Biogenesis Of Secretory And Membrane Proteins

蛋白质生物发生单位研究哺乳动物内质网（ER）分泌和膜蛋白的合成、转运和成熟的调节机制。内质网上有一个复杂的大分子组装体，称为转座子，作为蛋白质传导通道，使底物进入分泌途径。正在进行的研究的主要目标是确定易位子的分子机制和组成部分，这些易位子识别其底物的初级序列中的信息，以介导其适当的载体运输，不对称拓扑形成和膜整合。通过描述正常与疾病相关的分泌蛋白和膜蛋白变异的生物合成过程中的步骤，关于早期分泌途径的特定疾病的分子基础的假设正在制定和体内测试。朊蛋白（PrP）是一种与多种神经退行性疾病有关的脑糖蛋白，已被证明是复杂和高度调控的易位的一个特别有指导意义的例子。先前的研究已经确定，PrP在内质网的生物发生是不寻常的，因为最初同质的新生PrP链产生了三种不同的拓扑形式：一种完全易位的形式（称为sec-PrP），以及两种跨膜形式，以相反的方向跨越膜（Ntm-PrP和Ctm-PrP）。体内研究表明，在小鼠模型系统和自然发生的人类疾病中，即使cm - prp拓扑形式的略微过度代表也会导致神经退行性疾病的发展。在过去的一年里，我们的工作重点是剖析将最初同质的新生PrP多肽引导成多种拓扑形式的机制，不仅可以深入了解某些形式的朊病毒疾病的机制，还可以深入了解分泌和膜蛋白生物发生的基本机制。

The Unit on Protein Biogenesis studies the mechanisms regulating the synthesis, translocation and maturation of secretory and membrane proteins at the mammalian endoplasmic reticulum (ER). A complex macromolecular assembly at the ER, termed the translocon, serves as a protein-conducting channel where substrates enter the secretory pathway. The principal goal of ongoing studies is to define the molecular mechanisms and components of the translocon that recognize the information in the primary sequence of its substrates to mediate their proper vectorial transport, asymmetric topogenesis, and membrane integration. By delineating the steps during the biosynthesis of normal versus disease-associated variants of secretory and membrane proteins, hypotheses regarding the molecular basis of particular diseases of the early secretory pathway are being formulated and tested in vivo. The prion protein (PrP), a brain glycoprotein involved in various neurodegenerative diseases, has proven to be a particularly instructive example of complex and highly regulated translocation. Previous studies have established that the biogenesis of PrP at the ER is unusual in that an initially homogeneous cohort of nascent PrP chains gives rise to three distinct topologic forms: a fully translocated form (termed sec-PrP), and two transmembrane forms that span the membrane in opposite orientations (Ntm-PrP and Ctm-PrP). In vivo studies have revealed that even a slight overrepresentation of the Ctm-PrP topologic form results in the development of neurodegenerative disease in both mouse model systems and naturally occurring human disease. During the past year, our efforts have been focused on dissecting the mechanisms that direct an initially homogeneous cohort of nascent PrP polypeptides into multiple topologic forms to gain insight into not only the mechanisms underlying certain forms of prion disease, but also the basic mechanisms of secretory and membrane protein biogenesis. We recently discovered that segregation of nascent PrP into different topologic forms is critically dependent on the precise timing of signal sequence-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated Ctm-PrP caused by disease-associated mutations in the transmembrane domain. Studies in transgenic mice are now being initiated to determine whether Ctm-PrP-mediated neurodegeneration can be averted by modulating this newly discovered step during PrP biogenesis. Parallel biochemical studies employing the solubilization, fractionation and reconstitution of ER membrane proteins have demonstrated that regulatory trans-acting factors are absolutely required for PrP to be synthesized in the proper ratio of its topologic forms. We have now purified two of these factors and identified them as the translocon-associated protein complex (TRAP) and protein disulfide isomerase (PDI). Analysis of PrP translocation intermediates suggests that TRAP and PDI act sequentially to facilitate translocation of PrP's N-terminus into the ER lumen, the decisive event in determining its topology. Ongoing studies are investigating the role of these newly discovered factors in the biogenesis of other substrates and their potential role in the pathogenesis of PrP-associated neurodegeneration.