Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, H-1117 Budapest, Hungary.
ELTE-Functional Nucleic Acid Motifs Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, H-1117 Budapest, Hungary.
Int J Mol Sci. 2023 Aug 29;24(17):13403. doi: 10.3390/ijms241713403.
The aggregation and amyloid formation of α-synuclein is associated with Parkinson's disease and other synucleinopathies. In its native, monomeric form α-synuclein is an intrinsically disordered protein represented by highly dynamic conformational ensembles. Inhibition of α-synuclein aggregation using small molecules, peptides, or proteins has been at the center of interest in recent years. Our aim was to explore the effects of cross-linking on the structure and aggregation/amyloid formation properties of α-synuclein. Comparative analysis of available high-resolution amyloid structures and representative structural models and MD trajectory of monomeric α-synuclein revealed that potential cross-links in the monomeric protein are mostly incompatible with the amyloid forms and thus might inhibit fibrillation. Monomeric α-synuclein has been intramolecularly chemically cross-linked under various conditions using different cross-linkers. We determined the location of cross-links and their frequency using mass spectrometry and found that most of them cannot be realized in the amyloid structures. The inhibitory potential of cross-linked proteins has been experimentally investigated using various methods, including thioflavin-T fluorescence and transmission electron microscopy. We found that conformational constraints applied by cross-linking fully blocked α-synuclein amyloid formation. Moreover, DTSSP-cross-linked molecules exhibited an inhibitory effect on the aggregation of unmodified α-synuclein as well.
α-突触核蛋白的聚集和淀粉样形成与帕金森病和其他突触核蛋白病有关。在其天然的单体形式中，α-突触核蛋白是一种固有无序的蛋白质，由高度动态的构象集合表示。近年来，使用小分子、肽或蛋白质抑制α-突触核蛋白聚集一直是研究的重点。我们的目的是探索交联对α-突触核蛋白结构和聚集/淀粉样形成特性的影响。对现有高分辨率淀粉样结构和代表性结构模型以及单体α-突触核蛋白的 MD 轨迹的比较分析表明，单体蛋白中的潜在交联与淀粉样形式大多不兼容，因此可能抑制纤维形成。使用不同的交联剂，在各种条件下对单体α-突触核蛋白进行了分子内化学交联。我们使用质谱法确定了交联的位置和频率，并发现它们中的大多数不能在淀粉样结构中实现。使用各种方法，包括硫黄素-T 荧光和透射电子显微镜，实验研究了交联蛋白的抑制潜力。我们发现，交联施加的构象约束完全阻止了 α-突触核蛋白的淀粉样形成。此外，DTSSP 交联的分子也对未修饰的 α-突触核蛋白的聚集具有抑制作用。
