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 found 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 M-phase 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.

Keywords: Huntington disease; biophysics; neurodegenerative disease; phase transitions; polyglutamine; protein aggregation.

Figure 1.

Experimental evidence for Htt-NTF phase behavior with multiple phase boundaries. a, Htt-NTF sequence architectures used in this study. i, full Htt exon 1, N17-Q_n-C38; ii, Q_n-C38; iii, C38; iv, P₁₁; v, N17-Q_n-K₂; vi, Q_n-K₂. Here, n = 40 in all experiments except for the in-cell FRET aggregation assay (see Fig. S1), where n = 72, and some experiments where n = 30 was compared with n = 40 (see Fig. 5e). Additionally, K₂ corresponds to two lysines. b, previously reported (19) saturation concentrations (denoted here as c_F) determined by measuring the concentration of soluble protein remaining in the supernatant (colorimetric micro-BCA assay) following quiescent incubation at 30 °C and centrifugation of the indicated Htt-NTF constructs. Consistent with the presence of a saturation concentration, the same concentration was arrived at for a given construct regardless of the starting concentration, so long as the starting concentration was above the saturation concentration indicated. The saturation concentration is modulated by the N17 and C38 sequence modules that flank polyQ in Htt-NTFs. Black bars, mean ± S.D. (error bars) of four independent experiments. The N17-Q₄₀-K₂ c_F was below the detection limit of the assay for all trials, so the quantity plotted is the detection limit and represents an upper bound for the N17-Q₄₀-K₂ c_F. Statistical significance was determined by one-way ANOVA with Tukey's range test for post hoc analysis. ***, p < 0.001. ‡, the N17-Q₄₀-K₂ c_F data were not included in the statistical significance analysis for the reason cited above. c, TEM image of Q₄₀-C38 fibrils observed at a concentration supersaturated with respect to c_F (14 μm). Two of the fibrils in the image are indicated with white arrows labeled F, and two examples of spherical aggregates are indicated with white arrows labeled S. See Fig. 4c for an additional example of fibrils and spheres. d, right-angle static light scattering of Q₄₀-C38 in 20 mm Tris, 5 mm EDTA, 1 mm DTT, pH 7.4, as a function of Q₄₀-C38 concentration. The discontinuity at ∼290 nm is indicative of a phase boundary. e, TEM image of Q₄₀-C38 monomers and small oligomers observed at a concentration subsaturated with respect to c_S (126 nm). This particular field of view contains monomers/small oligomers, two of which are indicated with white arrows. f, TEM image of Q₄₀-C38 spherical aggregates observed at a concentration supersaturated with respect to c_S but subsaturated with respect to c_F (295 nm). One of the spherical aggregates is indicated with a white arrow labeled S. All TEM images (c, e, and f) are at the same scale. Scale bar, 100 nm.

Figure 2.

Schematic representation of Htt-NTF phase behavior and polyphasic linkage. a, Htt-NTF phase behavior is described by three phases (M, S, and F phase), color-coded as red, blue, and green, respectively, and two phase boundaries (c_S and c_F) indicated by gray lines between the phases. The total monomer-equivalent Htt-NTF concentration (horizontal axis) with respect to these boundaries will determine whether monomers and oligomers, spherical aggregates, or fibrils form. b, the influence of a binding partner (profilin) on the Htt-NTF c_S phase boundary. The phase boundary is depicted as a solid gray line in the absence of profilin (i) and for three different profilin binding scenarios (ii–iv). The relative number of profilin (black circles) associated with each phase (M or S) indicates which phase is preferentially bound. The dotted gray line in ii and iii indicates the c_S in the absence of profilin, and the red and blue arrows indicate the direction of the shift in the c_S if the M or S phase is preferentially bound, respectively. In scenario iv, profilin binds both phases equally well, and thus no shift in c_S is observed. The orientation of b is rotated 90° with respect to a for formatting purposes. See “Results” for further description of polyphasic linkage.

Figure 3.

Profilin shifts the c_S of Q₄₀-C38 through polyphasic linkage. a, right-angle static light scattering of Q₄₀-C38 solution as a function of Q₄₀-C38 concentration, in the absence of profilin (black, reproduced from Fig. 1d) and in the presence of 1, 5, 10, or 20 μm profilin (blue, purple, red, and green, respectively) in 20 mm Tris, 5 mm EDTA, 1 mm DTT, pH 7.4. One representative trial at each profilin concentration is shown, and the plots are arbitrarily offset in the vertical direction for the sake of clarity. The values of c_S for Q₄₀-C38 in the presence of each of these concentrations of profilin are indicated by the discontinuity in slope, and each discontinuity is marked using an open circle. We note that the slope of the low-concentration arm of the 10 μm profilin data set differs from the other data sets and suspect that this is the result of an experimental artifact (see “Experimental procedures”). Regardless, there was good agreement between trials concerning the location of the discontinuity, as is evident in b. b, Q₄₀-C38 c_S measured in a plotted as a function of the profilin concentration. The intersections of “best-fit” lines from jackknife sampling for at least three trials at each profilin concentration are plotted (see supporting Methods). Black bars, mean ± S.D. (error bars). Statistical significance was determined by Welch's ANOVA with Games–Howell post hoc analysis. **, p < 0.01; ***, p < 0.001. Colors are the same as in a. Even the lowest concentration of profilin tested (1 μm) resulted in more than an order of magnitude increase in the c_S of Q₄₀-C38.

Figure 4.

The influence of profilin on Q₄₀-C38 fibril formation. a, ThT assay of Q₄₀-C38 aggregation kinetics at a fixed concentration of Q₄₀-C38 in the presence of various concentrations of profilin. ThT fluoresces upon binding to amyloid-like fibrils, thus providing a readout of fibril formation in the form of fluorescence intensity. b, the plateau ThT fluorescence intensity (ThT I_max) and the time to reach 50% I_max (t₅₀) (red and blue, respectively), are plotted as a function of profilin concentration. c, representative TEM image of a sample taken from an aggregation assay in the absence of profilin showing an abundance of long fibrils coexisting with spherical aggregates. d, representative TEM image of a sample taken from an aggregation assay in the presence of 5 μm profilin shows short fibrils few in number as well as spherical aggregates and oligomers or possibly monomers. His-tagged profilin was marked on the carbon-coated copper grid with 5-nm nickel-nitrilotriacetic acid-gold nanoparticles, which appear as black dots. Examples of gold nanoparticles are indicated with black arrows. Examples of fibrils and spheres are indicated with white arrows labeled F and S, respectively, in c and d.

Figure 5.

The effect of polyQ on profilin binding to Htt-NTFs. a, binding isotherms of 5 μm profilin with C38 (black) versus 5 μm profilin with Q₄₀-C38 (purple). The presence of the polyQ tract enhances binding at a given profilin concentration. b, binding isotherms of 1, 5, 10, and 20 μm profilin with Q₄₀-C38. c, apparent K_d values extracted from fits to the isotherms in a. A slight dependence of the K_d,app on profilin concentration is apparent, although a one-way ANOVA test of the data revealed that the difference is at the threshold of significance, with a p value of 0.0507. d, when the concentration of each binding isotherm is adjusted by the magnitude of the profilin-dependent shift in c_S (c_S,Pfn/c_S,intrinsic), then the binding isotherms collapse to a single curve, confirming that the observed dependence of K_d,app on the profilin concentration is due to profilin-dependent changes in partitioning of Q₄₀-C38 molecules between the M- and S-phases (see “Results” for details). e, comparison of apparent K_d values of C38 (black) and Q₄₀-C38 (purple) with Q₃₀-C38 (orange) and N17-Q₃₀-C38 (teal), all measured at 5 μm profilin. Statistical significance was determined by one-way ANOVA with Tukey's range test for post hoc analysis. ***, p < 0.001. Binding of profilin appears to correlate with aggregation propensity, which is dictated by polyQ length (Q₃₀ versus Q₄₀) and flanking sequence context (absence or presence of N17). Black bars in c and e indicate the mean ± S.D. (error bars) of three or four independent experiments.

Figure 6.

Coarse-grained model identifies an auxiliary interaction necessary for the experimentally observed profilin effect. a, visual representation of the architecture used for the increased local concentration model. Q₄₀-C38 and profilin are represented by a set of spheres or “beads” as defined in the top legend (see supporting Methods for model details). The primary interaction between profilin (Pfn) and the polyP segment of C38 is denoted by a green arrow. b, visual representation of the architecture used for the polyQ-Pfn auxiliary interaction model. This model is identical to the increased local concentration model, except an auxiliary interaction is added between the polyQ bead and a nonspecific region of the profilin molecule that does not overlap with the polyP interaction site (red arrow). c and d, fraction of profilin molecules bound to polyP segments observed in coarse-grained simulations of the increased local concentration model and the polyQ-Pfn auxiliary interaction model, respectively. Each simulation contained 210 Pfn molecules and 630 C38 stretches. Additionally, each simulation had a homogeneous distribution of cluster sizes, X (X = 1, 3, 6, 9, 15, 35, 70, 210), such that the number of clusters multiplied by X equaled 630. Each bar denotes results from simulations performed with a distinct cluster size as indicated by the bottom legend. A cluster size of zero denotes C38 without the polyQ domain. Gray beads in the legend denote beads that interact with profilin via the primary Pfn-PolyP interaction. Error bars, S.E. from five independent simulations.