The S100 protein family consists of structurally similar calcium-binding proteins that play diverse roles in regulating calcium homeostasis, cell growth, differentiation, cytoskeleton dynamics and response to inflammation\(^{1}\). In recent years, several members have been found to play significant roles in neurodegeneration by facilitating neuroinflammatory signalling and forming amyloid fibrils\(^{2}\). Furthermore, it has been shown that some S100 proteins can interact with Hsp70/Hsp90 multichaperone complex with S100A1 exhibiting the strongest interaction\(^{3}\). Another common feature of all S100 proteins is their ability to form homo- and heterodimers\(^{2}\). A potential candidate for this type of interaction with S100A1 is S100A8, since both proteins are found within amyloid plaques or astrocytes and play a role in the Alzheimer’s disease (AD) cascade by transmitting neuroinflammatory signals\(^{2}\).
Another characteristic of AD pathogenesis is oxidative stress, caused by reactive oxygen species (ROS) and liquid-liquid phase separation (LLPS), during which proteins form membraneless organelles\(^{4}\). ROS can lead to structural modifications of proteins and cause cellular damage\(^{5}\). While it is known that ROS can impair the functionality of S100 proteins\(^{6}\), how it affects S100 aggregation is not known. In addition, LLPS is an emerging area of research exploring the relationship between phase transitions and neurological disease. LLPS is linked to redox imbalance in cells\(^{4}\), but currently, there are no available LLPS studies of S100 proteins. Therefore, we decided to investigate how these cross-interactions, ROS and LLPS, affect S100A1 protein.
We monitored the aggregation of S100A1, S100A8 and S100A1/A8 proteins using amyloid-specific dye Thioflavin T (ThT) fluorescence and the resulting aggregates were analysed via Atomic Force (AFM) and Transmission electron (TEM) microscopies. A lysozyme chaperone activity test was performed to investigate whether S100A1 exhibits aggregation-inducing properties. Furthermore, we explored the aggregation of S100A1 under conditions of liquid-liquid phase separation (LLPS) and oxidation by measuring ThT fluorescence and turbidity.
Our research demonstrated that S100A1 and S100A8 form fibrillar aggregates in the presence of each other – a process chaperoned by calcium ions. AFM and TEM imaging confirmed that the interaction between these proteins led to the formation of worm-like fibrils. The lysozyme chaperone activity test revealed induction of lysozyme aggregation by S100A1 at low concentrations of calcium ions. These findings suggest that S100A1, when mixed with S100 or other proteins, can promote their aggregation. Finally, we showed that the highly stable S100A1 protein can form worm-like fibrils in the presence of crowding and oxidizing agents.