Biogenesis Of Secretory And Membrane Proteins

我们之前的研究表明，朊蛋白（PrP）进入内质网（ER）的初始易位改变可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，而且还展示了如何调节这一过程中的关键步骤来影响潜在神经毒性PrP形式的产生。特别是，我们已经发现PrP生物发生中最重要和最受严格调控的步骤是其信号序列与蛋白质转座子之间的相互作用。研究发现，这一步骤严重依赖于一种功能未知的四蛋白复合物（称为TRAP复合物），没有这种复合物，PrP就无法进入内质网。我们发现并非所有的信号序列都需要TRAP，这表明不同的底物被转座子识别的方式不同，最近分析信号序列和转座子组分之间交联的研究进一步支持了这一观点。更重要的是，我们现在已经表明，这种信号-转座子相互作用性质的改变对蛋白质定位和功能有重大影响。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-易位相互作用对于允许这种蛋白质存在于两个区室（内质网管和细胞质）至关重要，在那里它具有独立的功能。因此，过去一年的进展开始阐明一个潜在的细胞调控的新位点，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。

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.