Toxicity of Protein Oligomers Is Rationalized by a Function Combining

Jul 31, 2014 - ABSTRACT: The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. ...
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Toxicity of Protein Oligomers Is Rationalized by a Function Combining Size and Surface Hydrophobicity Benedetta Mannini,† Estefania Mulvihill,† Caterina Sgromo,† Roberta Cascella,† Reza Khodarahmi,‡ Matteo Ramazzotti,† Christopher M. Dobson,§ Cristina Cecchi,† and Fabrizio Chiti*,† †

Department of Biomedical Experimental and Clinical Sciences, Section of Biochemistry, University of Florence, 50134 Florence, Italy Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran § Department of Chemistry, University of Cambridge, CB2 1EW Cambridge, United Kingdom ‡

S Supporting Information *

ABSTRACT: The misfolding and aberrant assembly of peptides and proteins into fibrillar aggregates is the hallmark of many pathologies. Fibril formation is accompanied by oligomeric species thought to be the primary pathogenic agents in many of these diseases. With the aim of identifying the structural determinants responsible for the toxicity of misfolded oligomers, we created 12 oligomeric variants from the N-terminal domain of the E. coli HypF protein (HypF-N) by replacing one or more charged amino acid residues with neutral apolar residues and allowing the mutated proteins to aggregate under two sets of conditions. The resulting oligomeric species have different degrees of cytotoxicity when added to the extracellular medium of the cells, as assessed by the extent of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, apoptosis, and influx of Ca2+ into the cells. The structural properties of the oligomeric variants were characterized by evaluating their surface hydrophobicity with 8-anilinonaphthalene-1-sulfonate (ANS) binding and by measuring their size by means of turbidimetry as well as light scattering. We find that increases in the surface hydrophobicity of the oligomers following mutation can promote the formation of larger assemblies and that the overall toxicity correlates with a combination of both surface hydrophobicity and size, with the most toxic oligomers having high hydrophobicity and small size. These results have allowed the relationships between these three parameters to be studied simultaneously and quantitatively, and have enabled the generation of an equation that is able to rationalize and even predict toxicity of the oligomers resulting from their surface hydrophobicity and size. oligomers generate highly reproducible results.8−10,16 Finally, and very importantly, HypF-N oligomers are sufficiently stable, allowing a detailed biophysical and biological characterization of their structural properties and toxicity, respectively.8 Two protocols have been established to convert native HypF-N into stable oligomers, which were denoted type A and type B, respectively.8,9,11 These oligomeric forms share a similar size and morphology, as observed using atomic force microscopy (AFM), as both are roughly spherical, with a diameter of 2−6 nm. Both oligomeric forms also bind the dye thioflavin T (ThT), resulting in similar levels of fluorescence enhancement, though lower than is commonly observed for fully formed amyloid fibrils.8 In spite of their similar size, morphology and ThT binding properties, only type A oligomers have been found to display significant toxicity in cultured neuronal cells, cultured primary neurons, and whole animal models.8,9,11 We have shown, using ANS binding and

The aberrant self-assembly of protein molecules is associated with a group of highly debilitating medical conditions, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and type II diabetes.1 In such pathological conditions, it is thought that an important role is played by small protein oligomers. These oligomers are generally metastable and structurally heterogeneous, making them extremely challenging to isolate and characterize.2−5 Studies on the HypF-N protein have contributed to much of our knowledge of the properties of these putatively pathogenic oligomers.3,6−11 First, HypF-N readily forms long-lived spherical oligomers as well as amyloid-like fibrils in vitro.8,12−15 Second, the oligomers impair cell viability when added to the extracellular medium of cultured cells3,6,8−10 or when injected into rat brains.7,9 Third, the oligomers bind to cultured primary rat neurons and colocalize with postsynaptic densities, inhibit long-term potentiation (LTP) in rat hippocampal slices and induce cognitive impairment following their injection into rat brains.11 Such characteristics resemble those of the Aβ42 oligomers at the biochemical, molecular biological, electrophysiological, and animal model levels.11 Fourth, studies of the aggregation of HypF-N and the toxicity of the resulting © XXXX American Chemical Society

Received: June 25, 2014 Accepted: July 31, 2014

A

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site directed pyrene labeling, that the three main hydrophobic regions of the HypF-N sequence, spanning approximately residues 22−34, 55−59, and 75−87, are highly structured and buried in type B oligomers, whereas they are more flexible and solvent-exposed in type A oligomers.8 It has also been found that further assembly of type A oligomers into large aggregates could be mediated by molecular chaperones, resulting in suppression of the toxicity of the oligomeric species.10,16 These two reports indicate that the flexibility and solvent exposure of hydrophobic moieties, along with their small size, are important structural determinants of HypF-N oligomer toxicity. In the present study, we have used site-directed mutagenesis to increase the hydrophobicity of the three main regions of the HypF-N sequence. The mutations were found to affect the toxicity, the size and the solvent-exposed hydrophobicity of the resulting HypF-N oligomers to different extents, allowing the various parameters to be analyzed in combination with each other and in quantitative manner. We also show that the solvent-exposed hydrophobicity and the aggregate size can be combined in a three-dimensional analytical approach to rationalize the effects of both factors on oligomer toxicity and to provide further insights into the general and intrinsic structural determinants of protein oligomer toxicity.

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RESULTS AND DISCUSSION Effects of Hydrophobic Mutations on the Structure and Aggregation Kinetics of HypF-N. Five variants of HypF-N, namely, R23L, E55L, E87L, R23L/E55L, and R23L/ E87L, were designed in which glutamate or arginine residues were replaced by leucine residues (Figure 1A) within the three

Figure 1. Structure of HypF-N variants. (A) Structure of wt HypF-N (PDB entry 1GXU) showing the residues mutated in the present study. (B) Far-UV CD spectra of the wt and mutational variants of HypF-N acquired under native conditions.

Figure 2. Aggregation of HypF-N variants. (A) Aggregation timecourses of wt and mutant HypF-N samples measured by Thioflavin T (ThT) fluorescence under conditions A (top panels) and B (bottom panels). All data points were blank-subtracted and normalized to the maximum fluorescence intensity. Solid lines represent the best fits of the data points to a single-exponential function (eq 3). (B) Apparent rate constants of aggregation (kagg) under conditions A (left) and B (right), obtained from the analysis of the best fits shown in part A. Error bars correspond to standard errors of the means of at least 4 independent experiments. The triple asterisks indicate p < 0.001 with respect to wt oligomers formed under corresponding conditions.

main hydrophobic regions of the HypF-N sequence (residues 22−34, 55−59, and 75−87). The far-UV circular dichroism (far-UV CD) spectra of the mutant and wt proteins in their native states are closely similar to that of the wt protein obtained in the present study (Figure 1B), and indeed to those reported previously,14,15 indicating that the introduction of the hydrophobic mutations does not alter significantly the native secondary structure of the protein. Twelve different types of oligomers were obtained by incubating individually the wt and the five mutational variants for 4 h at 25 °C under the two different experimental conditions, A and B, respectively (see Methods for details). The aggregation time courses of each variant under each condition were therefore measured using ThT fluorescence and fitted to a single exponential function, that is shown in eq 3 (Figure 2A).

Apparent rate constants for aggregation (kagg) were calculated from all the kinetic traces and are reported in Figure 2B for conditions A (left) and B (right). Under condition A, the kagg values measured for the variants are similar to that of the wt protein (0.0030 ± 0.0009), with the exception of the E87L protein, which has a substantially higher kagg value (0.017 ± 0.003). Under condition B, none of the variants has a significantly different kagg values from that of the wt protein. B

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the toxicity of the oligomers. The oligomers formed by the variants under condition B, however, showed no significant toxicity with the exception of the R23L mutant, which was observed to reduce slightly the cellular viability by 12.8 ± 2.7% (Figure 3). In control experiments, the native states of all the variants were tested and found in no case to cause detectable cellular dysfunction (Figure 3). The toxicities of the 12 oligomeric species were also evaluated using the apoptotic marker Hoechst 33342 in conjunction with fluorescence microscopy, as well as by measuring the levels of intracellular Ca2+ ions in association with confocal microscopy (Supporting Information Figures S1 and S2). The results are fully consistent with those obtained with the MTT reduction assay. Effect of the Mutations on the Surface Hydrophobicity of the Oligomers. To shed light on the link between the toxicity of the HypF-N oligomeric aggregates, described above, and their structural properties, we focused on defining the exposed hydrophobic surface areas and the sizes of the various oligomeric species. The former was probed by evaluating the ability of the fluorescent probe ANS to bind to the oligomers. ANS binds to solvent-exposed hydrophobic clusters, and this binding generates a large increase in its fluorescence emission intensity and a blue shift of its maximum emission wavelength (λmax).18 All the oligomers formed under condition A were found to bind to ANS, as an enhancement of its fluorescence emission was registered for all of them, though to different extents (Figure 4A). Thus, for example, the aggregates formed by the E55L and R23L/E55L variants showed an increase of their ANS emission relative to the wt oligomers, whereas those formed by the E87L and R23L/E87L variants resulted in ANS spectra similar to that of the wt oligomers. The behavior of the oligomers from the R23L variant is, however, quite distinct as its spectrum with ANS is dramatically reduced in intensity by a factor of 90.9 ± 0.4% relative to that of the wt oligomers. A notable blue shift of the ANS λmax value is observed for all the mutants compared to the wt oligomers, with the exception again of the R23L variant, which is characterized by a λmax value that is less blue-shifted than that of the wt protein (Figure 4C). Oligomers formed under condition B by all the variants were able to bind to ANS, and the spectra recorded for the single and the double variants showed, respectively, a very weak and a more prominent increase in fluorescence intensity relative to the type B oligomers of the wt protein (Figure 4B). These results correlate well with the observed ANS λmax values, as the oligomers formed by the single mutants had λmax values approximately comparable to those of the wt oligomers, whereas the aggregates formed by the double mutants showed λmax values with significantly larger blue-shifts (Figure 4D). These data show that the introduction of more highly hydrophobic residues by mutation generates a significant increase in the total solvent exposed hydrophobic surface areas of the oligomers formed under condition A, with the exception of the R23L mutation, which leads to a decrease in the exposed hydrophobic surface area. An increase of this magnitude is not observed in the case of the mutant oligomers formed under condition B, although a small but consistent trend toward a greater hydrophobic exposure is also observed under this condition. Effect of the Mutations on the Sizes of the Oligomers. To estimate the sizes of the type A and B oligomeric species, measurements of turbidimetry at 500 nm and of static light

These experiments also show that the aggregates formed by the wt and all the variant proteins bind to the amyloid-specific dye ThT, although to a lower extent compared to mature fibrils, but consistent with the presence in each case of the ordered intermolecular β-sheet structure. Moreover, the development of such structure in all the mutational variants follows a kinetic profile that is effectively a single exponential, with no evidence for a lag phase, consistently with oligomers that precede fibril formation17 and with previously characterized type A and B oligomers formed from the wt protein.8 Overall, therefore, the rate of the aggregation process does not appear to be significantly affected by the introduction of the hydrophobic mutations, which the single exception of the E87L variant under condition A. Effect of the Mutations on the Toxicity of the Oligomers. The toxicity of the oligomers formed by the hydrophobic variants under conditions A and B was assessed using human neuroblastoma SH-SY5Y cell cultures and evaluating cell viability using the MTT reduction inhibition assay, the Hoechst staining test and measuring the levels of intracellular Ca2+. For this purpose, the various aggregates were transferred from the solutions in which they were formed into a physiological medium and then added to the cell culture media before performing the MTT reduction assay. Type A and B oligomers generated by wt HypF-N caused a 29.1 ± 2.6% and a 2.6 ± 1.9% decrease in the viability of SH-SY5Y cells, respectively (Figure 3), consistent with previously reported

Figure 3. Toxicity of the oligomeric variants. 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay using SH-SY5Y cells treated with wt and mutant HypF-N proteins in their native states (light gray bars) and after aggregation under conditions A (dark gray bars) and B (black bars). Error bars correspond to standard errors of the means of 10 independent experiments. Single, double, and triple asterisks refer to p < 0.05, 0.05). Interestingly, however, the size of all the oligomers studied here correlates strongly with their surface hydrophobicity (Figure 6C, r = 0.89, p < 0.001). This statistically highly significant correlation reveals that the greater the overall hydrophobicity of the protein, the greater is the exposure of hydrophobic groups on the surface of the oligomers and the greater their size. Since the size and the extent of exposure of hydrophobic groups on the surface have been found to decrease and to increase, respectively, the toxicity of the HypF-N oligomers,8,10,16 no correlation is observed between the measured toxicity and either of these factors, when analyzed alone (Figure 6A and 6B). Indeed, a mutation that increases the surface hydrophobicity of the oligomers, and is thus expected to augment their toxicity, also increases their size; the latter increase is expected to result into a decrease of oligomer toxicity. To analyze concomitantly the three parameters of toxicity, size and surface hydrophobicity together, the graph of “size versus surface hydrophobicity” shown in Figure 6C was replotted in Figure 6D to distinguish between nontoxic and toxic oligomers in orange and purple, respectively. The nontoxic and toxic oligomers are all located above and below

R θ = 4.64 × 10−2 − 9.6 × 10−5λmax

(1)

where Rθ is the light scattering intensity expressed as the Rayleigh ratio (in cm−1 units) and λmax is the wavelength of the maximum fluorescence emission of ANS (in nm units). This equation partitions the data points into two regions of the graph indicating that a combination of high hydrophobicity and small size contributes to a high level of toxicity (data below the line). In Figure 6E and 6F, data describing the oligomeric variants are shown on a three-dimensional graph, viewed on different scales, which plots ANS λmax (x-axis), Rayleigh ratio (y-axis), and MTT reduction (z-axis). The data points were analyzed using a procedure of multiple linear regression in order to evaluate the possibility of predicting cell toxicity from a combination of both surface hydrophobicity and size. The resulting three-variable equation of best fit, describing a plane in the three-dimensional graph, has the form: E

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indeed influenced by both their surface hydrophobicity and their size and, most importantly, is the consequence of a fine balance between these two structural determinants. Further statistics on the procedure of best fitting are reported in Supporting Information Figure S3. The model was evaluated using bootstrap, jackknife and cross-validation methods and appears to be extremely robust; this finding indicates that eq 2 has predictive power for HypFN oligomers, as values of surface hydrophobicity (λmax) and size (Rθ) of a given HypF-N oligomer type can be used to predict its level of toxicity, at least under the ranges of λmax and Rθ investigated here (Supporting Information Tables S1 and S2 and Methods). It needs to be clarified, however, that eq 2 has accurate predictive value for the conditions used here, that is for the protein type, protein concentration and cell type used here. Indeed, it is widely accepted that cell toxicity also depends on protein concentration and cell type.3,19 Oligomer Size and Surface Hydrophobicity Together Determine Toxicity. Introduction of hydrophobic residues in the HypF-N sequence by mutagenesis has generated a set of oligomeric variants that are characterized by different degrees of toxicity, which provides an opportunity to explore how toxicity correlates with differences in the structures and morphologies of the aggregates in a quantitative manner. The structural characterization of the oligomers has been based on two parameters, both of which are recognized to play important roles as determinants of aggregate toxicity, namely, the degree of exposure of hydrophobic groups 8,20−27 and the size.2,10,24,28−31 In the present work, the selective replacement by mutation of hydrophilic by hydrophobic residues in the HypF-N sequence has been observed to increase the surface hydrophobicity of both the type A and B oligomers to different extents and, in addition, to generate an increase in the dimensions of the aggregates. This latter effect can be rationalized as a consequence of the increase in the exposure of hydrophobic groups to the solvent, facilitating the interactions between monomers such that the number of molecules contained in the most stable species is increased. It therefore follows that a correlation exists between solvent-exposed hydrophobicity and size in the group of 12 oligomeric species studied here, with large oligomers also having a higher surface hydrophobicity (Figure 6C). Despite this correlation, an increase in the degree of solventexposed hydrophobic surface and of the size of a misfolded protein oligomer will act as opposing factors in its ability to cause cellular dysfunction. Indeed, the data on the toxicity of misfolded protein oligomers obtained in this study cannot be explained adequately on the basis of either parameter considered individually. Thus, for example, the increase in size observed for type A oligomers of E55L and R23L/E55L is similar (Figure 5A and C), but the magnitude of the effects of the oligomers on cell viability is significantly different (Figure 3). Indeed, type A oligomers of E55L are not detectably toxic, whereas type A oligomers of R23L/E55L cause a 20% reduction of cell viability relative to untreated cells. However, type A oligomers of R23L/E55L have a higher degree of surface hydrophobicity than do type A oligomers of E55L (Figure 4A and C), which provides an explanation for its higher degree of toxicity. In Figure 6E and F, all the 12 oligomeric species studied here are plotted on just one graph correlating the value of their Rayleigh ratio, ANS λmax value (indicating size and the surface

Figure 6. Correlation of properties of the oligomeric species of wt and mutated HypF-N. (A) MTT reduction versus ANS λmax of type A (red) and type B (blue) oligomers. (B) MTT reduction versus light scattering intensity, expressed as the Rayleigh ratio of type A (red) and type B (blue) oligomers. (C) The Rayleigh ratio versus ANS λmax of type A (red) and type B (blue) oligomers. The solid line represents the best fit of the data points to a linear function, corresponding to eq 1. (D) As in part C, but the orange and purple squares correspond to oligomers characterized by MTT reduction values above 96% and ranging from 70% to 90%, respectively. (E) 3D graph showing ANS λmax (x-axis), Rayleigh ratio (y-axis) and MTT reduction values (zaxis) of the 12 oligomeric species studied here, which are highlighted again using the color scale corresponding to MTT reduction values and reported on the right. The red grid represents the plane of best fit to all the data points and corresponds to eq 2. (F) Enlargement in the vertical direction (MTT reduction) of part E.

RMTT = (4.13λmax ) + (3.9 × 104R θ) − 1.9 × 103

(2)

where RMTT is the observed MTT reduction (as percent of untreated cells). This equation is highly significant as indicated by the statistical parameters (Figure 6E, adjusted R2 = 0.86, p = 5.27 × 10−5). Interestingly, all the data points are close to the plane of best fit suggesting that the toxicity of the oligomers is F

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More generally, understanding the contribution of each structural determinant to the toxicity of specific oligomeric species is of vital importance for clarifying the generic molecular mechanisms by which misfolded oligomers can be harmful to cells and contribute to the pathogenesis of protein deposition diseases.

hydrophobicity, respectively) and ability to reduce cellular viability. A very strong correlation exists between the three parameters with all data points fitting closely on a downhill plane that relates oligomer toxicity to both their size and surface hydrophobicity. It is remarkable that a simple combination of the values of these two parameters can explain quantitatively the biological effects of the different oligomers and even have predictive power as indicated by the bootstrap, cross validation and jacknife methods. This result reveals yet again the striking ability of physical principles to explain in a quantitative manner the biological effects of the consequences of protein misfolding and aggregation.1,32 Size and Surface Hydrophobicity Are Generic Determinants of Misfolded Oligomer Cytotoxicity. Recent studies indicate that the size of protein misfolded aggregates plays a key role in defining the levels of their toxicity. The ability of Aβ40 and Aβ42 oligomers to decrease MTT reduction in cell cultures was found to correlate inversely with the molecular weight of the oligomers.2 Other studies have also found an inverse correlation between the size of Aβ42 oligomers and neuronal toxicity.24,29 In addition, certain agents, such as aromatic small molecules30 or molecular chaperones,10,31 have been found to promote the further assembly of toxic oligomers into larger aggregates, thereby suppressing the overall toxicity. By contrast, the fragmentation of fibrils formed by β2microglobulin, α-synuclein or hen egg white lysozyme was found to result in a toxicity increase.28 Larger assemblies are likely to be less harmful because they have a lower surface/ volume ratio, which reduces their potential to interact with specific cellular components such as membranes. Larger assemblies also have a lower diffusional mobility, which reduces their ability to reach their molecular targets and transmit toxicity within and between cells, perhaps limiting the spreading and intrusion within an organ such as the brain. As far as surface hydrophobicity is concerned, it has been observed in a range of studies that an increase in the exposure of hydrophobic groups on the surface of oligomers is accompanied by an increase in their ability to cause cellular dysfunction. An early report showed that a higher degree of surface hydrophobicity in Aβ40 aggregates is linked to an increased capacity to affect the fluidity of model membranes.20 Subsequently, a correlation was found between hydrophobicity and cytotoxicity for aggregates formed by a series of polypeptides including homopolymers of amino acids, HypFN, the E22G variant of the Aβ42 peptide, the NM region of yeast Sup35p and also rationally designed β-sheet proteins, indicating that this effect is a common or generic property of misfolded species.8,21,23,25−27 Conclusions. In conclusion, the biological activity of misfolded protein oligomers of a specific protein cannot be explained fully on the basis of either surface hydrophobicity or size if these two parameters are considered separately. By exploring the properties of protein oligomers where size and surface hydrophobicity have been changed concomitantly and to different extents by mutation of individual residues, however, it has been possible to interpret the toxicity of protein oligomers in a quantitative manner as a function of both parameters. This analysis has not only enabled the behavior of all the different mutational variants to be rationalized but also enables the effects of specific mutations to be predicted with great accuracy for a given protein, cellular system and set of conditions.

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METHODS

Preparation of HypF-N Oligomers. Mutations in the HypF-N gene were generated as described previously.8 Protein expression and purification were also carried out as described previously.8 The purified variants were stored at −20 °C in 20 mM phosphate buffer with 2 mM dithiothreitol (DTT) at pH 8.0. Oligomeric aggregates of HypF-N were prepared by incubating the wt or mutated proteins for 4 h at 25 °C and at a concentration of 48 μM under two different experimental conditions: (i) 50 mM acetate buffer, 12% (v/v) TFE, 2 mM DTT, pH 5.5 (condition A) and (ii) 20 mM TFA, 330 mM NaCl, pH 1.7 (condition B). For cell biology experiments, the oligomers were centrifuged at 16,100g for 10 min, dried under N2 and resuspended in cell culture media. Far-UV CD Measurements. Far-UV CD measurements were performed using a Jasco J-810 spectropolarimeter (Jasco) and a 1 mm path length quartz cell. The spectra were acquired at 19 μM protein concentration, 25 °C, in 20 mM potassium phosphate buffer, 2 mM DTT, pH 8.0. Thioflavin T Kinetics Assay. Samples of wt or mutated HypF-N were incubated at 48 μM under conditions A and B. At different time points, aliquots of each sample were added to a solution of 25 μM ThT (Sigma-Aldrich) dissolved in 25 mM phosphate buffer at pH 6.0, in order to keep a 3.7-fold molar excess of dye. The final protein concentration was 6 μM. The steady-state intensity of fluorescence emission at 485 nm (excitation at 440 nm) was recorded at 25 °C using 2 × 10 mm path-length cell and a PerkinElmer LS 55 spectrofluorimeter (PerkinElmer). The blank-subtracted ThT fluorescence emission intensity, F(t), is reported as a function of time, t, and fitted to the following single exponential function:

F(t ) = a + b e−kaggt

(3)

where a is the plateau value, b is the amplitude of the exponential phase, and kagg is the apparent rate constant of the protein aggregation process. For comparison, the data reported in Figure 2A were normalized to their plateau values and fitted again to eq 3. Cell Cultures. Human SH-SY5Y neuroblastoma cells (A.T.C.C.) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) F-12 Ham with 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) and NaHCO3 (1:1) supplemented with 10% fetal bovine serum (FBS), 1.0% glutamine, and 1.0% antibiotics. The cell cultures were maintained in a 5.0% CO2 humidified atmosphere at 37 °C and grown until 80% confluence for a maximum of 20 passages. MTT Assay. The toxic effects of the oligomer aggregates formed under conditions A and B by wt HypF-N and its mutational variants were assessed by the MTT assay, as described previously.16 Data are expressed as the mean ± SEM, and comparisons between the different groups were made by ANOVA followed by Bonferroni’s t test. P values