Alpha-B-Crystallin Effect on Mature Amyloid Fibrils: Different Degradation Mechanisms and Changes in Cytotoxicity

Abstract

Given the ability of molecular chaperones and chaperone-like proteins to inhibit the formation of pathological amyloid fibrils, the chaperone-based therapy of amyloidosis has recently been proposed. However, since these diseases are often diagnosed at the stages when a large amount of amyloids is already accumulated in the patient’s body, in this work we pay attention to the undeservedly poorly studied problem of chaperone and chaperone-like proteins’ effect on mature amyloid fibrils. We showed that a heat shock protein alpha-B-crystallin, which is capable of inhibiting fibrillogenesis and is found in large quantities as a part of amyloid plaques, can induce degradation of mature amyloids by two different mechanisms. Under physiological conditions, alpha-B-crystallin induces fluffing and unweaving of amyloid fibrils, which leads to a partial decrease in their structural ordering without lowering their stability and can increase their cytotoxicity. We found a higher correlation between the rate and effectiveness of amyloids degradation with the size of fibrils clusters rather than with amino acid sequence of amyloidogenic protein. Some external effects (such as an increase in medium acidity) can lead to a change in the mechanism of fibrils degradation induced by alpha-B-crystallin: amyloid fibers are fragmented without changing their secondary structure and properties. According to recent data, fibrils cutting can lead to the generation of seeds for new bona fide amyloid fibrils and accelerate the accumulation of amyloids, as well as enhance the ability of fibrils to disrupt membranes and to reduce cell viability. Our results emphasize the need to test the chaperone effect not only on fibrillogenesis, but also on the mature amyloid fibrils, including stress conditions, in order to avoid undesirable disease progression during chaperone-based therapy.

Trypsin Induced Degradation of Amyloid Fibrils

“…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 2 D and Figure 4 E).…”

“…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 3 A–I). The morphology of the prepared amyloid fibrils was analyzed by TEM ( Figure 3 A).…”

“…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 ].…”

“…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 ].…”

“…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 ].…”