Baronaite, I. | bioRxiv Science

bioRxiv2026

Liquid-liquid phase separation and amyloid aggregation in the 14-3-3 protein family

The 14-3-3 protein family, with over 1300 binding partners, is one of the largest regulators of protein-protein interactions (PPIs) in eukaryotic cells. They recognise and bind to phosphorylation-related motifs in their partner proteins, creating scaffolds to stabilise proteins and allowing or inhibiting kinases access to their targets. 14-3-3 consists of seven isoforms ({beta}, {gamma}, {varepsilon}, {zeta}, , {theta} and {sigma}), which are expressed across all tissues. Given their important role in PPIs, their dysregulation can contribute to a variety of diseases, such as cancer or neurodegenerative disorders like Creutzfeldt-Jakobs, Parkinsons or Alzheimers diseases. Pathological effects can arise due to loss of function, aberrant interactions or protein aggregation. Notably, 14-3-3 aggregates have been detected in Lewy bodies or cerebrospinal fluid, mirroring the presence of amyloid proteins, such as -synuclein (-syn) or {beta}-amyloid. Toxic amyloid aggregation signals the onset of neurodegeneration, which can occur through misfolding of proteins or liquid-liquid phase separation (LLPS) - a process during which proteins condense into membraneless organelles. Unlike other amyloidogenic proteins, there is little information on the conditions under which the 14-3-3 protein family members undergo LLPS or their relationship with amyloid aggregation. To address this gap, we examined all isoforms of 14-3-3 in vitro and observed the formation of amyloid aggregates and phase-separated droplets. Our results revealed that the {varepsilon} and {theta} isoforms form amyloid-like fibrils that can accelerate -syn aggregation. Furthermore, molecular crowding conditions promoted phase separation and aggregation in most 14-3-3 proteins. Finally, 14-3-3s incorporated together with -syn to generate heterotypical droplets.

biochemistry↗

bioRxiv2024

Calcium-mediated amyloid co-aggregation of S100A1 and S100A8 proteins

The S100 family consists of calcium binding proteins that are largely known for their contribution to the neuroinflammatory processes. They are associated with various cardiac and neurological functions as well as related diseases. A few S100 proteins can form unspecific or amyloid aggregates in neuropathologies and thus play a part in dementia pathogenesis. Among all S100 proteins, S100B and S100A9 aggregation properties are the most investigated, however, there is a lack of studies regarding other S100 members. In particular, S100A1 and S100A8 are also associated with neuropathies, but their interactions or aggregation are poorly understood. Therefore, in this study, we explored whether S100A1 and S100A8 proteins can form heterodimers, interact or co-aggregate. Our results revealed that S100A1 and S100A8 interactions and amyloid aggregation are driven by calcium ions. We observed that while S100A1 remains mostly stable, S100A8 forms various types of spherical or unspecific aggregates. While they do not form stable heterodimers like calprotectin, their transient interactions facilitate the formation of worm-like amyloid fibrils and the process is regulated by different calcium ion concentrations. At calcium ions saturation, both proteins are stabilized leading to inhibition of aggregation. Overall, by employing a diverse range of techniques from amyloid and protein-specific fluorescence detection to electron-electron double resonance spectroscopy, we elucidated interactions between S100 proteins that might otherwise be overlooked, enhancing our understanding of their aggregation behaviour.

biochemistry↗