镜来观察完整的核糖体-转位复合体。通过制备和分析缺少或含有特定成分的转位子复合体，如TRAP复合体，我们能够确定组成转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分析方法来识别、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转位调节的探针，以了解这一过程在正常和病理细胞生理学中的作用。 我们还对疾病相关PrP突变体的生物合成、运输和代谢进行了系统分析。这些研究的目的是准确地确定启动疾病过程的PrP错误折叠的细胞位置和机制。我们目前的分析已经将事件缩小到ER后的位置，这一发现值得注意，因为它是分泌和膜蛋白使用的细胞质量控制的主要位置。在平行研究中，正在研究PrP错误折叠和聚集的下游后果，以确定这些事件导致细胞功能障碍的机制。我们现在发现，这些聚集体招募了各种细胞因子，从而耗尽了它们的功能可用性。其中一个因素特别重要，因为它在小鼠身上的破坏直接导致神经退化表型，让人想起PrP引起的疾病。 最后，我们一直在研究膜蛋白插入内质网的分子机制和机制。我们现在已经确定了一种新的靶向因子，它高度保守，表达广泛，在插入一大类具有重要生理意义的膜蛋白中起着关键作用。该因子似乎与细胞质中新生的膜蛋白底物相互作用，并将它们传递到内质网膜上的一个尚未确定的受体。该因子的作用机制和该途径中其他成分的鉴定目前正在研究中。

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.