2.1. Mechanism of Amyloid Fibril Degradation Induced by Trypsin: Fragmentation, Disordering, and Monomerization
Model lysozyme amyloid fibrils, the accumulation of which leads to the development of hereditary lysozyme systemic amyloidosis (ALys amyloidosis, [
28]), were selected as one of the objects of study. Literature data indicate that trypsin exhibits proteolytic activity in a narrow range of neutral pH [
31,
32,
33]. In this regard, lysozyme amyloid fibrils prepared using the standard protocol (see Materials and Methods) were transferred to a solution with pH 7.4 to analyze the action of trypsin. Previously, we showed that mature lysozyme fibrils, transferred to physiological conditions, were stable for at least a week [
34]. Based on the literature, the ratio of trypsin and amyloid-forming protein in the experiment was chosen equal to 1: 125 [
35,
36,
37].
Considering the fact that trypsin is susceptible to autolysis, first of all, we checked the rate of this process in phosphate buffer with pH 7.4 at 37 °C using fluorescence spectroscopy and SDS-PAGE (
Figure S1). This allowed us to determine the duration of the experiment on fibril degradation. It turned out that, under the chosen conditions, trypsin actually underwent autolysis, and after 24 h, the band corresponding to this protein on SDS-PAGE became indistinguishable (
Figure S1A). Similar results, indicating the autolysis of trypsin during the day, were obtained when analyzing changes in the intrinsic tryptophan fluorescence intensity and parameter
A of the protease (parameter
A is the ratio of fluorescence intensities at wavelengths of 320 and 365 nm, sensitive to the position and shape of the fluorescence spectrum) [
38,
39]. Thus, it was concluded that, under the chosen conditions, trypsin could be active for at least 24 h. For example, residual activity of trypsin was observed in a concentrated solution of BSA even after prolonged incubation (
Figure S2). Since the weight ratio of trypsin to amyloidogenic protein in the sample was 1 to 125, it is highly likely that trypsin predominantly affects the amyloid-forming protein, and not itself. It is important to note that trypsin was not previously incubated in neutral buffer separately but was added directly into the fibril suspension. In this regard, it was decided to study the effect of trypsin on fibrils for at least 24 h and while detectable changes are observed.
Next, we studied trypsin binding to fibrils. For this aim, amyloid fibrils after trypsin adding and the enzyme alone (control) were incubated in PBS (pH 7.4) for 10 min at 37 °C. Then, trypsin was inactivated using phenylmethylsulfonyl fluoride (PMSF), and the samples were centrifuged at high speed (18,000 rpm) for 1 h. Supernatants of both samples were collected and analyzed using SDS-PAGE under denaturing conditions (
Figure S3). The results indicated the absence of trypsin in the supernatant of the sample with amyloid fibrils, although in the supernatant of the control (without fibrils), a bright band corresponding to the protease molecular weight was clearly seen (
Figure S3). This means that in the sample with amyloids, all trypsin molecules remained in the pellet in a bound to fibrils state. Thus, the trypsin binding to amyloid fibrils was experimentally confirmed.
In order to study trypsin’s effect on amyloid stability, we detected changes in the intrinsic photophysical characteristics of fibrils and the characteristics of specific fluorescent probes bound to them (
Figure 1). It was noted that the most significant change in the recorded characteristics occurred in the first few hours after the start of the experiment, and then smoother changes were observed over the next five days. Further, we investigated changes in the structure and properties of amyloid fibrils after them being exposed to trypsin, but we do not claim that trypsin hydrolyzes the fibrils during all five days of the experiment. These changes can both directly be caused by the enzyme present in the fibril sample (during the first day) and can be a delayed effect of short-term exposure to trypsin after its autolysis (for example, as a result of fibrils’ destabilization, which promotes their further gradual degradation).
Visualization of structural changes in amyloid fibrils by TEM at 20 min, 4 h, and 5 days after the trypsin addition was performed (
Figure 1A). We observed that after the addition of the protease to amyloids suspension, defragmentation of fibrils took place; however, the formed fragments of fibrils remained ordered (for 20 min); after that, the structure of these fragments was “fluffed” (with an increase in the incubation time to 4 h). Our results indicate that the samples of fibrils five days after the addition of trypsin contain non-fibrillar aggregates of various sizes (from several tens of monomeric subunits to the size of a mature amyloid fibril). These aggregates can be a result of fibril degradation induced by trypsin, as well as association of the products of proteolytic degradation of fibrils with each other. It should be noted that along with the products of proteolytic degradation full-length mature amyloid fibrils were retained in the sample.
We also checked whether degradation of amyloids induced by some external proteolytic activity (not coming from the trypsin) occurs in the control sample with no trypsin added. Tested samples were incubated at 37 °C in phosphate buffer. Intact amyloids were stable during at least a one-week period, as confirmed by TEM (
Figure S4).
To assess the degree of protein aggregates degradation, samples before and five days after the addition of protease were centrifuged at high speeds (2 h, 18,000 rpm), after which the supernatant was collected and analyzed. Analysis of the supernatant sample untreated with protease showed the absence of fibril fragments and disordered aggregates as confirmed by TEM (
Figure 2A, TEM data), as well as by the closeness to 0 of its optical density. This indicates the stability of the studied aggregates and the absence of processes of their spontaneous degradation. According to TEM data (
Figure 2A), degraded fibrils fragments, unstructured protein aggregates, were present in the supernatant of the sample after trypsin exposure. Using absorption spectroscopy, it was shown that the proportion of the degraded fraction in supernatant is about 20% of the initial concentration of amyloid fibrils (
Figure 2B). It should be noted that degraded aggregates partially precipitated during centrifugation and large non-fibrillar associates comparable in size to amyloid fibrils were found in the pellet. Thus, the real fraction of fibril degradation products substantially exceeds 20%. We also show that increase in trypsin concentration led to an increase in the proportion of the degraded fraction in the sample (
Figure S5).
The effect of trypsin on lysozyme amyloid fibrils was also analyzed by SDS-PAGE under denaturing conditions (
Figure 2C). Samples of trypsin in PBS at the same concentration as in samples with fibrils, as well as amyloid fibrils in the absence of trypsin, were used as controls. Surprisingly, we did not observe a band corresponding to the molecular weight of the protease in the analyzed sample immediately after the mixing fibrils with trypsin, in contrast to the control sample (freshly prepared trypsin only) (
Figure 2C, 0 h). Similar results were obtained earlier in the study of the interaction of Abeta-peptide amyloids with trypsin [
19]. This indicates that the affinity of trypsin binding to fibrils is so high that this interaction does not destroy either the effect of ionic detergent (SDS) or boiling the sample (for 5 min). It should be noted that the band corresponding to trypsin did not appear in the samples with fibrils for five days. At the same time, after the addition of trypsin to the fibril suspension, the content of monomeric lysozyme first increased and then decreased (according to the analysis of the monomeric fraction ratio in the samples before and after trypsin exposure, shown in
Figure S6), which confirmed the effect of protease on amyloids. We did not observe a band corresponding to the molecular weight of monomeric lysozyme five days after the addition of trypsin, which indicated an almost complete proteolytic degradation of lysozyme monomers released under the action of trypsin. In addition, a decrease in the number of protein fragments that are formed immediately after the addition of trypsin can be noted, pointing at their degradation.
Interestingly, according to the literature, compact globular lysozyme in its native state is not hydrolyzed by a number of proteolytic enzymes, including trypsin, chymotrypsin, and papain, which indicates the localization of protease cleavage binding sites within the protein globule [
40]. However, these enzymes can hydrolyze denatured lysozyme [
41]. We were convinced of the stability of monomeric native lysozyme exposed to trypsin using SDS-PAGE under denaturing conditions: the intensity of the band corresponding to monomeric lysozyme did not change five days after the addition of trypsin to protein (
Figure S7). The fact that during this time there is a significant degradation of amyloid fibrils formed from this protein allows us to conclude that amyloid cleavage sites are more exposed than in monomeric protein.
In order to reveal the structural changes of the studied lysozyme amyloid fibrils induced by trypsin, we analyzed the results obtained using various spectroscopic methods, such as intrinsic UV fluorescence of proteins and CD spectroscopy of fibrils in the far UV region, and also an approach based on the analysis of the interaction of amyloid fibrils with fluorescent probes. The amyloid-specific dye thioflavin T (ThT) [
42,
43,
44] and the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) [
45,
46,
47] were used, which are widely known probes to detect the formation of amyloid fibrils and study their structure and stability [
48,
49,
50,
51].
After the treatment of amyloid fibrils with trypsin, a significant decrease in the values of their photophysical characteristics such as parameter
A [
38,
39] (
Figure 1E) and fluorescence anisotropy (
Figure 1F) as well as a shift in the maximum of the spectrum of intrinsic UV fluorescence of the sample to longer wavelengths was observed (
Figure 1D). In addition, after trypsin exposure, an increase in the integral fluorescence intensity of tryptophan residues of amyloid fibrils was found (
Figure 1C). Such changes in photophysical characteristics are usually observed during protein denaturation [
49,
52]. As already noted, five days after the addition of trypsin to fibrils, significant changes in the values of the recorded parameters were no longer observed (
Figure 1). However, the values of these parameters did not reach the values characteristic of lysozyme in the unfolded state, in which all tryptophan amino acid residues of the protein are exposed to water, which determines the low value of the parameter
A (
A = 0.45) and fluorescence anisotropy (
r365 = 0.05), as well as the long wavelength position of the fluorescence spectrum maximum (
λmax = 350 nm) of the sample [
53,
54,
55,
56,
57]. The observed difference in the values of the photophysical characteristics of the studied fibrils, measured five days after the addition of trypsin, from the values characteristic of the protein in the unfolded state, may be due to the following facts: (1) the sample still contains a significant amount of mature intact amyloid fibrils that have not been exposed to protease and also (2) the monomers of the amyloid-forming protein in the aggregates are not completely denatured after trypsin exposure. To understand the reasons for the observed differences, we compared the samples of amyloid fibrils five days after the addition of trypsin at trypsin/protein ratios of 1:125 and 1:1. It turned out that the characteristics of the sample treated with higher concentration of trypsin were even closer to the characteristics of the denatured protein. Following that, we sedimented amyloid fibrils and large aggregates by centrifugation and evaluated the photophysical characteristics of the protein that remained in the supernatant. As we expected, these characteristics practically coincided with the characteristics of the fully denatured protein (
Figure S8).
Four hours after the addition of protease to the sample with amyloids, a change in the shape of the CD spectrum of fibrils in the region of 190 nm was observed (
Figure 1B). An even more significant change in the shape of the CD spectra (not only in the short-wavelength region, but also in the region of the minimum at 220 nm) was detected 24 h and five days after adding trypsin to the sample with amyloids. The obtained data indicate that the secondary structure of lysozyme subunits in the amyloid fibril changes significantly after treatment of the samples with trypsin. In order to assess the nature of these changes, we analyzed the content of various elements of the secondary structure in the sample using special software CDPro [
58]. It was shown that the addition of trypsin to the studied amyloid fibrils led to an increase in the proportion of disordered structure due to a decrease in the proportion of ordered α-helical structure and β-strands in amyloid-forming proteins (
Figure S9A). Considering the fact that β-strands form the backbone of the amyloid fibril, the observed changes confirm the assumptions about the degradation of amyloid fibrils. In this case, the addition of trypsin leads to a change in the structure of not only amyloidogenic (β-folded) but also non-amyloidogenic protein fragments.
Trypsin treatment of the studied amyloid fibrils was also accompanied by a decrease in the fluorescence intensity of specific dyes ThT (
Figure 1H) and ANS (
Figure 1I) bound to fibrils. According to the literature, ThT incorporates into the grooves formed by the side chains of amino acids of the amyloid fibril backbone along the fiber axis perpendicular to the beta-sheets [
59], and ANS is a hydrophobic probe interacting with protein associates [
45,
46]. Thus, the dyes are likely to bind to different sites of amyloids, which is in line with the results of the work [
48]. We believe that it explains the difference of kinetic dependences of fibril degradation monitored by ANS and ThT fluorescence. Different ANS and ThT binding kinetics was observed in the process of fibrillogenesis previously [
60]. It can be assumed that the change in ThT fluorescence indicates degradation of amyloid fibers, and ANS fluorescence mainly reflects the fibril declasterization and degradation of non-fibrillar protein aggregates. This is in good agreement with TEM data indicating fragmentation and decreased ordering of amyloids in the presence of trypsin (
Figure 1A). The significant reduction in Rayleigh light scattering (RLS) of amyloid fibril samples after the addition of trypsin confirms the disintegration of fibrillar clots and destruction of the fibrillar core (
Figure 1G).
To assess the effect of trypsin on the cytotoxicity of amyloid fibrils, the metabolic activity of
Hela cells was determined using the MTT test in the presence of amyloids before and five days after the addition of protease. First of all, trypsin at the used concentration was shown to be non-toxic to cells (
Figure S10). It turned out that the level of reduced MTT was the same for samples of amyloid fibrils exposed to trypsin and for intact amyloids (data not shown). This indicates that the action of trypsin on fibrils did not change their toxicity to cells. A significant increase in the proportion of degraded fraction of amyloid fibrils in the sample with a 17-fold increase in trypsin concentration also did not lead to a change in toxicity (
Figure 2D), despite a significant decrease of intact amyloids in the sample. In order to explain these results, the metabolic activity of
Hela cells was determined in the presence of monomeric lysozyme and its fragments obtained as a result of proteolytic degradation of the protein (
Figure S10) (to obtain protein fragments, protein was preliminary denatured). It turned out that monomeric lysozyme and its fragments did not affect cell viability at a concentration equal to the concentration of fibrils in the experiment. It means that if only these protein fractions were the products of degradation, the total cytotoxicity of the sample after trypsin exposure should have decreased. It can be assumed that, along with lysozyme fragments and monomers, proteolytic degradation of fibrils also results in the formation of non-fibrillar aggregates that are toxic to cells (probably even more cytotoxic than amyloid fibrils themselves). At the same time, the quantitative ratio of cytotoxic and non-cytotoxic protein fractions is such that the cytotoxic effect of non-fibrillar aggregates is leveled and, as a result, the viability of cells before and after their treatment with trypsin remains unchanged.
We tried to estimate the size of aggregates formed after trypsin exposure. We analyzed the aggregates, formed after treatment of amyloid fibrils with trypsin (trypsin/protein ratio of 1:7.5), using pseudo-native SDS-PAGE in 8% polyacrylamide gel [
61]. Obtained results indicate the absence in the sample of fractions with molecular weights less than 220 kDa (
Figure S11). Increase in the concentration of trypsin in the sample (trypsin/protein ratio of 1:1) led to similar results. Our rough estimate of the size of aggregates visualized by TEM (
Figure 2A and
Figure S12) allowed us to conclude that they consist of 20 or more monomeric protein subunits (which may explain their absence on SDS-PAGE) and have a broad size range. The linear dimensions of some aggregates are comparable to the size of intact amyloid fibrils (
Figure S12). A higher cytotoxicity of such large aggregates, representing degraded amyloids, relative intact fibrils have been already demonstrated by us previously for the same fibrils treated with a protein with a chaperone activity of alpha-B-crystallin [
34].
Based on the obtained results, it is concluded that trypsin-induced degradation of the studied lysozyme amyloid fibrils occurs according to the following mechanism: (1) first, amyloid clots are declustering, and amyloid fibers are fragmented; (2) then, the structure of shortened fibril fragments “decompacts” and becomes less ordered, (3) after which these aggregates degrade to monomers, which, in turn, degrade into short fragments. In this case, despite the fact that proteolytic cleavage of fibrils to short non-toxic protein fragments occurs, the total cytotoxicity of the sample after trypsin exposure does not decrease, which may indicate the presence of highly toxic non-fibrillar aggregates in the sample.
2.2. Relationship of Amyloid Fibrils Degradation Induced by Trypsin and Their Polymorphism
Analysis of the lysozyme amino acid sequence indicates that the protein has 17 trypsin cleavage sites located in different parts of the protein (
Scheme 1).
Taking into account that the conformation of lysozyme significantly affects its stability, we can suggest that the mechanism of protease action on fibrils may depend on the secondary and tertiary structures of amyloid-forming protein, that is, on different localization of proteolytic cleavage sites. In order to understand this issue, we investigated the effect of protease on lysozyme amyloids formed under alternative conditions (at acidic pH) [
30,
34].
Lysozyme amyloid fibrils prepared under acidic conditions (pH 2.0), as well as fibrils previously prepared under neutral conditions in the presence of GdnHCl, were transferred to a buffer with pH 7.4 and incubated at 37 °C. Previously, it was shown that under these conditions, fibrils retained their structure and stability for a long time [
34] that corresponds to our results (
Figure S4), which made it possible to observe the change in their characteristics induced by trypsin within a week (
Figure 3A–I). The morphology of the prepared amyloid fibrils was analyzed by TEM (
Figure 3A). It turned out that lysozyme fibrils, obtained under acidic conditions, were thin long fibers, visually similar to fibrils formed from the same protein at neutral pH of the solution in the presence of denaturant (
Figure 1A and
Figure 3A). However, TEM data suggested that amyloids prepared under different conditions have different tendencies to clustering. In order to prove this assumption, we analyzed fibril samples using confocal microscopy in the presence of a ThT fluorescent probe. Analysis of confocal microscopy results (
Figure 4A), as well as the RLS values of the studied aggregates (
Figure 1G and
Figure 3G), confirmed our assumption about the smaller size of fibrillar clots in the sample obtained in the acid buffer in comparison to the aggregates formed in the alternative conditions.
It is noteworthy that the values of the parameter
A and fluorescence anisotropy of lysozyme fibrils prepared at acidic pH (
Figure 3E,F) are lower than the values of the corresponding characteristics for lysozyme fibrils obtained at neutral pH (
Figure 1E,F). At the same time, the proportion of β-folded structure in lysozyme monomers, which form fibrils prepared at acidic pH, is significantly lower than in fibrils obtained at neutral pH, while the proportion of α-helical elements, on the contrary, is higher (
Figure S9A,B). These data indicate that the structure of lysozyme molecules in amyloid fibrils prepared under different conditions is not identical.
The observed polymorphism of lysozyme amyloid fibrils affects their cytotoxicity, which is in good agreement with our earlier data [
34,
49]. Cell viability in in vitro experiments when cells were treated with lysozyme fibrils prepared at acidic pH turned out to be higher than in the presence of lysozyme fibrils obtained at neutral pH, as evidenced by the difference in the level of reduced MTT in the presence of different types of fibrils by about 20% (
Figure 2D and
Figure 4E).
The results of our experiments showed that, when trypsin was added to the suspension of fibrils obtained at acidic pH, after 10 min, the enzyme in a soluble form was not detected in the buffer (as in the case of lysozyme fibrils obtained at neutral pH) (
Figure S3). This means that all trypsin molecules in the sample are in a fibril-bound state. Thus, the change in the structure of lysozyme fibrils did not break their ability to interact with trypsin.
Next, we examined whether the mechanism of action of trypsin changes with the change of fibrils structure. The experiment was carried out for five days, as in the case of fibrils obtained under alternative conditions. According to TEM data, fragmentation of lysozyme amyloid fibrils formed under acidic conditions begins as early as 20 min after the addition of the protease (that is, earlier than for amyloid fibrils prepared at neutral pH) (
Figure 3A). These results are consistent with the data obtained by SDS-PAGE under denaturing conditions (
Figure 4B). It turned out that already 20 h after the addition of trypsin to fibrils obtained at acidic pH, a significant decrease in the intensity of the band corresponding to monomeric lysozyme was observed. At the same time, the intensity of this band did not change even after 24 h after adding trypsin to fibrils prepared at neutral pH.
It can be assumed that the lower rate of degradation of lysozyme fibrils obtained at physiological pH is due to a higher degree of clustering, i.e., to a lower availability of proteolytic cleavage sites. Fragmentation of these fibrils with trypsin probably requires preliminary disaggregation of fibrillar clots. However, despite the different rates of degradation, the mechanism of action of trypsin on different types of lysozyme fibrils turned out to be identical: with the time, the fragments of fibrils were “decompacted” and lost their rigid structure. This mechanism of trypsin action was also confirmed by a decrease in the fluorescence intensity of ANS (
Figure 3I) and ThT (
Figure 3H) in the sample with amyloid fibrils.
It is important to note that the value of the fluorescence intensity of bound dyes (
Figure 3H,I), as well as the scale of their RLS (
Figure 3G) recorded five days after the addition of trypsin to lysozyme amyloid fibrils prepared under acidic conditions, is comparable in magnitude with the degree of changes in these characteristics obtained for lysozyme amyloid fibrils prepared under neutral conditions. (
Figure 1G–I). The proportion of the degraded fibril fraction five days after the addition of trypsin to different types of lysozyme amyloids (
Figure 2A and
Figure 4C) determined using the samples after centrifugation is also similar (
Figure 2B and
Figure 4D). It was shown that an increasing of trypsin concentration can lead to differences in proportion of this fraction in the case of different amyloids (
Figure S5). At the same time, the analysis of aggregates, formed after the treatment of different types of lysozyme amyloid fibrils with trypsin in different concentrations using pseudo-native SDS-PAGE in 8% polyacrylamide gel [
61], showed similar results. Obtained results indicate the absence in the samples of fractions with molecular weights less than 220 kDa (
Figure S11).
The results of the assessment of cell viability in the presence of amyloid fibrils indicate that the cytotoxicity of both types of amyloids after their treatment with trypsin (including, in the case of an increase in its concentration by 17 times) practically does not change compared to intact fibrils (
Figure 4E), which indicates similar protein species formed as a result of proteolytic cleavage of the studied amyloids, which does not contradict the TEM data (
Figure S12).
It is interesting that despite a similar mechanism of lysozyme amyloid fibrils degradation under the action of trypsin, the recorded change in the intrinsic characteristics of tryptophan fluorescence of amyloid fibrils obtained at acidic pH turned out to be less pronounced (
Figure 3C–F) than in the case of lysozyme fibrils prepared at neutral pH (
Figure 1C–F). In addition, only a slight decrease in the CD signal was observed without a significant change in the shape of the spectrum for amyloid fibrils prepared at acidic pH after the trypsin effect (
Figure 3B). However, the assessment of the content of secondary structure elements showed that trypsin’s influence on lysozyme fibrils prepared at acidic pH, as in the case of fibrils obtained at neutral pH, still led to the transformation of the ordered secondary structure of lysozyme into a disordered one (
Figure S9B). It can be assumed that the different effect of trypsin on the photophysical properties of lysozyme fibrils obtained under different conditions is due to the different structure of the protein in amyloid fibrils. The absence of a pronounced effect of proteolytic degradation of lysozyme fibrils obtained at acidic pH on the secondary structure of the amyloid-forming protein and the microenvironment of tryptophan amino acid residues in this protein is probably due to the structural features of lysozyme subunits in these fibrils.
As in the case of lysozyme amyloid fibrils prepared at pH 7, for these amyloids, we compared the samples five days after the addition of trypsin at trypsin/protein ratios of 1:125 and 1:1. It turned out that the characteristics of the sample treated with higher concentration of trypsin were significantly closer to the characteristics of the denatured protein. Evaluation of the photophysical characteristics of the protein remained in the supernatant after sedimentation of amyloid fibrils, and large aggregates by centrifugation showed that these characteristics practically coincided with the characteristics of the fully denatured protein (
Figure S8).
Based on the obtained results, it was concluded that polymorphism of amyloid fibrils originating from the same protein (differing in the degree of clustering, secondary and tertiary structures of the protein in amyloid fibrils) could affect the rate of trypsin-induced degradation of amyloid fibrils but had no impact on the mechanism of this process and the cytotoxicity of degraded amyloid aggregates. The discovered relationship between the structure of fibrils and the rate of their degradation by trypsin can become the basis for the development of a new express method for the analysis of amyloid polymorphism.
2.3. Relationship of Amyloid Fibrils Degradation Induced by Trypsin and Primary Structure of Amyloid-Forming Protein
In order to determine whether the amino acid sequence of the amyloidogenic protein affects the mechanism of trypsin action, we prepared amyloid fibrils on the basis of another amyloidogenic protein, beta-2-microglobulin, the accumulation of which leads to the development of dialysis-related amyloidosis (DRA, [
29]). Analysis of the primary structure of beta-2-microglobulin indicates that the protein has 13 trypsin cleavage sites located in different parts of the protein (
Scheme 1). SDS-PAGE results of beta-2-microglobulin sample after adding trypsin indicate that the band corresponding to the monomeric protein is noticeably weakened within 30 min after the addition of protease to the sample, and after 24 h it is no longer detected (
Figure S7). Thus, it was shown that beta-2-microglobulin in native state, in contrast to lysozyme, was not resistant to the action of trypsin.
Beta-2-microglobulin amyloid fibrils were prepared under acidic conditions (pH 2.0), like lysozyme fibrils, after which they were transferred to neutral conditions and incubated at 37 °C as a control (
Figure S4). As in the case of previously studied amyloid fibrils, the structure and stability of the prepared protein aggregates after the adding of trypsin were analyzed using a wide range of physicochemical approaches (
Figure 5A–I). The morphology of the prepared amyloid fibrils was analyzed by TEM (
Figure 5A). It turned out that beta-2-microglobulin fibrils in their morphology and degree of clustering were more similar to lysozyme amyloid fibrils obtained under acidic conditions (
Figure 3A and
Figure 5A) than to lysozyme fibrils obtained under neutral conditions (
Figure 1A). This was also confirmed by the RLS value of beta-2-microglobulin amyloids (
Figure 5G). At the same time, the content of β-strands that form the fibril core in the sample with beta-2-microglobulin fibrils turned out to be rather high (closer to the values observed for lysozyme amyloid fibrils obtained at neutral pH in the presence of GdnHCl) (
Figure S9C). Thus, the morphology of beta-2-microglobulin fibrils is more similar to lysozyme fibrils obtained at acidic pH, and their secondary structure is more similar to lysozyme fibrils prepared under neutral conditions.
According to TEM data, fragmentation of beta-2-microglobulin amyloid fibrils begins as early as 20 min after adding trypsin (as in the case of lysozyme fibrils formed at acidic pH) (
Figure 5A). These results are in good agreement with the data obtained by SDS-PAGE under denaturing conditions (
Figure 6A). A significant decrease in the intensity of the band of monomeric beta-2-microglobulin was observed already 24 h after the addition of trypsin, as in the case of lysozyme fibrils obtained under the same conditions. In contrast, in the case of lysozyme fibrils prepared at neutral pH, the intensity of the band of monomeric lysozyme did not change significantly 24 h after the addition of trypsin. The obtained results confirm the previously made the assumption that the rate of amyloid fibrils degradation is influenced by their morphology and the degree of clustering.
SDS-PAGE of amyloid fibrils formed from beta-2-microglobulin untreated with trypsin showed a band with a molecular weight of 23–24 kDa, corresponding to the molecular weight of the dimeric form of the protein (
Figure S13). SDS-PAGE of the sample of beta-2-microglobulin fibrils mixed with trypsin, the proteolytic activity of which was quenched by PMSF, revealed two separate bands with close molecular weights (about 23–24 kDa), corresponding to the molecular weight of beta-2-microglobulin dimer found in amyloid fibrils untreated with trypsin, and to the molecular weight of trypsin (
Figure S13). Beta-2-microglobulin dimer exhibited a lower electrophoretic mobility compared to trypsin. SDS-PAGE of amyloid beta-2-microglobulin fibrils treated with trypsin showed a single band with a molecular weight of 23–24 kDa (
Figure 6A), corresponding to the molecular weight of trypsin, and not to the dimeric form beta-2-microglobulin (which appears to have been degraded immediately after the addition of trypsin). Thus, in the case of beta-2-microglobulin fibrils in contrast to lysozyme fibrils, trypsin can be detected in the sample using SDS-PAGE.
TEM data indicate that, after adding trypsin to the sample, the fibril fragments lose their rigid fiber structure, as in the case of various types of lysozyme amyloid fibrils (
Figure 5A and
Figure S12). This mechanism of trypsin action is also confirmed by a decrease in the fluorescence intensity of ANS (
Figure 5I) and ThT (
Figure 5H) in the sample. Thus, the mechanism of action of trypsin is not affected by either the morphology or the structure (primary and secondary) of the amyloidogenic protein.
The trypsin-induced change in the fluorescence intensity of the dyes bound to beta-2-microglobulin amyloid fibrils (
Figure 5H,I), and the decrease in the RLS value measured for them five days after the start of the experiment (
Figure 5G) were more pronounced than the change in these characteristics for both types of lysozyme amyloid fibrils (
Figure 1G–I and
Figure 5G–I). In addition, the proportion of the degraded fraction of fibrils five days after the addition of trypsin in the case of beta-2-microglobulin amyloids (
Figure 2A,
Figure 4C and
Figure 6B) was more than 30% and was noticeably higher than in the case of both types of lysozyme amyloid fibrils (
Figure 2B,
Figure 4D and
Figure 6C). This indicates that the efficiency of proteolytic degradation of amyloid fibrils depends on the primary structure of the protein.
After treatment of beta-2-microglobulin amyloid fibrils with trypsin, a significant decrease in the values of parameter
A (
Figure 5E) and fluorescence anisotropy (
Figure 5F) was observed, as well as a red shift in the maximum of the fluorescence spectrum of the sample (
Figure 5D). In addition, a significant decrease in the integral fluorescence intensity of tryptophan residues of amyloid fibrils (
Figure 5C) after adding trypsin was found. The shape of the CD spectrum of beta-2-microglobulin amyloid fibrils differed from the shape of the CD spectrum of the sample measured 20 min after adding trypsin (
Figure 5B). The change in the shape of the CD spectrum of the sample recorded 24 h after adding trypsin was even more pronounced. According to the assessment of the content of various types of secondary structure in the samples, the trypsin addition to the studied amyloid fibrils leads to an increase in the proportion of disordered structure due to a decrease in the proportion of β-strands in the amyloid-forming protein (
Figure S9C). The observed changes confirm the assumption about the degradation of amyloid fibrils.
Trypsin can be detected in a sample with beta-2-microglobulin amyloid fibrils no more than a day after its addition by SDS-PAGE (
Figure 6A). The most pronounced changes in the photophysical characteristics and morphology of these amyloid fibrils after trypsin adding were observed in the same period (
Figure 5). Further moderate changes either may be associated with the immediate action of a small amount of trypsin molecules remaining in the sample, which avoid detection by SDS-PAGE, or may be the result of processes triggered by trypsin on the first day after its addition (leading to the decreased stability of fibrils).
The results obtained in the study of the cytotoxicity of beta-2-microglobulin amyloid fibrils after trypsin addition were quite unexpected. We not only did not observe a decrease but observed an increase in the cytotoxicity of amyloids of this protein after their treatment with trypsin (
Figure 6D). It can be assumed that, in the case of beta-2-microglobulin fibrils, the cytotoxicity of the forming amyloid aggregates (fragments of amyloid fibrils or non-fibrillar amyloid aggregates) is higher than in the case of amyloid fibrils of lysozyme. Another reason for the observed effect may be the greater number of formed toxic non-fibrillar aggregates (
Figure S12) in relation to non-toxic protein monomers and their fragments (
Figure S10) than in the case of lysozyme fibrils.
Thus, the results obtained on the example of two amyloidogenic proteins (and three polymorphic structures) made it possible to assume that the mechanism of fibril degradation under the action of trypsin did not depend on the morphology or primary and secondary structure of the amyloidogenic protein. At the same time, the rate of proteolytic degradation of amyloids depends on the morphology and degree of clustering of fibrils. In addition, it was suggested that the cytotoxicity of trypsin-degraded amyloid fibrils might depend on the ratio of highly toxic and low toxic/non-toxic protein species in the sample after such exposure.