Pandit Esha, Das Lopamudra, Das Anoy Kumar, Dolui Sandip, Saha Saumen, Pal Uttam, Mondal Animesh, Chowdhury Joydeep, Biswas Subhas C, Maiti Nakul C
Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India.
Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Kolkata, India.
Front Chem. 2023 May 26;11:1145877. doi: 10.3389/fchem.2023.1145877. eCollection 2023.
Parkinson's disease is an age-related neurological disorder, and the pathology of the disease is linked to different types of aggregates of α-synuclein or alpha-synuclein (aS), which is an intrinsically disordered protein. The C-terminal domain (residues 96-140) of the protein is highly fluctuating and possesses random/disordered coil conformation. Thus, the region plays a significant role in the protein's solubility and stability by an interaction with other parts of the protein. In the current investigation, we examined the structure and aggregation behavior of two artificial single point mutations at a C-terminal residue at position 129 that represent a serine residue in the wild-type human aS (wt aS). Circular Dichroism (CD) and Raman spectroscopy were performed to analyse the secondary structure of the mutated proteins and compare it to the wt aS. Thioflavin T assay and atomic force microscopy imaging helped in understanding the aggregation kinetics and type of aggregates formed. Finally, the cytotoxicity assay gave an idea about the toxicity of the aggregates formed at different stages of incubation due to mutations. Compared to wt aS, the mutants S129A and S129W imparted structural stability and showed enhanced propensity toward the α-helical secondary structure. CD analysis showed proclivity of the mutant proteins toward α-helical conformation. The enhancement of α-helical propensity lengthened the lag phase of fibril formation. The growth rate of β-sheet-rich fibrillation was also reduced. Cytotoxicity tests on SH-SY5Y neuronal cell lines established that the S129A and S129W mutants and their aggregates were potentially less toxic than wt aS. The average survivability rate was ∼40% for cells treated with oligomers (presumably formed after 24 h of incubation of the freshly prepared monomeric protein solution) produced from wt aS and ∼80% for cells treated with oligomers obtained from mutant proteins. The relative structural stability with α-helical propensity of the mutants could be a plausible reason for their slow rate of oligomerization and fibrillation, and this was also the possible reason for reduced toxicity to neuronal cells.
帕金森病是一种与年龄相关的神经疾病，该疾病的病理学与α-突触核蛋白（α-synuclein，aS）的不同类型聚集体有关，α-突触核蛋白是一种内在无序的蛋白质。该蛋白的C末端结构域（第96 - 140位氨基酸残基）高度波动，具有随机/无序的卷曲构象。因此，该区域通过与蛋白质的其他部分相互作用，在蛋白质的溶解性和稳定性方面发挥着重要作用。在当前的研究中，我们研究了野生型人aS（wt aS）中第129位C末端残基处的两个人工单点突变体的结构和聚集行为，该残基在野生型中为丝氨酸残基。进行了圆二色性（CD）和拉曼光谱分析，以分析突变蛋白的二级结构，并将其与wt aS进行比较。硫黄素T测定和原子力显微镜成像有助于了解聚集动力学和形成的聚集体类型。最后，细胞毒性测定揭示了由于突变在不同孵育阶段形成的聚集体的毒性情况。与wt aS相比，突变体S129A和S129W具有结构稳定性，并表现出对α-螺旋二级结构更强的倾向。CD分析表明突变蛋白倾向于α-螺旋构象。α-螺旋倾向的增强延长了原纤维形成的延迟期。富含β-折叠的纤维化的生长速率也降低了。对SH-SY5Y神经细胞系的细胞毒性测试表明，S129A和S129W突变体及其聚集体的潜在毒性可能低于wt aS。用wt aS产生的寡聚体（可能是新制备的单体蛋白溶液孵育24小时后形成的）处理的细胞，平均存活率约为40%，而用突变蛋白获得的寡聚体处理的细胞，平均存活率约为80%。突变体具有α-螺旋倾向的相对结构稳定性可能是其寡聚化和纤维化速率缓慢的合理原因，这也是其对神经细胞毒性降低的可能原因。
