Understanding Structural Destabilization and Amyloid Aggregation in ALS-Related Neurodegenerative Disorder: An In Silico and Experimental Analysis of SOD1 Variants

Protein misfolding has been reported as a common symptom in many neurodegenerative diseases, leading to the formation of protein aggregates. Metal ions (holo form) are critical for the folding and function of WT-SOD1, whereas their absence (apo form) can lead to aggregation and misfolding under physiological conditions. Therefore, this study investigates the role of mutations/metal deficiencies in the metal binding loop and how the mutations affect the SOD1 aggregation process in amyotrophic lateral sclerosis through an experimental and computational approach. Molecular dynamic (MD) simulation results show a significant difference in apo-SOD1 compared to holo-SOD, which is consistent with experimental studies. Dictionary of Secondary Structure in Proteins (DSSP), Fourier-transform infrared (FTIR), and Circular dichroism (CD) results confirmed a tendency for increased β-sheet formation in the apo-SOD1 form, which can be attributed to protein aggregation. The observed conformational changes under amyloidogenic conditions suggest that the hydrophobic pockets in apo-SOD1 are more exposed compared to holo-SOD1, as confirmed by ANS fluorescence. Thermodynamic investigations with GdnHCl demonstrated that mutation/metal deficiency are necessary to trigger the misfolding and aggregation of SOD1. Our results show that apo/holo SOD1 variants induce the formation of aggregated species under physiological conditions. These aggregates are detected by Congo red and ThT fluorescence and further validated by transmission electron microscopy (TEM) imaging. Overall, mutations in loop IV and structural abnormalities such as mutation/metal deficiency and reduced disulfide bonds synergistically lead to reduced thermodynamic stability of SOD1 variants, facilitating the formation of amyloid/amorphous aggregates. Ultimately, this study could serve as a basis for new research to develop new treatments for neurological disorders, and help to better understand the role of mutation in the formation of amyloid aggregates and identify different factors in ALS disease.