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

朊病毒蛋白聚集体后内质网质量控制机制的研究

• 批准号: RGPIN-2014-04839
• 负责人: Gilch, Sabine
• 金额: $ 2.54万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=651878
• 关键词: Investigations; post; endoplasmic; reticulum; quality

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.

维持正确的蛋白质折叠对于细胞存活至关重要，并且几种神经退行性疾病与错误折叠蛋白质的积累有关。其中包括朊病毒疾病，这是动物和人类的致命传染性疾病。它们是独特的，因为它们是由朊病毒引起的，朊病毒是仅由称为PrPSc的细胞朊病毒蛋白PrPc的错误折叠同种型组成的病原体。* 我们小组的长期目标是阐明神经变性的机制，并通过研究朊病毒-宿主细胞相互作用和朊病毒蛋白的运输来发现抑制朊病毒繁殖和扩散的新靶点。我的研究计划的一个中心问题是如何正确的折叠PrPc是控制在神经元细胞内。PrPc是位于细胞表面的糖蛋白。在这里，PrPc被发现在脂筏，膜微区与高含量的胆固醇和鞘脂。在再循环和最终内化后，PrPc沿着内吞途径转运，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位于内质网（ER）中。然而，在急诊室之外会发生什么？是否存在类似的机制，确保蛋白质质量控制后ER隔室，如高尔基体网络？* 在我以前发表的工作中，我描述了一种新的ER后PrPc质量控制机制。我证明了用萘基脲化合物苏拉明处理培养的细胞会诱导PrPc聚集。这些聚集体被保留在细胞内，并被阻止转运到细胞表面。相反，它们从trans-Golgi网络（TGN）重新路由到溶酶体进行降解。与致病性PrPSc聚集体相反，它们是非感染性的，并且可以被细胞降解，其半衰期与PrPc相似。我进一步的缺失突变体分析表明，重新路由，但不聚集，是依赖于前octarepeat结构域（aa 23-50）的PrP氨基末端的存在。值得注意的是，其他人也对致病性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，并且如我所描述的药物诱导的PrP聚集体，在细胞内重新路由到溶酶体进行降解。此外，蛋白酶弗林蛋白酶在聚集后进行ER后质量控制，并且聚集体被转运至溶酶体。这表明蛋白质聚集体从TGN到溶酶体的重新路由是一种不仅与PrP运输相关的细胞机制。* 相反，充分研究ER蛋白质的质量控制机制，只有很少有人知道后ER蛋白质的质量控制在哺乳动物细胞。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，以获得新的机制的见解后ER在哺乳动物细胞中的质量控制。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ER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元细胞中的聚集。我希望获得新的见解，到目前为止，在哺乳动物细胞中的ER后质量控制机制研究不足。从我的研究中获得的结果将推进细胞的能力，以抵消蛋白质错误折叠不仅朊病毒蛋白，但也通过分泌途径旅行的其他蛋白质的理解。从长远来看，我对蛋白质错误折叠的基础研究将产生改善蛋白质折叠的新型药物靶点，这将激发应用健康研究行业的转化方法。