Lysozyme amyloidosis is caused by an amino acid substitution in the sequence of this protein. In our study, we described a clinical case of lysozyme amyloidosis in a Russian family. In our work, we ...described in detail the histological changes in tissues that appeared as a result of massive deposition of amyloid aggregates that affected almost all organ systems, with the exception of the central nervous system. We determined the type of amyloidosis and mutations using mass spectrometry. Using mass spectrometry, the protein composition of tissue samples of patient 1 (autopsy material) and patient 2 (biopsy material) with histologically confirmed amyloid deposits were analyzed. Amino acid substitutions p.F21L/T88N in the lysozyme sequence were identified in both sets of samples and confirmed by sequencing of the lysozyme gene of members of this family. We have shown the inheritance of these mutations in the lysozyme gene in members of the described family. For the first time, we discovered a mutation in the first exon p.F21L of the lysozyme gene, which, together with p.T88N amino acid substitution, led to amyloidosis in members of the studied family.
To date, some scientific evidence (limited proteolysis, mass spectrometry analysis, electron microscopy (EM)) has accumulated, which indicates that the generally accepted model of double-stranded of ...filamentous actin (F-actin) organization in eukaryotic cells is not the only one. This entails an ambiguous understanding of many of the key cellular processes in which F-actin is involved. For a detailed understanding of the mechanism of F-actin assembly and actin interaction with its partners, it is necessary to take into account the polymorphism of the structural organization of F-actin at the molecular level. Using electron microscopy, limited proteolysis, mass spectrometry, X-ray diffraction, and structural modeling we demonstrated that F-actin presented in the EM images has no double-stranded organization, the regions of protease resistance are accessible for action of proteases in F-actin models. Based on all data, a new spatial model of filamentous actin is proposed, and the F-actin polymorphism is discussed.
There are different insulin analogues with various pharmacokinetic characteristics, such as, rapid-acting, long-acting, or intermediate-acting analogues. Since insulin tends to form amyloid ...aggregates, it is of particular interest to measure characteristic times of formation of amyloid aggregates and compare those to action times for insulin and its analogues. For the study we have chosen one of the insulin analogues - insulin Lispro, which is a fast acting insulin analog. It is usually thought of amyloid aggregation as a nucleation-dependent process. We have estimated the size of the primary nucleus to be one monomer and the size of the secondary nucleus to be around zero in both insulin and Lispro insulin aggregation processes. The main structural element of insulin and Lispro insulin amyloid fibrils is a rounded ring oligomer of about 6-7 nm in diameter, about 2-3 nm in height and about 2 nm in diameter of the hole. Fibrils of several μm in length are produced due to interaction of such oligomers. The packing of ring oligomers in fibrils differs because of the difference in their orderliness. Though the initial stages of fibril formation (monomer, oligomer) are similar, the further process depends on the unique sequence of each peptide. Namely the sequence affects the final morphology of mature amyloids. These observations allow us to conclude that formation of fibrils by short peptides occurs via and by means of oligomer ring structures. Such an important issue as the nature of polymorphism of insulin amyloid fibrils has been settled by us. The role of early oligomeric aggregates in such processes as nucleation and aggregation of amyloid fibrils has been examined.
The article presents a clinical case describing a complex differential diagnosis of cardiac amyloidosis types and verification of the diagnosis of AL-amyloidosis.
This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric ...A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.
The data presented in this article are related to the research article entitled “One of the possible mechanisms of amyloid fibrils formation based on the sizes of primary and secondary folding nuclei ...of Aβ40 and Aβ42” (Dovidchenko et al., 2016) 1. Aβ peptide is one of the most intensively studied amyloidogenic peptides. Despite the huge number of articles devoted to studying different fragments of Aβ peptide there are only several papers with correct kinetics data, also there are a few papers with X-ray data, especially for Aβ42. Our data present X-ray diffraction patterns both for Aβ40 and Aβ42 as well for Tris–HCl and wax. Moreover, our data provide kinetics of amyloid formation by recombinant Аβ40 and synthetic Аβ42 peptides by using electron microscopy.
To identify the key stages in the amyloid fibril formation we studied the aggregation of amyloidogenic fragments of Aβ peptide, Aβ(16–25), Aβ(31–40), and Aβ(33–42), using the methods of electron ...microscopy, X-ray analysis, mass spectrometry, and structural modeling. We have found that fragments Aβ(31–40) and Aβ(33–42) form amyloid fibrils in the shape of bundles and ribbons, while fragment Aβ(16–25) forms only nanofilms. We are the first who performed 2D reconstruction of amyloid fibrils by the Markham rotation technique on electron micrographs of negatively stained fragments of Aβ peptide. Combined analysis of the data allows us to speculate that both the fibrils and the films are formed via association of ring-shaped oligomers with the external diameter of about 6 to 7 nm, the internal diameter of 2 to 3 nm, and the height of ∼3 nm. We conclude that such oligomers are the main building blocks in fibrils of any morphology. The interaction of ring oligomers with each other in different ways makes it possible to explain their polymorphism. The new mechanism of polymerization of amyloidogenic proteins and peptides, described here, could stimulate new approaches in the development of future therapeutics for the treatment of amyloid-related diseases.
The article presents a clinical case describing a complex differential diagnosis of cardiac amyloidosis types and verification of the diagnosis of AL-amyloidosis.
In the presented paper, theoretical as well as electron microscopy and X-ray diffraction experimental approaches were employed for studding the process of Aβ amyloid formation. Using quantitative ...estimates of a number of monomers which form the nuclei of amyloid fibrils the sizes of folding nuclei of amyloid fibrils for Aβ40 and 42 have been determined for the first time. We have shown that the size of the primary nucleus of Aβ42 peptide fibrils corresponds to 3 monomers, the size of the secondary nucleus for this peptide is 2 monomers. Applying the same analysis to Aβ40 we conclude that the size of the primary nucleus is 2 monomers, and the size of the secondary nucleus is one monomer. Summation of our theoretical and experimental results has allowed us to propose a new model of the structural organization of amyloid fibrils. Our model suggests that the generation of fibrils takes place along the following simplified pathway: a monomer→a ring oligomer→a mature fibril consisting of ring oligomers. These data shed more light upon our understanding of what sizes of the oligomers could represent main targets for future therapies (tetramers for Aβ42 and trimers for Aβ40), and aid in the development of inhibitors of Aβ40 and 42 oligomer formation.
It has been demonstrated using Aβ40 and Aβ42 recombinant and synthetic peptides that their fibrils are formed of complete oligomer ring structures. Such ring structures have a diameter of about 8-9 ...nm, an oligomer height of about 2- 4 nm, and an internal diameter of the ring of about 3-4 nm. Oligomers associate in a fibril in such a way that they interact with each other, overlapping slightly. There are differences in the packing of oligomers in fibrils of recombinant and synthetic Aβ peptides. The principal difference is in the degree of orderliness of ring-like oligomers that leads to generation of morphologically different fibrils. Most ordered association of ring-like structured oligomers is observed for a recombinant Aβ40 peptide. Less ordered fibrils are observed with the synthetic Aβ42 peptide. Fragments of fibrils the most protected from the action of proteases have been determined by tandem mass spectrometry. It was shown that unlike Aβ40, fibrils of Aβ42 are more protected, showing less ordered organization compared to that of Aβ40 fibrils. Thus, the mass spectrometry data agree with the electron microscopy data and structural models presented here.
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