Abstract
Amyloids are insoluble fibrous protein aggregates, and their accumulation is associated with amyloidosis and many neurodegenerative diseases, including Alzheimer's disease. In the present study, we report that smooth muscle titin (SMT; 500 kDa) from chicken gizzard forms amyloid aggregates in vitro. This conclusion is supported by EM data, fluorescence analysis using thioflavin T (ThT), Congo red (CR) spectroscopy and X-ray diffraction. Our dynamic light scattering (DLS) data show that titin forms in vitro amyloid aggregates with a hydrodynamic radius (Rh) of approximately 700–4500 nm. The initial titin aggregates with Rh approximately 700 nm were observed beyond first 20 min its aggregation that shows a high rate of amyloid formation by this protein. We also showed using confocal microscopy the cytotoxic effect of SMT amyloid aggregates on smooth muscle cells from bovine aorta. This effect involves the disorganization of the actin cytoskeleton and result is cell damage. Cumulatively, our results indicate that titin may be involved in generation of amyloidosis in smooth muscles.
Highlights
Amyloids are insoluble fibrous protein aggregates sharing specific structural features: high content of β-sheet structure, ability to bind to Congo red (CR) and thioflavin T (ThT), birefringence in polarized light, insolubility in most solvents and resistance to proteases [1,2,3]
Amyloid was found in cross-striated muscles, for example formed from such proteins as immunoglobulin (Ig) light chain, Ig heavy chains, transthyretin, serum amyloid A, apolipoprotein AIV, fibrinogen α chain and atrial natriuretic factor, which contribute to the development of ‘amyloid cardiomyopathy’ or ‘cardiac amyloidosis’ [5]
Amyloid deposits were found in blood vessels [7,8]; in particular, it was demonstrated that aggregates of serum amyloid A (SAA) and its fragments can accumulate in the intima and medial arterioles and beneath the venular endothelium [7]
Summary
Amyloids are insoluble fibrous protein aggregates sharing specific structural features: high content of β-sheet structure, ability to bind to Congo red (CR) and thioflavin T (ThT), birefringence in polarized light, insolubility in most solvents and resistance to proteases [1,2,3]. There is a group of proteins whose amyloid properties were demonstrated in vitro, that are not associated with known diseases [3,4]. Amyloid was found in cross-striated muscles, for example formed from such proteins as immunoglobulin (Ig) light chain, Ig heavy chains, transthyretin, serum amyloid A, apolipoprotein AIV, fibrinogen α chain and atrial natriuretic factor, which contribute to the development of ‘amyloid cardiomyopathy’ or ‘cardiac amyloidosis’ [5]. Amyloid depositions containing the Aβ peptide were detected upon generation of inclusion-body myositis in skeletal muscles. It was found that myosin subfragment-1 from rabbit skeletal muscles can form in vitro spherical oligomers with amyloid-like dye-binding properties [6]. Medin amyloids (50-aa-long peptides of lactadherin) were detected at aorta amyloidosis [8]
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