Natural variants of apolipoprotein A1 L99P and R173P exhibited a high degree of amyloid fibril formation: Implications for high-density lipoprotein functions and disease pathogenesis.

Apolipoprotein A1 (ApoA1), the primary protein component of high-density lipoprotein (HDL), plays a crucial role in cholesterol transport and cardiovascular protection. However, specific mutations in ApoA1 are associated with hereditary amyloidosis, a condition marked by protein misfolding and deposition of amyloid fibrils in various tissues. This study aimed to experimentally characterize the structural and functional consequences of four disease-associated ApoA1 mutations (G26R, L84P, L99P, and R173P), previously predicted to be amyloidogenic via computational analysis. Recombinant ApoA1 variants were expressed in Bacillus subtilis, purified, and characterized using a combination of biophysical, structural, and cellular assays. The L99P and R173P mutants exhibited significant structural destabilization, increased exposure of hydrophobic surfaces, enhanced β-sheet formation, and a strong tendency to aggregate. These properties were associated with the formation of elongated fibrils and a marked increase in cytotoxicity in HEK293 cells. In contrast, the L84P variant formed spherical aggregates with relatively preserved secondary structure and showed minimal cytotoxic effects, suggesting a distinct, possibly less pathogenic aggregation pathway. G26R displayed intermediate behavior, with notable amyloidogenic potential and cellular toxicity. This study provides experimental validation for the amyloidogenic nature of select ApoA1 mutations and highlights the structural heterogeneity underlying their aggregation behavior.