拥挤环境中气味结合蛋白纤维化形成的调控

文献信息

中文摘要

具有β-桶拓扑结构的蛋白质的淀粉样聚集正日益被认为与人类病理过程相关。然而，密集堆积的细胞内和细胞外环境（大分子拥挤条件）对这些高度结构化蛋白质纤维化形成的影响仍研究不足。为填补这一空白，我们将分析重点放在气味结合蛋白（OBPs）上，因为近期研究表明其淀粉样生成与嗅觉功能障碍有关，尤其是在神经退行性疾病中。在牛OBP（bOBP）模型中，使用多种拥挤剂——聚乙二醇（PEG-8k和PEG-12k）和葡聚糖（Dex-15-25k和Dex-70k）——以及一系列全面的物理化学方法，证明了拥挤条件对β-桶蛋白纤维化形成具有阶段特异性和聚合物依赖性效应。具体而言，加入PEG缩短甚至消除了纤维化形成的滞后期，这是通过加速初级成核实现的。同时，所有拥挤剂均减弱了纤维化形成指数期的过程。拥挤剂的这些效应归因于：1）抑制β-桶的整体结构转变，以及2）增强已形成聚集体的簇集，从而抑制纤维断裂等次级过程。拥挤剂的联合作用减少了成熟淀粉样蛋白的形成，促进了高度疏水且具有细胞毒性的前纤维化前体的积累，并产生了结构异质性聚集体的簇。我们的发现揭示，大分子拥挤影响β-桶蛋白形成的淀粉样聚集体的纤维化形成轨迹和性质，在分析这些过程在体内的生物学后果时必须加以考虑。

英文摘要

Amyloid aggregation of proteins with β-barrel topology is increasingly being considered in relation to pathological processes in humans. Nevertheless, the influence of the densely packed intracellular and extracellular environments (macromolecular crowding conditions) on the fibrillogenesis of these highly structured proteins remains insufficiently studied. Addressing this gap, we focused our analysis on odorant-binding proteins (OBPs), as recent studies suggest the involvement of their amyloidogenesis in olfactory dysfunction, particularly in neurodegenerative diseases. Within the bovine OBP (bOBP) model, a stage-specific and polymer-dependent effect of crowding conditions on β-barrel protein fibrillogenesis was demonstrated using various crowding agents, polyethylene glycol (PEG-8k and PEG-12k) and dextran (Dex-15-25k and Dex-70k), and a comprehensive range of physicochemical methods. Specifically, the addition of PEGs shortened and even eliminated the lag phase of fibrillogenesis by accelerating primary nucleation. Concurrently, all crowding agents attenuated the processes of the exponential phase of fibrillogenesis. These effects of crowding agents were attributed to: 1) inhibition of global structural transformations in the β-barrel, and 2) enhanced clustering of formed aggregates, which suppresses secondary processes like fibril fragmentation. The combined action of crowding agents reduced mature amyloid formation, promoted the accumulation of highly hydrophobic and cytotoxic prefibrillar precursors, and generated clusters of structurally heterogeneous aggregates. Our findings reveal that macromolecular crowding influences the fibrillogenesis trajectory and the properties of amyloid aggregates formed from β-barrel proteins, which must be considered when analyzing the biological consequences of these processes in vivo.