Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T

Sulatskaya, Anna I.; Rodina, Natalia P.; Polyakov, Dmitry; Sulatsky, Maksim I.; Артамонова, Т. О.; Khodorkovskii, M. A.; Shavlovsky, M. M.; Kuznetsova, Irina М.; Turoverov, Konstantin K.

doi:10.3390/ijms19092762

Cited by 21 publications

(22 citation statements)

References 50 publications

(74 reference statements)

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“…For the preparation of amyloid fibrils from beta-2-microglobulin, the protocol described in the work [ 64 ] was used. The samples of recombinant beta-2-microglobulin were received from Mikhail M. Shavlovsky, Dmitry S. Polyakov, and Rodion G. Sakhabeev (Department of Molecular Genetics, Institute of Experimental Medicine).…”

Section: Methodsmentioning

confidence: 99%

Trypsin Induced Degradation of Amyloid Fibrils

Stepanenko

Sulatsky

Михайлова

et al. 2021

IJMS

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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. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

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Section: Methodsmentioning

confidence: 99%

Trypsin Induced Degradation of Amyloid Fibrils

Stepanenko

Sulatsky

Михайлова

et al. 2021

IJMS

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“…Lysozyme (ALys amyloidosis) and beta-2-microglobulin (DRA), as proteins with significantly different secondary and tertiary structures and stability, were chosen by us as target proteins. Amyloid fibrils formed from these proteins were prepared by previously developed approaches [ 42 , 43 ].…”

Section: Resultsmentioning

confidence: 99%

“…To prepare amyloid fibrils from beta-2-microglobulin, we followed the special method used in the work [ 42 ]. The recombinant beta-2-microglobulin was kindly provided by Mikhail M. Shavlovsky, Dmitry S. Polyakov and Rodion G. Sakhabeev (Department of Molecular Genetics, Institute of Experimental Medicine).…”

Section: Methodsmentioning

confidence: 99%

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

Stepanenko

Sulatsky

Михайлова

et al. 2020

IJMS

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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.

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“…Generally, we observed that Thioflavin T fluorescence reached a maximum, and then slowly declined. This pattern has been attributed to matting of fibrils, which can interfere with binding of the dye to fibrils, to selfquenching of ThT and related fluors, and to complexities of multiple aggregation products [42][43][44][45] . Fibrils obtained through successful seeding were examined by SSNMR (patients 1, 2, and 3) and XRD (the same three patients, and patient 4).…”

Section: Seeding Of Aβ40 Fibril Growth By Parenchymal and Vascular Ismentioning

confidence: 99%

Atomic-level differences between brain parenchymal- and cerebrovascular-seeded Aβ fibrils

Scherpelz

Wang

Pytel

et al. 2021

Sci Rep

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Alzheimer’s disease is characterized by neuritic plaques, the main protein components of which are β-amyloid (Aβ) peptides deposited as β-sheet-rich amyloid fibrils. Cerebral Amyloid Angiopathy (CAA) consists of cerebrovascular deposits of Aβ peptides; it usually accompanies Alzheimer’s disease, though it sometimes occurs in the absence of neuritic plaques, as AD also occurs without accompanying CAA. Although neuritic plaques and vascular deposits have similar protein compositions, one of the characteristic features of amyloids is polymorphism, i.e., the ability of a single pure peptide to adopt multiple conformations in fibrils, depending on fibrillization conditions. For this reason, we asked whether the Aβ fibrils in neuritic plaques differed structurally from those in cerebral blood vessels. To address this question, we used seeding techniques, starting with amyloid-enriched material from either brain parenchyma or cerebral blood vessels (using meninges as the source). These amyloid-enriched preparations were then added to fresh, disaggregated solutions of Aβ to make replicate fibrils, as described elsewhere. Such fibrils were then studied by solid-state NMR, fiber X-ray diffraction, and other biophysical techniques. We observed chemical shift differences between parenchymal vs. vascular-seeded replicate fibrils in select sites (in particular, Ala2, Phe4, Val12, and Gln15 side chains) in two-dimensional 13C-13C correlation solid-state NMR spectra, strongly indicating structural differences at these sites. X-ray diffraction studies also indicated that vascular-seeded fibrils displayed greater order than parenchyma-seeded fibrils in the “side-chain dimension” (~ 10 Å reflection), though the “hydrogen-bond dimensions” (~ 5 Å reflection) were alike. These results indicate that the different nucleation conditions at two sites in the brain, parenchyma and blood vessels, affect the fibril products that get formed at each site, possibly leading to distinct pathophysiological outcomes.

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Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T

Cited by 21 publications

References 50 publications

Trypsin Induced Degradation of Amyloid Fibrils

Trypsin Induced Degradation of Amyloid Fibrils

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

Atomic-level differences between brain parenchymal- and cerebrovascular-seeded Aβ fibrils

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