R&D, Orion Pharma, Orion Corporation, Turku, Finland.
Faculty of Medicine, Institute of Biomedicine, University of Turku, Turku, Finland.
PLoS One. 2023 Aug 17;18(8):e0290179. doi: 10.1371/journal.pone.0290179. eCollection 2023.
Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.
凝胶蛋白片段的聚集是遗传性系统性疾病——肌联蛋白淀粉样变性的标志。与其他蛋白质错误折叠疾病一样，迫切需要针对肌联蛋白淀粉样变性的有效疾病修饰治疗方法。通过在体外孵育致病的淀粉样变性 8 kDa 多肽、70 个残基的肌联蛋白片段、AGelD187N 173-242，可以复制淀粉样的形成，并通过硫黄素 T 染料监测该过程。然而，对于筛选潜在的聚集抑制剂，所需的蛋白量很大，并且生物技术生产淀粉样蛋白具有许多挑战。相反，使用较短的 AGelD187N 173-242 合成区域并不能模拟全长片段的体内聚集动力学，因为它们具有不同的聚集倾向。在这项研究中，我们提出了一种体外全长 AGelD187N 173-242 聚集测定法，该方法通过固相化学合成并经过仔细单体化来制备。化学合成使我们能够高效且低成本地生产大量全长片段。我们证明了所产生的聚集体具有纤维状性质，以及纯度、末端修饰、初始聚集体和接种如何影响合成肌联蛋白片段的聚集动力学。我们还为初始单体化合成多肽提供了足够的质量标准。
