  Liquid liquid phase separation (LLPS) of proteins is an intracellular process
that is widely used by cells for many purposes. In living cells (in vivo), LLPS
occurs in complex and crowded environments. Amino acids (AAs) are vital
components of such environments, occupying a significant fraction of the
cellular volume. In this work, we studied the effects of proline and other
proteinogenic AAs on the LLPS of proteins, both in test tubes (in vitro) and in
cells (in vivo). The effects of proline on the protein-protein interaction
(PPI) and LLPS of both bovine serum albumin (BSA, a folded protein) and the
low-complexity domain of fused in sarcoma (FUS267, an intrinsically disordered
protein) is first established in vitro. Then, the effects of proline and other
proteinogenic AAs on the formation of stress granules (SGs) by LLPS in U2OS and
HeLa cells are studied. We find that the presence of AAs renders the net
interaction between proteins more repulsive (i.e. stabilizes protein solution),
thus suppressing protein phase separation in vitro and in vivo. We also show
that the formation of SGs is suppressed by AAs using both immunofluorescence
and live-cell microscopy. Our study reveals an underappreciated role of
cellular AAs in modulating intracellular phase separation. It may find
biomedical applications, especially in the treatment of protein aggregation
diseases.
