Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils

Abstract

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

Figure 1. Size distributions of proteins reported to form non-amyloid deposits in human pathology (left) and of proteins described to form extracellular amyloid fibrils or intracellular inclusions with amyloid-like characteristics in human disease (right).

Each data point represents one peptide or protein with its size described as a logarithm of the number of amino acid residues (y axis). A list of all the proteins reported in the graph and of their sizes is shown in Tables S1 and S2. The scatter of data points on the x axis has no meaning and it has been introduced to separate the data points in the graph. The horizontal and vertical lines indicate the mean values and the associated standard deviations for both populations. The size distributions are significantly different (p<0.001).

Figure 2. H-induced unfolding of YHKB.

(A) pH-induced unfolding of YHKB monitored by the fluorescence emission intensity at the maximum wavelength (▪) and [θ]₂₂₂ (□). (B) Spectral diagram of pH-induced equilibrium unfolding of YHKB, reporting on F₃₄₀ versus [θ]₂₂₂.

Figure 3. Far-UV CD spectra of native YHKB in 50 mM phosphate buffer, pH 7.0 (▪), in 20 mM TFA, pH 1.7 without salts (□), in 20 mM TFA, pH 1.7 with 360 mM NaCl after 5 min (▴) and under the same conditions after 120 min (Δ).

Figure 4. ThT and CR binding of YHKB aggregates at low pH.

(A) ThT fluorescence emission spectra obtained after addition of YHKB samples pre-incubated at pH 1.7, 360 mM NaCl for 5, 30, 60 and 120 minutes. All spectra were obtained after subtraction of the corresponding spectra obtained with ThT in the absence of YHKB. The spectrum of ThT alone and that obtained after addition of the native protein are also shown for comparison. (B) Difference absorbance spectra of CR obtained with YHKB samples pre-incubated as described in panel (A). In each case, the difference spectrum was obtained by subtracting the spectra acquired for the protein alone and for CR alone from the spectrum recorded for the protein in the presence of CR.

Figure 5. TM-AFM images (height data) of YHKB aggregates obtained in 20 mM TFA, 360 mM NaCl, pH 1.7.

In each frame the corresponding aggregation time t is reported. Scan size 1 µm; Z range 20 nm (A–C) and 30 nm (D).

Figure 6. TFE-induced unfolding of YHKB monitored by [θ]₂₂₂ in the absence (▪) and/or presence (□) of 1 mM α-cyclodextrin.

Figure 7. Far-UV CD and X-ray diffraction analysis of YHKB aggregates in TFE.

(A) Far-UV CD spectra of native YHKB in 50 mM phosphate buffer, pH 7.0 (▪), in 50 mM acetate buffer, pH 5.5 with 30% (v/v) TFE after 5 minutes (▴), 30 minutes (□), 60 minutes (♦) and 120 minutes (Δ). (B) X-ray diffraction diagram of YHKB aggregates grown in 50 mM acetate buffer, pH 5.5 with 30% (v/v) TFE for 120 minutes. The diagram was collected on sedimented aggregates and shows the characteristic cross-β spacings at 4.6 and 10 Å.

Figure 8. ThT and CR binding of YHKB aggregates in TFE.

(A) ThT fluorescence emission spectra obtained after addition of YHKB samples pre-incubated at pH 5.5, 30% (v/v) TFE for 5, 30, 60 and 120 minutes. All spectra were obtained after subtraction of the corresponding spectra obtained with ThT in the absence of YHKB. The spectrum of ThT alone and the spectrum obtained after addition of the native protein are also shown for comparison. (B) Difference absorbance spectra of CR obtained with YHKB samples pre-incubated as described in panel (A). In each case, the difference spectrum was obtained by subtracting the spectra acquired for the protein alone and for CR alone from the spectrum recorded for the protein in the presence of CR.

Figure 9. TM-AFM images (height data) of YHKB aggregates obtained in 30% (v/v) TFE, 50 mM acetate buffer, pH 5.5.

In each frame the corresponding aggregation time t is reported. Scan size 1 µm; Z range 10 nm (A, B), 30 nm (C) and 20 nm (D).

Figure 10. Cytotoxicity of YHKB aggregates.

Cell viability was determined by the MTT reduction test in SH-SY5Y cells exposed to 2 µM native YHKB (bar 1), YHKB aggregates incubated for 5, 30 and 120 min in 30% (v/v) TFE at pH 5.5 (bars 2–4), YHKB aggregates incubated for 5 min, 30 min, 120 min and 1 month at pH 1.7, 360 mM NaCl (bars 5–8). The reported values are representative of two independent experiments, each carried out four times.