scholarly journals Trypsin Induced Degradation of Amyloid Fibrils

2021 ◽  
Vol 22 (9) ◽  
pp. 4828
Author(s):  
Olga V. Stepanenko ◽  
Maksim I. Sulatsky ◽  
Ekaterina V. Mikhailova ◽  
Olesya V. Stepanenko ◽  
Irina M. Kuznetsova ◽  
...  

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.

1998 ◽  
Vol 258 (1) ◽  
pp. 61-67 ◽  
Author(s):  
Vittorio Bellotti ◽  
Monica Stoppini ◽  
Palma Mangione ◽  
Margaret Sunde ◽  
Carol Robinson ◽  
...  

2018 ◽  
Vol 19 (9) ◽  
pp. 2762 ◽  
Author(s):  
Anna Sulatskaya ◽  
Natalia Rodina ◽  
Dmitry Polyakov ◽  
Maksim Sulatsky ◽  
Tatyana Artamonova ◽  
...  

The persistence of high concentrations of beta-2-microglobulin (β2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of β2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length β2M (β2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6β2m and ΔN10β2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of β2M amyloid fibrils with affinity ~104 M−1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with β2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-β2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10β2m fibrils from other β2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between β2m and ΔN6β2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the β2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit β2M fibrillogenesis for the treatment of dialysis-related amyloidosis.


2019 ◽  
Vol 9 (Suppl_1) ◽  
pp. S33-S34
Author(s):  
Anna Sulatskaya ◽  
Maksim Sulatsky ◽  
Evgeny Pichkur ◽  
Irina Kuznetsova ◽  
Konstantin Turoverov

1992 ◽  
Vol 263 (3) ◽  
pp. F540-F545 ◽  
Author(s):  
S. M. Moe ◽  
S. M. Sprague

beta 2-Microglobulin (beta 2M) polymerizes to form amyloid fibrils that deposit and cause destructive bone lesions in patients on chronic dialytic therapy. beta 2 M is mitogenic to osteoblasts; however, its effect on bone mineralization is unknown. To determine whether beta 2M causes bone demineralization, neonatal mouse calvariae were incubated with and without beta 2M, and net calcium flux was calculated. Following a 48-h but not 3- or 24-h incubation, beta 2M (10(-8)-10(-6) M) induced a net calcium efflux. The efflux was similar to that observed with 10(-10) M parathyroid hormone (PTH) but less than that observed with 10(-8 M PTH. Devitalizing the calvariae resulted in a net calcium influx that was unaffected by the addition of beta 2M, indicating a cell-mediated phenomenon. The release of beta-glucuronidase, an osteoclast enzyme, increased after a 48-h but not a 24-h incubation with beta 2M. Calcitonin, an osteoclast inhibitor, blocked the beta 2M-induced calcium efflux and beta-glucuronidase release, suggesting osteoclast involvement. Thus beta 2M induces a dose- and time-dependent, cell-mediated calcium efflux from neonatal mouse calvariae that involves osteoclast stimulation.


2019 ◽  
Vol 20 (8) ◽  
pp. 656-664 ◽  
Author(s):  
Yi Da ◽  
K. Akalya ◽  
Tanusya Murali ◽  
Anantharaman Vathsala ◽  
Chuen-Seng Tan ◽  
...  

Background: : Drug-induced Acute Kidney Injury (AKI) develops in 10-15% of patients who receive nephrotoxic medications. Urinary biomarkers of renal tubular dysfunction may detect nephrotoxicity early and predict AKI. Methods:: We prospectively studied patients who received aminoglycosides, vancomycin, amphotericin, or calcineurin inhibitors, and collected their serial urine while on therapy. Patients who developed drug-induced AKI (fulfilling KDIGO criteria) were matched with non-AKI controls in a 1:2 ratio. Their urine samples were batch-analyzed at time-intervals leading up to AKI onset; the latter benchmarked against the final day of nephrotoxic therapy in non- AKI controls. Biomarkers examined include clusterin, beta-2-microglobulin, KIM1, MCP1, cystatin-C, trefoil-factor- 3, NGAL, interleukin-18, GST-Pi, calbindin, and osteopontin; biomarkers were normalized with corresponding urine creatinine. Results:: Nine of 84 (11%) patients developed drug-induced AKI. Biomarkers from 7 AKI cases with pre-AKI samples were compared with those from 14 non-AKI controls. Corresponding mean ages were 55(±17) and 52(±16) years; baseline eGFR were 99(±21) and 101(±24) mL/min/1.73m2 (all p=NS). Most biomarker levels peaked before the onset of AKI. Median levels of 5 biomarkers were significantly higher in AKI cases than controls at 1-3 days before AKI onset (all µg/mmol): clusterin [58(8-411) versus 7(3-17)], beta-2-microglobulin [1632(913-3823) versus 253(61-791)], KIM1 [0.16(0.13-0.76) versus 0.07(0.05-0.15)], MCP1 [0.40(0.16-1.90) versus 0.07(0.04-0.17)], and cystatin-C [33(27-2990) versus 11(7-19)], all p<0.05; their AUROC for AKI prediction were >0.80 (confidence intervals >0.50), with average accuracy highest for clusterin (86%), followed by beta-2-microglobulin, cystatin-C, MCP1, and KIM1 (57%) after cross-validation. Conclusion: : Serial surveillance of these biomarkers could improve the lead time for nephrotoxicity detection by days.


1993 ◽  
Vol 67 (1) ◽  
pp. 589-592 ◽  
Author(s):  
L Fiette ◽  
C Aubert ◽  
M Brahic ◽  
C P Rossi

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