S100A8 and S100A9 are members of the S100 calcium-binding protein family. They are established as biomarkers of inflammation with various extracellular and intracellular functions [1]. Both of the proteins can be heavily upregulated in Alzheimer's disease (AD) patients' brain tissues [2]. S100A9 forms neurotoxic amyloid fibrils [3], which may contribute to the pathology of AD [4]. Differently from S100A9, S100A8 assembles into non-fibrillar aggregates in the brain tissues of transgenic APP or ageing wild-type mice [5]. Moreover, S100A8 and S100A9 are able to form heterodimeric complex (calprotectin (CP)). Calcified amyloidogenic fibrils of calprotectin can be found in the ageing prostate, which additionally emphasizes the connection between inflammation and amyloid deposits formation [6].
Assembly into amyloids is related to the changes in the protein's secondary structure. During the aggregation process, a transformation from the native state into cross β-sheet structures occurs [7]. However, existing evidence implies that S100A9 has a distinct aggregation pathway as S100A9 fibrils retain a significant amount of α-helical motives alongside β-sheets [3]. Thus, S100A9 amyloid fibrils structural propensities remain unclear. Moreover, there are no detailed studies of S100A8 and CP aggregation in vitro. Therefore, in this study, we were investigating S100A8 and S100A9 fibril's secondary structures and morphologies, as well as their aggregation rate dependence on protein concentration. Additionally, we examined CP formation influence on S100A8 and S100A9 aggregation.
In order to evaluate S100A8 and S100A9 aggregation propensities, Thioflavin T Fluorescence Assays were conducted under 37 °C. Aggregation kinetics results indicated that both proteins have a short lag-phase period and half-time values of the amyloid formation kinetics elevate notably in a concentration-dependent manner. However, distinct characteristics of S100A8 and S100A9 aggregation curves were observed, hence, formed aggregates were examined using Atomic Force Microscopy (AFM). AFM imaging indicated S100A8 aggregates as spherical oligomers, differently from S100A9 short worm-like fibrils. Lastly, we observed that CP formation inhibits S100A9 assembly into fibrils in vitro. In summary, our conducted research provides important and previously unknown insights into S100A8 and S100A9 aggregation, which is tightly connected to various pathologies.