Profilin reduces aggregation and phase separation of huntingtin N-terminal fragments by preferentially binding to soluble monomers and oligomers

Abstract

Huntingtin N-terminal fragments (Htt-NTFs) with expanded polyglutamine tracts form a range of neurotoxic aggregates that are associated with Huntington's disease. Here, we show that aggregation of Htt-NTFs, irrespective of polyglutamine length, yields at least three phases (designated M, S, and F) that are delineated by sharp concentration thresholds and distinct aggregate sizes and morphologies. We find that monomers and oligomers make up the soluble M-phase, ~25 nm spheres dominate in the soluble S-phase, and long, linear fibrils make up the insoluble F-phase. Previous studies showed that profilin, an abundant cellular protein, reduces Htt-NTF aggregation and toxicity in cells. We confirm that profilin achieves its cellular effects through direct binding to the C-terminal proline-rich region of Htt-NTFs. We show that profilin preferentially binds to Htt-NTF M-phase species and destabilizes aggregation and phase separation by shifting the concentration boundaries for phase separation to higher values through a process known as polyphasic linkage. Our experiments, aided by coarse-grained computer simulations and theoretical analysis, suggest that preferential binding of profilin to the Mphase species of Htt-NTFs is enhanced through a combination of specific interactions between profilin and polyproline segments and auxiliary interactions between profilin and polyglutamine tracts. Polyphasic linkage may be a general strategy that cells utilize to regulate phase behavior of aggregation-prone proteins. Accordingly, detailed knowledge of phase behavior and an understanding of how ligands modulate phase boundaries may pave the way for developing new therapeutics against a variety of aggregation-prone proteins.Many diseases are associated with protein misfolding and aggregation (1,2). The aggregation process is often characterized by the presence of one or more threshold concentrations at which a sharp, discontinuous change to some aspect of the assembly state (e.g., size, conformational characteristics, material properties) occurs (3-6). Such a change can be described using the concepts of phase transitions. Phase separation, a subcategory of phase transitions, has recently received considerable attention due to increasing recognition of its importance in cell biology (7)(8)(9)(10)(11)(12)(13). Phase separation refers to aggregation-related changes in molecular density that give rise to the coexistence of dilute macromolecule-deficient phases and dense macromolecule-rich phases (3,14,15). Examples of multiple coexisting phases have been observed in biological contexts (15)(16)(17)(18), and these phases can be liquid, solid, or semisolid (e.g., a gel) (10,(19)(20)(21)(22)(23)(24)(25) Modulation of Htt-NTF aggregation via polyphasic linkage2 separation are quantified in terms of saturation concentrations (14,19,26). For a given two-phase system, the saturation concentration is the bulk concentration of the protein beyond which the solution separates into two coexisting phases. The lower the saturation concentration, t...

Inhibition of amyloid beta fibril formation by monomeric human transthyretin

“…It is possible that the species that are preferentially recognized by M-TTR are analogous to the recently observed S-phase species of Httex1. 48 The fact that monomeric TTR (as opposed to the genetically engineered constitutive monomer M-TTR used in this study) can inhibit fibril formation by Ab and possibly other amyloid precursors suggests that there may be a general ability for aggregation-prone proteins to interact in a heterotypic manner, whereas conformational specificity is required for nucleation and templating that leads to fibril formation via homotypic associations. 52 We conjecture that oligomers of M-TTR might display peptide structures that resemble Ab sequences to interfere with the specific interactions required for fibrils, generating aggregates that are less likely to form fibrils.…”

“…In this regard, it is worth noting that species in the 25–50 nm range have been observed for huntingtin exon 1 (Httex1) spanning fragments and have been designated as S‐phase species. It is possible that the species that are preferentially recognized by M‐TTR are analogous to the recently observed S‐phase species of Httex1 …”

“…39 The concept of polyphasic linkage was recently demonstrated in the context of profilin binding to exon1 spanning constructs of the protein hutingtin. 48 The FCS analysis presented here using labeled M-TTR and Ab42 provides insights regarding the interactions that cause Ab42 to supersaturate the soluble phase. Since M-TTR is larger than Ab42 monomer (13.8 vs. 4.5 kDa), the stronger Ab42 signal in the complexes indicates that high Ab42/M-TTR molar ratios are most consistent with larger soluble aggregates of Ab42 being bound by small oligomers (or monomers) of M-TTR.…”

“…Polysteric linkage is a specialized circumstance of polyphasic linkage, where the former refers to preferential binding to specific species without necessarily altering phase boundaries, whereas polyphasic linkage refers to preferential binding that leads to changes in the positions of phase boundaries . The concept of polyphasic linkage was recently demonstrated in the context of profilin binding to exon1 spanning constructs of the protein hutingtin …”

Huntingtin fibrils with different toxicity, structure, and seeding potential can be reversibly interconverted

“…If the structural differences among the different fibril types are directly linked to toxicity, one would also expect that the PRD could be a contributor to HTTex1 toxicity. Intrabodies or proteins such as profilin, which bind to the PRD region have been shown to reduce HTTex1 aggregation and toxicity (Southwell et al, 2009(Southwell et al, , 2008Burnett et al, 2008;Shao et al, 2008;Posey et al, 2018). One of the factors that could contribute to the different toxicities of the HTTex1 fibril types is their seeding ability.…”