Investigations on post-endoplasmic reticulum quality control mechanisms for prion protein aggregates

维持正确的蛋白质折叠对细胞生存至关重要，一些神经退行性疾病与错误折叠的蛋白质积累有关。其中包括Pron病，这是一种致命的动物和人类传染性疾病。它们是独一无二的，因为它们是由PrPc引起的，PrPc是一种病原体，只由细胞PrPc蛋白的一个错误折叠的异构体组成，称为PrPSc。 我的团队的长期目标是阐明神经退行性变的机制，并通过研究Pron与宿主细胞的相互作用和Pron蛋白的运输来发现抑制Pron繁殖和扩散的新靶点。我的研究计划的一个核心问题是，如何在神经细胞内控制PrPc的正确折叠。PrPC是一种位于细胞表面的糖蛋白。在这里，PrPc在脂筏中被发现，膜微区含有高含量的胆固醇和鞘磷脂。在循环和最终内化时，PrPc沿着内吞途径运输，在酸性囊泡中降解。 在哺乳动物细胞中，最具特征的蛋白质质量控制机制存在于内质网(ER)。然而，在急诊室之外会发生什么？是否存在类似的机制来确保ER后隔室的蛋白质质量控制，如高尔基网络？ 在我之前发表的工作中，我描述了一种新的PrPC的ER后质量控制机制。我证明了用萘脲化合物苏拉明处理培养的细胞可以诱导PrPc聚集。这些聚集体被保留在细胞内，并阻止它们向细胞表面的运输。相反，它们被从跨高尔基网络(TGN)重新路由到溶酶体进行降解。与致病PrPSc聚集体不同，它们是非传染性的，可以被细胞降解，半衰期类似于PrPc。我进一步的缺失突变分析表明，重新路由，而不是聚集，取决于PrP氨基末端的前八重复域(aa23-50)的存在。值得注意的是，其他人也对致病PrP突变体进行了类似的观察。这些突变体具有更高的聚集倾向，正如我所描述的药物诱导的PrP聚集体一样，它们在细胞内被重新路由到溶酶体进行降解。此外，蛋白酶Furin在聚集时受到ER后质量控制，聚集体被运输到溶酶体。这表明蛋白质聚集体从TGN重新路由到溶酶体是一种细胞机制，不仅与PrP运输有关。 与已被充分研究的ER蛋白质量控制机制相比，对哺乳动物细胞中ER蛋白后质量控制知之甚少。我提出的研究的目标是使用药物诱导的PrP聚集体作为模型，以获得对哺乳动物细胞ER后质量控制的新的机制见解。这将通过三个具体目标来实现： 1.通过sirna文库筛选鉴定参与内质网后质量控制的蛋白质 2.分析糖基化在识别PrP聚集体中的作用，以及 3.研究PrP在非药物处理的神经细胞中的聚集情况。 我期望对哺乳动物细胞中迄今未被研究的ER后质量控制机制有新的见解。我的研究结果将促进人们理解细胞对蛋白质错误折叠的能力，不仅是Pron蛋白质，而且是通过分泌途径传播的其他蛋白质。从长远来看，我对蛋白质错误折叠的基础研究将产生新的药物靶点，以改善蛋白质折叠，这将启发应用健康研究行业的翻译方法。

Sustaining correct protein folding is critically important for cell survival and several neurodegenerative disorders are associated with the accumulation of misfolded proteins. Among these are prion diseases, which are fatal transmissible disorders of animals and humans. They are unique because they are caused by prions, pathogens which are composed solely of a misfolded isoform of the cellular prion protein PrPc, termed PrPSc. The long-term goals of my group are to elucidate mechanisms of neurodegeneration and to discover novel targets for inhibiting prion propagation and spreading by studying prion-host cell interactions and trafficking of prion proteins. One central question of my research program is how correct folding of PrPc is controlled within neuronal cells. PrPc is a glycoprotein located at the cell surface. Here, PrPc is found in lipid rafts, membrane microdomains with a high content of cholesterol and sphingolipids. Upon recycling and final internalization, PrPc is transported along the endocytic pathway for degradation in acidic vesicles. In mammalian cells, the best characterized protein quality control machinery resides in the endoplasmic reticulum (ER). However, what happens beyond the ER? Are there similar mechanisms existing that ensure protein quality control in post-ER compartments, such as the Golgi network? In my previously published work I described a novel post-ER quality control mechanism for PrPc. I demonstrated that treatment of cultured cells with the naphthyl-urea compound suramin induces PrPc aggregation. These aggregates are intracellulary retained and their transport to the cell surface is prevented. Instead, they are re-routed from the trans-Golgi network (TGN) to lysosomes for degradation. In contrast to pathogenic PrPSc aggregates, they are non-infectious and can be degraded by the cells with a half-life similar to that of PrPc. My further deletion mutant analysis revealed that re-routing, but not aggregation, is dependent on the presence of the pre-octarepeat domain (aa 23-50) of the PrP amino-terminus. Of note, similar observations were made by others for pathogenic PrP mutants. These mutants had a higher propensity to aggregate and were, as the drug-induced PrP aggregates described by myself, intracellularly re-routed to lysosomes for degradation. Furthermore, the protease furin is subjected to post-ER quality control upon aggregation, and aggregates are transported to lysosomes. This indicates that re-routing of protein aggregates from TGN to lysosomes is a cellular mechanism not only relevant for PrP trafficking. In contrast to the well investigated ER protein quality control mechanisms, only little is known about post-ER protein quality control in mammalian cells. The goal of my proposed studies is to use drug-induced PrP aggregates as a model to gain novel mechanistic insights into post-ER quality control in mammalian cells. This will be achieved by three specific aims: 1. to identify proteins that are involved in post-ER quality control by siRNA library screens 2. to analyse the role of glycosylation in recognition of PrP aggregates, and 3. to investigate PrP aggregation in non-drug treated neuronal cells. I expect to obtain new insights into so far under-investigated post-ER quality control mechanisms in mammalian cells. Results obtained from my study will advance the understanding of the cellular capacity to counteract protein misfolding not only of the prion protein, but also of other proteins travelling through the secretory pathway. In a long term, my fundamental research on protein misfolding will result in novel drug targets to improve protein folding which will inspire translational approaches by the applied health research industry.