Institute of Protein Research RAS, 142290 Pushchino, Russia.
Biomolecules. 2021 Oct 30;11(11):1608. doi: 10.3390/biom11111608.
The development of many severe human diseases is associated with the formation of amyloid fibrils. Most of the available information on the process of amyloid formation has been obtained from studies of small proteins and peptides, wherein the features of complex proteins' aggregation remain insufficiently investigated. Our work aimed to research the amyloid aggregation of a large model protein, bovine carbonic anhydrase B (BCAB). It has previously been demonstrated that, when exposed to an acidic pH and elevated temperature, this protein forms amyloid fibrils. Here, we show that, under these conditions and before amyloid formation, BCAB undergoes fragmentation by acid hydrolysis to give free individual peptides and associated peptides. Fragments in associates contain a pronounced secondary structure and act as the main precursor of amyloid fibrils, wherein free peptides adopt mostly unstructured conformation and form predominantly irregular globular aggregates. Reduced acidity decreases the extent of acid hydrolysis, causing BCAB to form amorphous aggregates lacking the thioflavin T binding β-structure. The presented results provide new information on BCAB amyloid formation and show the importance of protein integrity control when working even in mildly acidic conditions.
许多严重人类疾病的发展都与淀粉样纤维的形成有关。大多数关于淀粉样形成过程的现有信息都是从小蛋白和肽的研究中获得的，而复杂蛋白聚集的特征仍未得到充分研究。我们的工作旨在研究大型模型蛋白牛碳酸酐酶 B (BCAB)的淀粉样聚集。先前已经证明，当暴露于酸性 pH 值和高温时，该蛋白会形成淀粉样纤维。在这里，我们表明，在这些条件下并且在形成淀粉样纤维之前，BCAB 通过酸水解进行片段化，得到游离的单个肽和相关肽。在相关物中的片段含有明显的二级结构，并且作为淀粉样纤维的主要前体，其中游离肽主要采用无规卷曲构象，并且形成主要为不规则的球形聚集物。降低酸度会降低酸水解的程度，导致 BCAB 形成缺乏硫黄素 T 结合β-结构的无定形聚集物。所呈现的结果提供了有关 BCAB 淀粉样形成的新信息，并表明即使在温和酸性条件下工作时，控制蛋白质完整性的重要性。
