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Cytotoxic aggregation and amyloid formation by the myostatin precursor protein.

Starck CS, Sutherland-Smith AJ - PLoS ONE (2010)

Bottom Line: The mechanism for how MstnPP contributes to disease pathogenesis is unknown.Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts.These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer's and Parkinson's diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer's amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by beta-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.

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Characterisation of MstnPP soluble aggregates by negative-stain transmission electron microscopy (TEM) and ThT binding.(A–B) TEM of MstnPP soluble aggregates and C. insulin positive control containing both protofibrils (black arrow) and mature fibrils (white arrow). D. ThT binding of MstnPP soluble aggregates (green), compared to an insulin positive control (red), the MstnPP dimer (yellow) and a buffer (50 mM Tris-HCl pH 8.5, 150 mM NaCl) blank (blue).
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pone-0009170-g003: Characterisation of MstnPP soluble aggregates by negative-stain transmission electron microscopy (TEM) and ThT binding.(A–B) TEM of MstnPP soluble aggregates and C. insulin positive control containing both protofibrils (black arrow) and mature fibrils (white arrow). D. ThT binding of MstnPP soluble aggregates (green), compared to an insulin positive control (red), the MstnPP dimer (yellow) and a buffer (50 mM Tris-HCl pH 8.5, 150 mM NaCl) blank (blue).

Mentions: Transmission electron microscopy (TEM, Fig. 3A and B) revealed that MstnPP soluble aggregates exhibit a morphology and size similar to that documented for amyloid protofibrils from a number of other proteins such as lysozyme [59], [60] and HypF-N [61] as well as the insulin protofibrils generated as a positive control (Fig. 3C) using a well-established protocol [39], [62], [63]. A range of structures, including spheres and oligomers of associated spheres (diameter 15.6±1.6 nm SD), can be seen, and examples of increased elongation of the aggregates are apparent (Fig. 3B).


Cytotoxic aggregation and amyloid formation by the myostatin precursor protein.

Starck CS, Sutherland-Smith AJ - PLoS ONE (2010)

Characterisation of MstnPP soluble aggregates by negative-stain transmission electron microscopy (TEM) and ThT binding.(A–B) TEM of MstnPP soluble aggregates and C. insulin positive control containing both protofibrils (black arrow) and mature fibrils (white arrow). D. ThT binding of MstnPP soluble aggregates (green), compared to an insulin positive control (red), the MstnPP dimer (yellow) and a buffer (50 mM Tris-HCl pH 8.5, 150 mM NaCl) blank (blue).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820090&req=5

pone-0009170-g003: Characterisation of MstnPP soluble aggregates by negative-stain transmission electron microscopy (TEM) and ThT binding.(A–B) TEM of MstnPP soluble aggregates and C. insulin positive control containing both protofibrils (black arrow) and mature fibrils (white arrow). D. ThT binding of MstnPP soluble aggregates (green), compared to an insulin positive control (red), the MstnPP dimer (yellow) and a buffer (50 mM Tris-HCl pH 8.5, 150 mM NaCl) blank (blue).
Mentions: Transmission electron microscopy (TEM, Fig. 3A and B) revealed that MstnPP soluble aggregates exhibit a morphology and size similar to that documented for amyloid protofibrils from a number of other proteins such as lysozyme [59], [60] and HypF-N [61] as well as the insulin protofibrils generated as a positive control (Fig. 3C) using a well-established protocol [39], [62], [63]. A range of structures, including spheres and oligomers of associated spheres (diameter 15.6±1.6 nm SD), can be seen, and examples of increased elongation of the aggregates are apparent (Fig. 3B).

Bottom Line: The mechanism for how MstnPP contributes to disease pathogenesis is unknown.Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts.These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular BioSciences, Massey University, Palmerston North, New Zealand.

ABSTRACT
Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer's and Parkinson's diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer's amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by beta-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.

Show MeSH
Related in: MedlinePlus