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。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。 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内质网中蛋白质转位机制的组成部分。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。

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.