To assess the thermostability of PSMα1 and PSMα3 fibrils, we conducted heat shock (HS) experiments followed by visualization using transmission electron microscopy (TEM) (Figure 2A). After incubation at 37°C for 2 days, both PSMα1 and PSMα3 fibrils were subjected to HS at 80°C for 10 min. TEM micrographs revealed the presence of abundant PSMα1 fibrils before and after HS, indicating their resistance to heat. However, the HS-treated PSMα1 fibrils appeared thinner and denser, suggesting a potential influence on fibrillation rate and morphology. In contrast, TEM images of PSMα3 fibrils indicated their lack of thermostability, as the fibrils had disaggregated following HS treatment.

To further confirm the HS-induced disassembly of PSMα3 fibrils, we analyzed the mobility of PSMα3 using a cationic native gel (Figure 2B). Initially, freshly dissolved PSMα3 exhibited a band corresponding to soluble species that migrated on the gel. After incubation, PSMα3 fibrils became immobile on the gel, indicating their aggregated state. However, HS treatment resulted in a band similar to that of freshly dissolved PSMα3, providing additional evidence for the disaggregation of PSMα3 fibrils back into soluble species.

Overall, our findings demonstrate the contrasting thermostability of PSMα1 and PSMα3 fibrils, with PSMα1 exhibiting resistance to HS-induced changes in structure and PSMα3 undergoing disaggregation upon heat shock treatment.

The size distribution of PSMα1 and PSMα3 was determined using dynamic light scattering (DLS) in different states: freshly dissolved, incubated before and after heat shock. The particle size distribution was plotted against volume, dividing the particles into three size ranges: <500 nm, 500–5,000 nm, and >5,000 nm (Figure 3; Table 1).

For PSMα1, the size population distribution remained relatively stable before and after incubation, with a slight increase in the average particle size from approximately 492 nm–651 nm. The percentage of large particles (>5,000 nm) also increased, which correlates with the formation of mature fibrils observed in the electron micrographs (Figure 2), although they probably represent a small fraction of the population of species. After HS, there was a further increase in the percentage of large particles at the expense of small particles, leading to an overall average particle size of approximately 1,254 nm. These results suggest that PSMα1 fibrillation is temperature-driven and indicate the thermostability of the fibrils.

In contrast, PSMα3 exhibited a distinct behavior compared to PSMα1. Incubation caused a substantial increase in the average particle size from approximately 444 nm–1,362 nm for PSMα3, accompanied by a decrease in the percentage of small-sized particles (<500 nm) from 89% to 38%. However, following HS, there was a notable shift towards smaller particles, with particles smaller than 500 nm constituting approximately 93.5% of the population. The total average size decreased to 160 nm, the smallest among all tested samples. These findings align with the TEM images and native gel results, which demonstrate the HS-induced breakdown of PSMα3 fibrils (Figure 2).

These results provide comprehensive information on the size distribution of PSMα1 and PSMα3 fibrils and confirm the thermostability of PSMα1 fibrils while demonstrating the sensitivity of PSMα3 fibrils to heat shock.

Heat shock treatment has distinct effects on the secondary structure of PSMα1 and PSMα3 fibrils, as determined by attenuated total internal reflection Fourier transform infrared (ATR-FTIR) spectroscopy (Figure 4). The secondary derivative of each FTIR spectrum was calculated to identify the major peaks contributing to the overlapping signals. Peaks in the regions of 1,611–1,630 cm⁻¹ and ∼1,685–1,695 cm⁻¹ indicate rigid cross-β fibrils, while peaks in the region of 1,637–1,645 cm⁻¹ represent disordered species (Goormaghtigh et al., 1990; Zandomeneghi et al., 2004; Sarroukh et al., 2013; Moran and Zanni, 2014). Additionally, peaks in the region of 1,646–1,662 cm⁻¹ suggest the presence of α-helices, but may overlap with random coil structures, especially for broad peaks (Barth and Zscherp, 2002; Cracchiolo et al., 2022).

PSMα1, after 2 h of incubation, exhibited a dominant helical population with a peak at 1,649 cm⁻¹. After 2 days of incubation, a minor cross-β population emerged alongside the major random/α-helical population. Heat shock treatment had a significant impact on both the 2-h and 2-day incubated samples, leading to a structural transition towards a predominantly cross-β conformation with a peak at 1,620 cm⁻¹ (Figure 4).

PSMα3, after 2 h or 2 days of incubation, displayed peaks at 1,651 cm⁻¹ and 1,649 cm⁻¹, respectively, indicating a predominant helical population, as previously shown (Tayeb-Fligelman et al., 2020; Zaman and Andreasen, 2020). In contrast to PSMα1, heat shock induced a changed into disordered morphology in both PSMα3 samples, with a peak at 1,645 cm⁻¹ (Figure 4). These findings are consistent with the results obtained from DLS (Figure 3), TEM, and native gel analyses (Figure 2), suggesting that heat shock disaggregates PSMα3 α-helical fibrils into soluble and less ordered species.

To assess the recovery of PSMα3 fibril formation after heat shock-induced disaggregation, thioflavin-T (ThT) fluorescence kinetics and TEM visualization were employed (Figure 5). Freshly dissolved PSMα3 displayed the characteristic amyloid lag time followed by rapid aggregation, consistent with previous findings (Tayeb-Fligelman et al., 2020; Zaman and Andreasen, 2020). After incubation and heat shock, PSMα3 exhibited a similar fibrillation curve, albeit with a slightly longer lag time. TEM micrographs confirmed the initial formation of PSMα3 fibrils (Figure 5B), the heat shock-induced disaggregation (Figure 5C), and subsequent reformation of PSMα3 fibrils with a similar morphology (Figure 5D). These results indicate the reversible nature of fibril formation following heat stress.

The cytotoxicity of PSMα1 and PSMα3 against T2 cells was assessed by measuring lactate dehydrogenase (LDH) release. The cytotoxicity was evaluated for freshly dissolved peptides, peptides incubated for 2 days, and incubated fibrils before and after heat shock (Figure 6).

When freshly dissolved, both PSMα1 and PSMα3 exhibited high toxicity, with 10 μM concentration resulting in 70% and 100% cell death, respectively. Heat shock of the freshly dissolved samples did not significantly alter their toxicity, indicating the stability of the peptides at 80°C. After 2 days of incubation, PSMα1 showed a slight reduction in cytotoxicity. However, heat shock of the incubated samples further decreased the cytotoxicity of PSMα1. At a concentration of 5 μM, cell death reduced to only 4% after heat shock, compared to 49% for the fresh sample. At 10 μM, cytotoxicity decreased to 26% (compared to 70%). In contrast, PSMα3 cytotoxicity was abolished after 2 days of incubation, but almost fully restored after heat shock (Figure 6).

PSMα1 and PSMα3 (>98% purity) were purchased from GL Biochem (Shanghai) Ltd. The peptides were dissolved at 5 mM in 20% dimethyl sulfoxide (DMSO) and 80% double-distilled water (DDW) and incubated at 37°C for 2 h or 2 days. Heat shock treatment was induced by incubating the samples at 80°C for 10 min.

The cytotoxicity assay was performed using human lymphoblast T2 cells (174 x CEM.T2) obtained from ATCC® (CRL-1992™). The cells were cultured in the assay medium, namely, RPMI 1640 medium with L-glutamine from Sigma (Israel) and supplemented with penicillin (100 U/mL), streptomycin (0.1 mg/mL), and 10% heat-inactivated fetal calf serum from Biological Industries (Israel). The cells were maintained at 37°C in a 5% CO2 environment. For the cytotoxicity assay, freshly dissolved peptides and samples incubated for 2 days at 37°C, with and without subsequent heat shock treatment, were used. The peptide samples were diluted to a concentration of 1 mM in DDW and then further diluted to 160 μM in the assay medium.

Serial twofold dilutions of the peptides were prepared in the assay medium, and 50 μL of each dilution was added in triplicate to a 96-well plate. The plate was incubated for 15 min at room temperature. The T2 cells were washed and resuspended in the assay medium to a concentration of 0.15 × 106 cells/mL, and 50 μL of the cell suspension was added to the diluted peptides in the plate. The plate was then incubated for 30 min at 37°C in a 5% CO₂ environment. Serial twofold dilutions of the peptides were prepared in the assay medium, and 50 μL of each dilution was added in triplicate to a 96-well plate. The plate was incubated for 15 min at room temperature. The T2 cells were washed and resuspended in the assay medium to a concentration of 0.15 × 106 cells/mL, and 50 μL of the cell suspension was added to the diluted peptides in the plate. The plate was then incubated for 30 min at 37°C in a 5% CO₂ environment.

The quantification of cell lysis was performed using the lactate dehydrogenase (LDH) release assay according to the manufacturer’s instructions. The LDH release was measured using the LDH Cytotoxicity Detection Kit Plus from Roche Applied Science (Germany), including all recommended controls. The absorbance at 490 nm and 690 nm was measured using a plate reader (FLUOstar Omega, BMG Labtech, Germany). The absorbance values at 690 nm were subtracted from the absorbance values at 490 nm, and the average absorbance values of triplicate samples and controls were calculated after subtracting the background. The experiment was repeated three times on different days. The percentage of cytotoxicity for each sample was determined by averaging the data from all replicates and repeats. Error bars were included to represent the standard errors of the mean, providing a measure of the variability in the results. The p-value was calculated using a t-test to compare the % cytotoxicity of the freshly dissolved peptide with the incubated peptide, and of the incubated samples before and after heat shock, at each concentration, as displayed in Figure 6.

ThT is a widely used “gold standard” stain for identifying and studying the kinetics of amyloid fibril formation, both in vivo and in vitro. Fibrillation curves in the presence of ThT typically show a lag time for the nucleation step, followed by rapid aggregation. To ensure the presentation of the fibrillation lag time, PSMα3 was pre-treated prior to the experiment by dissolving in 1:1 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and Trifluoroacetic acid (TFA) to a concentration of 1 mg/mL, followed by 10 min bath-sonication, at room temperature. The organic solvent was evaporated using a mini-rotational vacuum concentrator (Christ, Germany) at 1,000 rpm for 2 h, at room temperature. For the experiment, fresh PSMα3 peptides and incubated PSMα3 after HS were diluted to 100 μM in phosphate buffer, pH 7.5, containing filtered ThT diluted from the stock prepared in UPddw. The final concentrations for each reaction were 100 µM peptide and 200 µM ThT. Blank solutions containing all components except from the peptides/fibrils were also prepared for each reaction. The reaction was carried out in black 96-well flat-bottom plates (Greiner bio-one) covered with a thermal seal film (EXCEL scientific) and incubated in a plate reader (OMEGA) at a temperature of 37°C with shaking at 500 rpm for 85 s before each reading cycle, and up to 1,000 cycles of 6 min each. Measurements were performed in triplicate. Fluorescence was measured by excitation at 438 ± 20 nm and emission at 490 ± 20 nm over a period of approximately 100 h. All triplicate values were averaged, appropriate blanks were subtracted, and the resulting values were plotted against time. Calculated standard errors of the mean are shown as error bars. The whole experiment was repeated at least three times on different days.

Samples (4–5 µL) of incubated PSMα1 and PSMα3, with and without HS treatment were applied directly onto glow-discharged (easiGlow; Pelco, Clovis, CA, United States, 15 mA current; negative charge; 25 s time) 400 mesh copper grids, with a grid hole size of 42 μm, stabilized with Formvar/carbon (Ted Pella, Inc.). Samples were allowed to adhere for 60 s. Samples were then stained with 1% uranyl acetate solution (Electron Microscopy Science, 22400-1) for 60 s. Samples were examined in the Ilse Katz Institute for Nanoscale Science and Technology, Ben Gurion University of the Negev, Israel, with a ThermoFisher Scientific (FEI) Talos F200C transmission electron microscope operating at 200 kV, equipped with a Ceta 16M CMOS camera.

PSMα1 and PSMα3 were subjected to HFIP pretreatment according to the method described for the ThT experiment. The lyophilized peptides were then dissolved in 5 mM hydrochloric acid (HCl) at a concentration of 1 mg/mL and sonicated for 5 min at room temperature using a bath sonicator. Subsequently, the peptide solution was frozen in liquid nitrogen and lyophilized overnight to ensure complete drying. This process was repeated twice to ensure the removal of any residual TFA, as TFA exhibits a strong FTIR signal in the amide I′ region of the spectra. Finally, the peptides were dissolved in deuterium oxide (D2O) at a concentration of 1 mg/mL, frozen in liquid nitrogen, and lyophilized overnight to achieve complete dryness. This double procedure was repeated. The dried peptides were then dissolved in D2O to a concentration of 5 mM, and samples were prepared for measurement after 48 h of incubation, both with and without heat shock.

A volume of 5 μL of each sample was spread onto the surface of the ATR module and allowed to dry. Absorption spectra were recorded using a Tensor 27 FTIR spectrometer (Bruker Optics) on the dried samples. Measurements were conducted in the wavelength range of 400–4,000 cm⁻¹ with a step size of 2 cm⁻¹, and an average of 32 scans was taken. Background (air) and blank (D2O) absorbances were measured and found to have a negligible signal. Peaks were selected by the instrument based on the second derivative using OPUS software.

PSMα1 and PSMα3 peptides were dissolved in a solution containing 20% DMSO and 80% DDW at a concentration of 5 mM. They were then incubated at 37°C for 2 and 48 h. The freshly dissolved peptides underwent pretreatment by dissolving them in HFIP at a concentration of 1 mg/mL, followed by a 10-min bath sonication at room temperature. The organic solvent was evaporated using a mini-rotational vacuum concentrator (Christ, Germany) at 1,000 rpm for 2 h at room temperature. The treated peptides were also dissolved in a solution containing 20% DMSO and 80% DDW at a concentration of 5 mM.

Freshly dissolved samples, and incubated ones with and without HS treatment (20–30 μL), were mounted using 1.0 × 1.0 mm disposable cuvette capillaries with a thickness of 200 μm (Malvern, ZSU0003), which were then sealed with clay. The capillaries were placed within a low-volume disposable sizing cell Kitholder (Malvern, ZSU1002). Light scattering readings were measured using a ZetaSizer Ultra (Ultra ZS; Malvern). Hydrodynamic radii (Rh) were determined using backscattered light at a fixed angle of 90°. A 633 nm wavelength He–Ne laser was used. The cell holder was maintained at 37°C throughout the measurement. The scattering data were obtained from two (fresh samples) or three (incubated samples) different measurements performed on different days, each consisting of nine and thirteen consecutive scans, respectively.

For each peptide, the particle size distribution was plotted against intensity or volume and each representation provides a different perspective. In particular, even for particles with a perfect spherical shape and smooth morphology, the intensity distribution for a fixed mean size shifts towards larger sizes with increasing polydispersity (Borah and Verbruggen, 2022). Therefore, we chose to represent the particle size distribution plotted against volume, to be able to better observe smaller particles. The mean volume intensities of the particles were divided into three subpopulations based on their size: 0–500 nm, 500–5,000 nm, and greater than 5,000 nm. To calculate the average size of particles in each subpopulation, the sum of the multifaction of different particles sizes in the corresponding percentage of volume intensity for each size was divided by the sum to the total percentage of volume intensity for the range. To calculate the error presented in Figure 3 and Table 1, the standard deviation was divided by 100, and the square root of the number of measurements.

To determine the average size of particles within each subpopulation presented in Figure 3 and Table 1, the following calculation was performed: the sum of the product of different particle sizes and their corresponding percentage of volume intensity was divided by the total percentage of volume intensity within the size range. To calculate the error, the standard deviation was divided by the square root of the number of measurements and by 100.

Native gel was prepared as follows: The separating gel consisted of 385 mM acetate-KOH pH-4.3, 11% glycerol, 10% acrylamide 35.5:1, 0.12% ammonium persulfate (APS) and 0.15% tetramethylethylenediamine (TEMED). The stacking gel consisted of 62.5 mM acetate-KOH pH = 6.8, 8% acrylamide 35.5:1, 0.1% APS and 0.1% TEMED. The separating gel solution was dispensed into the gel template, covered with isopropanol and incubated for polymerization for 1 h at room temperature. After polymerization, residual isopropanol was removed and stacking gel was added for 1 h to allow complete polymerization on top of the separating gel. The running buffer contained 0.35 M β-alanine and 0.14 M acetate at pH 4.3. PSMα3 samples of freshly dissolved peptide and after incubation, with and without HS treatment, were loaded in X5 sample buffer containing 37.2% glycerol, 62.5 mM acetate KOH pH 6.8% and 0.01% methyl green. The gel was run on Bio-Rad mini-PROTEAN tetra system units with reverse polarity at a constant voltage of 150 V.