Rotation 1: Computational design of antibodies targeting GABAA receptor subunits

第 1 轮：针对 GABAA 受体亚基的抗体的计算设计

• 批准号: 2643479
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2643479
• 关键词: Rotation; Computational; design; antibodies; targeting

BBSRC strategic theme: Bioscience for an integrated understanding of healthProtein misfolding and aggregation in vivo is associated with a wide range of human disorders including Alzheimer's disease, Parkinson's disease and systemic amyloidosis. The pathological hallmark of these diseases is amyloid fibrils that are formed when soluble proteins undergo multiple diverse and ever-changing conformations over time resulting in proteinaceous aggregates. As the world's population is ageing, the impact of "amyloid diseases" is an increasingly prevalent health concern; therefore, it is critical to understand the mechanisms by which disease-associated peptides and proteins behave aberrantly. Although the mechanisms of protein misfolding and aggregation are well studied in vitro using purified proteins and controlled conditions, this process occurs in a complex cellular environment that will highly influence protein misfolding and disease progression. Amyloid-B peptide (AB) deposits in the brain are a hallmark of Alzheimer's disease (AD), however, a large number of AD patients also develop cerebral amyloid angiopathy (CAA) with deposits of AB in the walls of cerebral arteries and capillaries. As well, studies have shown that in cognitively healthy subjects, high baseline plasma AB1-42 levels may be associated with increased future risk of AD; therefore, looking at factors within blood homeostasis that impacts on the behaviour of AB is important for understanding the disease in a cellular context. Platelets are the main cellular component of haemostasis. Upon stimulation, platelets expose phosphatidylserine (PS) and release PS-exposing extracellular vesicles (EVs) that circulate through the blood. The circulating level of these PS-exposing EVs is increased in many inflammatory and cardiovascular diseases. Intriguingly, artificial lipid membrane bilayers containing PS have been reported to affect the oligomerisation and fibril growth of AB. Our first research question therefore focuses on the EVs themselves: to what extent do these physiologically and pathologically relevant circulating EVs modulate the aggregation of disease-relevant proteins? Activated platelets also release a wide array of biomolecules. To date, several extracellular chaperones (ECs) such as clusterin and alpha-2-macroglobulin, have been isolated from blood plasma, are released by activated platelets, and found to potently inhibit AB aggregation. Interestingly, clusterin has been shown to decrease neuroinflammatory effects of AB aggregates. Our second research question therefore focuses on the factors released by platelets: do activated platelets release biomolecules that may act as enhancers or inhibitors of AB aggregation? We will isolate EVs from stimulated platelets and erythrocytes and characterise them by flow cytometry and biophysical techniques (such as dynamic light scattering). The impact of the isolated extracellular factors on protein aggregation will be assessed using fibril forming kinetics assays to gain (with the Kumita lab) and identification of EV components will be probed using immunoblotting and proteomics-based methods. Isolation and detailed biochemical studies of the inhibitory effects of putative modulators will be done, using cell-based assays to understand their impact on neuroinflammation caused by AB aggregates.

BBSRC战略主题：生物科学用于对健康的综合理解体内蛋白质错误折叠和聚集与广泛的人类疾病有关，包括阿尔茨海默病，帕金森病和系统性淀粉样变性。这些疾病的病理学标志是淀粉样蛋白原纤维，当可溶性蛋白质随时间经历多种多样且不断变化的构象时形成淀粉样蛋白原纤维，从而导致蛋白质聚集体。随着世界人口的老龄化，“淀粉样疾病”的影响是一个日益普遍的健康问题;因此，了解疾病相关肽和蛋白质行为异常的机制至关重要。尽管蛋白质错误折叠和聚集的机制在体外使用纯化的蛋白质和受控条件进行了充分研究，但该过程发生在复杂的细胞环境中，这将高度影响蛋白质错误折叠和疾病进展。淀粉样蛋白B肽（AB）在脑中的沉积是阿尔茨海默病（AD）的标志，然而，大量AD患者还发展为脑淀粉样蛋白血管病（CAA），其中AB在脑动脉和毛细血管壁中沉积。此外，研究表明，在认知健康的受试者中，高基线血浆AB 1 -42水平可能与AD的未来风险增加有关;因此，研究影响AB行为的血液稳态内的因素对于在细胞背景下理解疾病很重要。血小板是止血的主要细胞成分。在刺激时，血小板暴露磷脂酰丝氨酸（PS）并释放PS暴露的细胞外囊泡（EV），其通过血液循环。这些PS暴露EV的循环水平在许多炎症和心血管疾病中增加。有趣的是，含有PS的人工脂质膜双层已被报道影响AB的寡聚化和原纤维生长。因此，我们的第一个研究问题集中在EV本身：这些生理和病理相关的循环EV在多大程度上调节疾病相关蛋白质的聚集？活化的血小板还释放出大量的生物分子。迄今为止，已经从血浆中分离出几种细胞外伴侣（EC），如簇蛋白和α-2-巨球蛋白，它们由活化的血小板释放，并被发现有效地抑制AB聚集。有趣的是，clusterin已被证明可以降低AB聚集体的神经炎症作用。因此，我们的第二个研究问题集中在血小板释放的因子上：活化的血小板释放的生物分子是否可以作为AB聚集的增强剂或抑制剂？我们将从刺激的血小板和红细胞中分离EV，并通过流式细胞术和生物物理技术（如动态光散射）对其进行鉴定。分离的细胞外因子对蛋白质聚集的影响将使用原纤维形成动力学测定进行评估，以获得（与Kumita实验室一起），并使用免疫印迹和基于蛋白质组学的方法对EV组分的鉴定进行探索。将使用基于细胞的测定法对推定的调节剂的抑制作用进行分离和详细的生物化学研究，以了解它们对AB聚集体引起的神经炎症的影响。