Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids.
Figure 3. Cytotoxicity of amyloids formed from different proteins. (A-C) The electron micrographs, (D-F) CD spectra and (G-I) dose-dependent inhibition effect on HeLa cells (determined by MTT assay) of amyloid fibrils formed from lysozyme at pH7 (A, D, G) and pH2 (B, E, H) and from beta-2-microglobulin (C, F, I) . Scale bars at the electron micrographs are equal to 1 μm. At the Panels (G-I) data are expressed as medians and interquartile ranges, whiskers denote 1.5 × IQR. ANOVA, Tukey post hoc test, * р < 0.01.
Binding of proteases to amyloids and proteolytic degradation of fibrils, at first glance, seems to be a positive effect in terms of amyloidosis treatment; however, this is actually not entirely true. The interaction of trypsin with abnormal aggregates amyloid fibrils, which was shown in our work, as well as in the work, can lead to the inability of the enzyme interaction with other substrates. For example, trypsin is required for Aβ-peptide catabolism, and its interaction with Aβ-peptide amyloids can reduce clearance of soluble Aβ-peptide, and an increased concentration of Aβ-peptide enhances its aggregation. A similar conclusion was reached in the study of the trypsin binding to fibrillar Aβ-peptide. This assumption is also in a good agreement with the results according to which mice deficient of proteolytic enzymes exhibited elevated levels of Aβ while overexpression of neprilysin in APP-transgenic mice could reduce the deposition of fibrillar Aβ.
In addition, we showed that proteolytic degradation of amyloids did not reduce cytotoxicity of the protein aggregates for Hela cells, as might be expected (at the Figure 3 G-I). We attribute this to the formation of highly toxic non-fibrillar aggregates along with the formation of non-toxic protein monomers and their fragments induced by trypsin. A high cytotoxicity of large disordered aggregates forming after exposure of the lysozyme and beta-2-microglobulin fibrils to the protein with chaperone activity alpha-B-crystallin was already demonstrated by us earlier.
In conclusion, we show that the proteolytic enzyme of the pancreas, trypsin, can induce degradation of amyloid fibrils, and the mechanism of this process is qualitatively the same for amyloids with different structure and morphology (Figure 4). At the same time, the efficiency and rate of fibril degradation are dependent on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils, which determines the number, localization, and availability of protease cleavage sites. Proteolytic degradation of amyloids not only does not reduce, as might be expected, but, in some cases, even increases cytotoxicity of the amyloid fibrils, presumably due to the formation of more toxic non-fibrillar aggregates.
Figure 4. Mechanism of trypsin induced degradation of amyloids formed from different proteins: 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. The cytotoxicity of amyloids treated with trypsin not only does not decrease, but can even increase (this has been demonstrated for beta-2-microglobulin fibrils).