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

Abstract 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 (β, γ, ε, ζ, η, θ and σ), 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-Jakob’s, Parkinson’s or Alzheimer’s 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 α-synuclein (α-syn) or β-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 ε and θ isoforms form amyloid-like fibrils that can accelerate α-syn aggregation. Furthermore, molecular crowding conditions promoted phase separation and aggregation in most 14-3-3 proteins. Finally, 14-3-3s incorporated together with α-syn to generate heterotypical droplets. Competing Interest Statement The authors have declared no competing interest. Footnotes This version of the manuscript has been revised to include new data about alpha-synuclein and 14-3-3 aggregation and LLPS.