Delineating the Aggregation-Prone Hotspot Regions (Peptides) in the Human Cu/Zn Superoxide Dismutase 1

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal, incurable neurodegenerative disease described by progressive degeneration of motor neurons. The most common familial form of ALS (fALS) has been associated with mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Mutation-induced misfolding and aggregation of SOD1 is often found in ALS patients. In this work, we probe the aggregation properties of peptides derived from the SOD1. To examine the source of SOD1 aggregation, we have employed a computational algorithm to identify four peptides from the SOD1 protein sequence that aggregates into a fibril. Aided by computational algorithms, we identified four peptides likely involved in SOD1 fibrillization. These four aggregation-prone peptides were ¹⁴VQGIINFE21, ³⁰KVWGSIKGL38, ¹⁰¹DSVISLS¹⁰⁷, and ¹⁴⁷GVIGIAQ¹⁵³. In addition, the formation of fibril propensities from the identified peptides was investigated through different biophysical techniques. The atomic structures of two fibril-forming peptides from the C-terminal SOD1 showed that the steric zippers formed by ¹⁰¹DSVISLS¹⁰⁷ and ¹⁴⁷GVIGIAQ¹⁵³ vary in their arrangement. We also discovered that fALS mutations in the peptide ¹⁴⁷GVIGIAQ¹⁵³ increased the fibril-forming propensity and altered the steric zipper's packing. Thus, our results suggested that the C-terminal peptides of SOD1 have a central role in amyloid formation and might be involved in forming the structural core of SOD1 aggregation observed in vivo.

Figure 1

(A) Three-dimensional structure of SOD1 dimer (PDB code: 2C9V) indicating the fibrillogenic peptides shown in red. (B) Calculated Rosetta energies for each six-residue sequence were obtained from the Zipper-DB program. Peptides predicted to form amyloid fibrils are highlighted in red.

Figure 2

Secondary structure of SOD1 amyloidogenic peptides were analyzed by far-UV CD spectroscopy before (black line) and after incubation at 37 °C with shaking of 200 rpm for 5 min (red line), 30 min (green line), and 60 min (yellow line). Structural transformation of (A) P1, (B) P2, (C) P3, (D) P4, (E) P4^T, and (F) P4^R peptides.

Figure 3

ThT binding to SOD1 fibrillogenic peptides. ThT fluorescence intensity of (A) P1, (B) P2, (C) P3, (D) P4, (E) P4^T, and (F) P4^R incubated at 37 °C with shaking of 200 rpm as a function of incubation time for 5 min (black line), 30 min (red line), and 60 min (green line).

Figure 4

Secondary structure profiles of SOD1 peptide fibrils examined by ATR–FTIR (1000–2000 cm^–1) of amyloid fibrils formed in vitro. (A) P1, (B) P2, (C) P3, (D) P4, (E) P4^T, and (F) P4^R suspension of peptide fibrils.

Figure 5

XRD pattern of SOD1 preformed fibrils. (A) P1, (B) P2, (C) P3, (D) P4, (E) P4^T, and (F) P4^R peptides. The reflection at ∼ 5.0 Å corresponds to the repeat distance of β-strands aligned perpendicularly to the fiber axis, whereas the reflection at ∼10.0 Å is attributed to the repetitive distance between packed β-sheets.

Figure 6

AFM images of different aggregates formed en route to fibrilization. The area of measurement is 3 × 3 μM, and scale bar represents 1 μM length. The area corresponding to each stage has been magnified for a clearer view. (A–E) P3 incubated at 37 °C and shaking of 200 rpm for 30 min, 1 h, 4 h, 24 h, and 48 h, respectively. (F–J) P4 incubated at 37 °C and shaking of 200 rpm for 30 min, 1 h, 4 h, 24 h, and 48 h, respectively.