Peer-reviewed Publication by Science CRO

This study integrates high-resolution native PAGE, detergent ultracentrifugation, reductive discrimination, and Python-based kinetic modeling to resolve SOD1 nucleation phases in human cells, revealing radical-dependent polymerization relevant to ALS.

Methodological details

Time-course aggregation in HEK293FT cells was quantified using detergent ultracentrifugation (S1/P2 fractions) with superior detection sensitivity relative to size exclusion methods.

High-resolution colorless native PAGE (hrcN-PAGE) with in-gel βME/SDS reduction delineated oxidized versus reduced intra-disulfide states and resolved monomers, trimers, hexamers, and higher multimers under controlled pH and migration conditions.

Aggregation kinetics were modeled programmatically using logistic, autocatalytic, and linear functions with goodness-of-fit testing, enabling discrimination of lag, nucleation, growth, and plateau phases.

Critical findings

• • Nucleation proceeds via intra-disulfide–reduced monomers, trimers, and hexamers, defining a mechanistic threshold preceding insoluble aggregate growth.
• • Aggregation displays pseudo-zeroth order or autocatalytic behavior depending on variant, with statistically validated logistic fits.
• • Superoxide exposure via paraquat initiates trimer formation and increases aggregation propensity, implicating radical substrate in polymer initiation.
• • Aggregation propensity is concentration dependent and variant specific, establishing a quantitative cellular framework for ALS-linked gain-of-function toxicity.