| dc.description.abstracteng | The discovery of phase-separated bodies in cells has revealed an additional mesoscale layer
of organisation in cellular and molecular biology. The enrichment of RNA in these
biomolecular condensates suggests that charge plays a key role in phase separation. Complex
coacervation—a type of polymer phase separation—occurs in solutions of oppositely
charged polyelectrolytes.
In this project, we investigated the role of naturally occurring, cellularly abundant
polyamines as cationic binding partners, given their close association with RNA biology and
their ability to induce complex coacervation with nucleic acids. Unlike RNA, many proteins
have non-uniform charge distributions and complex primary sequences, where the effects of
charge must be considered alongside other factors, such as hydrophobicity and charge
patterning. Whether these factors can similarly drive complex coacervation of intrinsically
disordered proteins (IDPs) remains unclear.
To explore this, we studied α-synuclein (αSyn) and G3BP1. G3BP1 is a stress granule
scaffold protein whose condensation is driven by a combination of homotypic and
heterotypic interactions. Its long acidic intrinsically disordered region (IDR1) suggests that
interactions with cationic species may be as critical as its RNA-binding motif. Similarly, the
role of αSyn’s acidic C-terminus in modulating phase separation and its connection to
amyloid aggregation remain poorly understood.
We find that PolyU RNA and spermine form stoichiometry-dependent complex coacervates
that are highly sensitive to temperature and ionic strength, consistent with a classical
complex coacervation process. Spermine also promotes phase separation of αSyn through
charge interactions with its acidic C-terminus. This process is strongly dependent on αSyn-
to-spermine stoichiometry, increases as the isoelectric point of αSyn is approached, and
decreases as polyamines lose charge. Phase separation is abolished at NaCl concentrations
above 200 mM, highlighting the electrostatically driven nature of the process. Importantly,
heterotypic phase separation accelerates αSyn aggregation. Polyamines with weaker net
charge relative to spermine interact more weakly with αSyn, resulting in reduced phase
separation.
IV
Our results identify the C-terminus of αSyn as the scaffold domain for phase separation and
show that multivalent polyamines can promote condensation at physiological
concentrations. In sub-saturated solutions, clusters are preferentially formed, which persist
within condensates, indicating electrostatic nanocluster formation and microstructural
organisation. RNA, which binds spermine more strongly than αSyn, can sequester spermine
from the protein, exposing the C-terminus and promoting miscibility. Finally, we show that
G3BP1 interacts with spermine via its acidic IDR1, lowering the protein concentration
required for phase separation.
These findings provide mechanistic insight into how electrostatic interactions and
multivalent cations regulate the phase separation of IDPs and stress granule scaffold proteins
in cells. | de |