Secondary structure of the nascent chain as regulatory element for translocation into the endoplasmic reticulum; mechanisms and pathophysiological relevance

真核细胞胞浆中合成的所有蛋白质中约有25-30%被整合到质膜中或被分泌出来。膜整合和进入内质网（ER）管腔主要是由一种称为Sec61复合物的异三聚体膜蛋白复合物介导的，该复合物与细菌中的SecY复合物同源。分泌蛋白的主要部分含有n端信号肽，介导新生链转运到Sec61易位并启动转运进入内质网管。不能将分泌蛋白或膜蛋白运送到内质网可能会导致致命的后果。首先，这些蛋白质不能到达它们的最终目的地（失去它们的生理功能）；其次，它们可能形成具有毒性活性的细胞质聚集体，这是基于疏水结构域的存在（获得毒性功能）。例如，在转基因小鼠和哺乳动物细胞培养模型中，将细胞朊病毒蛋白错定位于细胞质可诱导神经元细胞死亡。因此，质量控制途径已经发展到消除错标蛋白。我们的工作揭示了真核生物/细菌的Sec61/SecY复合体的内在损伤，以转运完全非结构化的分泌蛋白。该研究计划的重点是详细了解多肽链的结构特征调节易位过程的机制。此外，将讨论有缺陷的ER输入的病理生理后果。具体而言，我们将研究使哺乳动物易位子易位具有扩展非结构化结构域的客户蛋白的途径，并研究应激和传播神经毒性蛋白构象的细胞中不受调节的易位。最后，我们将研究非易位分泌蛋白的命运，以评估未裂解信号肽对降解、随后的运输和细胞毒性活性的影响。实验方法包括体外分析，细菌，酵母和哺乳动物细胞培养模型，包括初级神经元，以及神经退行性疾病的小鼠模型。

Around 25-30% of all proteins synthesized in the cytosol of eukaryotic cells are integrated into the plasma membrane or are secreted. Membrane integration and import into the lumen of the endoplasmic reticulum (ER) is mainly mediated by a heterotrimeric membrane protein complex called Sec61 complex, which is homologous to the SecY complex in bacteria. A major fraction of secretory proteins contains N-terminal signal peptides that mediate transport of the nascent chain to the Sec61 translocon and initiate translocation into the ER lumen. Failure to deliver secretory or membrane proteins to the ER can have fatal consequences. First, these proteins do not reach their final destination (loss of their physiological function) and second, they may form cytosolic aggregates with toxic activities based on the presence of hydrophobic domains (gain of toxic function). For example, mislocalization of the cellular prion protein to the cytosol can induce neuronal cell death in transgenic mice and mammalian cell culture models. Thus, quality control pathways have evolved to eliminate mistargeted proteins. Our work revealed an intrinsic impairment of the Sec61/SecY complex of eukaryotes/bacteria to translocate secretory proteins that are entirely unstructured. The research proposal focusses on a detailed understanding of the mechanisms by which structural features of the polypeptide chain modulate the translocation process. In addition, pathophysiological consequences of a defective ER import will be addressed. Specifically, we will investigate pathways that enable the mammalian translocon to translocate client proteins with extended unstructured domains and study a deregulated translocation during stress and in cells propagating neurotoxic protein conformers. Finally, we will investigate the fate of non-translocated secretory proteins to evaluate the impact of an uncleaved signal peptide on degradation, subsequent trafficking and cytotoxic activity. The experimental approaches include in vitro assays,bacterial, yeast and mammalian cell culture models, including primary neurons, and mouse models of neurodegenerative diseases.