Department of Neurology, Brain Research Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.
Analyst. 2023 May 16;148(10):2283-2294. doi: 10.1039/d3an00487b.
Deposition of aggregated proteins is a pathological feature in many neurodegenerative disorders such as Alzheimer's and Parkinson's. In addition to insoluble amyloid fibrils, protein aggregation leads to the formation of soluble oligomers, which are more toxic and pathogenic than fibrils. However, it is challenging to screen for inhibitors targeting oligomers due to the overlapping processes of oligomerization and fibrillization. Here we report a protein aggregation platform that uses intact and split TEM-1 β-lactamase proteins as reporters of protein aggregation. The intact β-lactamase fused with an amyloid protein can report the overall protein aggregation, which leads to loss of lactamase activity. On the other hand, reconstitution of active β-lactamase from the split lactamase construct requires the formation of amyloid oligomers, making the split lactamase system sensitive to oligomerization. Using Aβ, a protein that forms amyloid plaques in Alzheimer's disease, we show that the growth curves of bacterial cells expressing either intact or split lactamase-Aβ fusion proteins can report changes in the Aβ aggregation. The cell lysate lactamase activity assays show that the oligomer fraction accounts for 20% of total activity for the split lactamase-Aβ construct, but only 3% of total activity for the intact lactamase-Aβ construct, confirming the sensitivity of the split lactamase to oligomerization. The combination of the intact and split lactamase constructs allows the distinction of aggregation modulators targeting oligomerization from those targeting overall aggregation. These low-cost bacterial cell-based and biochemical assays are suitable for high-throughput screening of aggregation inhibitors targeting oligomers of various amyloid proteins.
聚集蛋白的沉积是许多神经退行性疾病（如阿尔茨海默病和帕金森病）的病理特征。除了不溶性淀粉样纤维外，蛋白质聚集还会导致可溶性寡聚物的形成，寡聚物比纤维更具毒性和致病性。然而，由于寡聚化和纤维化过程的重叠，筛选针对寡聚物的抑制剂具有挑战性。在这里，我们报告了一种蛋白质聚集平台，该平台使用完整和分裂的 TEM-1 内酰胺酶蛋白作为蛋白质聚集的报告蛋白。与淀粉样蛋白融合的完整内酰胺酶可以报告整体蛋白质聚集，这会导致内酰胺酶活性丧失。另一方面，从分裂的内酰胺酶构建体中重建有活性的内酰胺酶需要形成淀粉样寡聚物，这使得分裂的内酰胺酶系统对寡聚化敏感。使用 Aβ，一种在阿尔茨海默病中形成淀粉样斑块的蛋白质，我们表明表达完整或分裂的内酰胺酶-Aβ融合蛋白的细菌细胞的生长曲线可以报告 Aβ聚集的变化。细胞裂解物内酰胺酶活性测定表明，分裂的内酰胺酶-Aβ构建体的寡聚物部分占总活性的 20%，而完整的内酰胺酶-Aβ构建体的总活性仅占 3%，证实了分裂的内酰胺酶对寡聚化的敏感性。完整和分裂的内酰胺酶构建体的结合允许区分针对寡聚化的聚集调节剂与针对整体聚集的调节剂。这些基于廉价细菌细胞的生化测定法适合用于针对各种淀粉样蛋白的寡聚物的聚集抑制剂的高通量筛选。
