Abstract
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.
Highlights
Protein aggregation is a rather widespread process in cells of a living organism.Amyloid aggregation is of great interest for researchers throughout the world due to the wide occurrence of amyloidoses in man and animals
Were glycine–KOH, pH 7.0, at 4 ◦ C for 24 h. (f) A separately lying fibrillar filament and oligomers of skeletal myosin binding protein-C (sMyBP-C) were obtained obtained by placing it into a solution of 0.15 M glycine–KOH, pH ◦7.0, at 4 °C for 24 h
atomic force microscopy (AFM) of sMyBP-C incubated for 24 h in a solution containing 0.15 M glycine-KOH, pH 7.0, revealed aggregates of different morphology and size
Summary
Protein aggregation is a rather widespread process in cells of a living organism. Amyloid aggregation is of great interest for researchers throughout the world due to the wide occurrence of amyloidoses in man and animals. Research into the amyloid properties of multidomain muscle proteins, such as titin and myosin binding protein-C (MyBP-C), which consist of β-folded domains [12,13,14], can, in our mind, contribute to the insight into this problem. The paralog of cardiac MyBP-C (cMyBP-C) is expressed correspondingly in cardiac muscle MyBP-C (cMyBP-C) and is coded for by the gene MYBPC3 [12] These three paralogs consist of immunoglobulin-like (Ig) and fibronectin III-like (FnIII) domains (Figure 1) [12,13]. This work investigated in vitro aggregation features and amyloid-like properties of rabbit skeletal myosin binding protein-C. Studies of smooth-muscle titin reveal the ability of this protein to form amyloid aggregates of a cross-β quaternary structure in vitro [35]. Domains was drawn based on the PDB file https://www.uniprot.org/uniprot/Q00872
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