Covalent α-Synuclein Dimers: Chemico-Physical and Aggregation Properties

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

α-突触核蛋白（α-synuclein）聚集形成淀粉样原纤维，是帕金森病（Parkinson's disease）发病的关键步骤。α-突触核蛋白的淀粉样原纤维是路易小体（Lewy bodies）的主要组成成分，而路易小体是该疾病的组织病理学标志性特征。目前学界对α-突触核蛋白的聚集机制尚缺乏深入了解。在聚集过程中，α-突触核蛋白会形成被认为具有毒性的瞬时中间产物。α-突触核蛋白的二聚化可能是该蛋白聚集过程中的限速步骤。本研究对四种共价连接的α-突触核蛋白二聚体展开分析，这四种二聚体分别通过N端共价连接、C端共价连接、两段序列串联克隆，以及将1–104和29–140序列串联并列整合至同一蛋白中获得。通过圆二色谱（Circular Dichroism, CD）、傅里叶变换红外光谱（Fourier Transform Infrared Spectroscopy, FT-IR）与核磁共振波谱（Nuclear Magnetic Resonance, NMR）对其在溶液中的生物物理特性进行测定，同时研究了十二烷基硫酸钠（Sodium Dodecyl Sulfate, SDS）诱导的蛋白折叠过程。通过硫黄素T（Thioflavin T, ThT）荧光检测与偏振荧光分析法分析了原纤维的形成情况；借助透射电子显微镜（Transmission Electron Microscopy, TEM）以及基于原子力显微镜（Atomic Force Microscopy, AFM）的定量形态学分析考察了其形态特征。所有二聚体在生理条件下均不具有有序二级结构，但在与SDS相互作用时会发生α-螺旋构象转变。所有蛋白变体均能形成类淀粉样原纤维。二聚体构建体中α-突触核蛋白单体的相对取向会影响聚集过程的动力学，并据此确定了相对淀粉样生成倾向的排序。通过傅里叶变换红外光谱与原纤维核心的蛋白水解图谱开展的结构研究未发现各蛋白变体间存在显著差异，而形态学分析结果显示，相较于野生型α-突触核蛋白原纤维，二聚体形成的原纤维具有更低且更为多样化的组织层级。本研究表明，尽管α-突触核蛋白的二聚化并不意味着参与形成二聚体的单体获得了特定优势构象，但它能够显著影响分子的聚集特性。本研究结果凸显了单个单体的相对取向在决定原纤维高级结构层级中的核心作用。