Profilin Binding Modulates the Aggregation and Phase Separation of Huntingtin N-Terminal Fragments via Polyphasic Linkage

Abstract

Neurotoxic aggregates formed by N-terminal fragments of the mutant huntingtin protein (Htt-NTFs) are associated with the onset and progression of Huntington's disease. Htt-NTFs encompass expanded polyglutamine (polyQ) tracts that are flanked on the N-terminus by a 17-residue stretch and on the C-terminus by a 38-residue proline-rich stretch (C38). Htt-NTF aggregation and toxicity in cells is diminished by direct interactions with profilin, which is an abundant actin-binding protein that also binds polyproline ligands through a distinct binding site.Htt-NTF aggregation leads to the formation of soluble spherical aggregates (30-50 nm in diameter) and insoluble fibrillar deposits. These aggregates constitute distinct phases and are characterized by saturation concentrations, i.e., threshold concentrations that must be crossed in order for the corresponding phases to form. Using a combination of in vitro measurements we have demonstrated that profilin binding lowers the driving forces for Htt-NTF aggregation and phase separation via polyphasic linkage, a phenomenon whereby distinct saturation concentrations are shifted upwards by an order of magnitude. Accordingly, in the presence of profilin, a higher total concentration of Htt-NTFs is required to cross a given phase boundary. Our binding assays reveal that profilin binds preferentially to Htt-NTF monomers and small soluble oligomers. This involves specific interactions between profilin and the polyproline modules of C38 and possibly non-specific interactions between profilin and polyQ via an avidity effect.Our data provide a biophysical explanation for the observed cellular effects of profilin on Htt-NTF aggregation. Increases in saturation concentrations through polyphasic linkage might be a general mechanism for lowering the driving forces for aggregation and phase separation of aggregation-prone proteins, and may contribute to the protein homeostasis mechanisms used by cells to cope with protein aggregation stress.