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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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MstnPP aggregates and protofibrils (PF) are cytotoxic to C2C12 mouse myoblasts.C2C12 cytotoxicity assay using the WST-1 reagent where the difference in absorbance at 450 and 630 nm directly correlates to cell density after incubation with increasing concentrations of MstnPP soluble aggregates (SA, 5–25 µM), protofibrils (PF, 5–25 µM), 25 µM MstnPP dimer and 25 µM PF/F (fibrils). Concentrations are expressed as monomer equivalents. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Error bars represent the standard error of the mean for triplicate samples from two independent experiments. Statistical significance was calculated using a paired Student's t-test where ** P<0.01 and * P<0.05.
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pone-0009170-g008: MstnPP aggregates and protofibrils (PF) are cytotoxic to C2C12 mouse myoblasts.C2C12 cytotoxicity assay using the WST-1 reagent where the difference in absorbance at 450 and 630 nm directly correlates to cell density after incubation with increasing concentrations of MstnPP soluble aggregates (SA, 5–25 µM), protofibrils (PF, 5–25 µM), 25 µM MstnPP dimer and 25 µM PF/F (fibrils). Concentrations are expressed as monomer equivalents. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Error bars represent the standard error of the mean for triplicate samples from two independent experiments. Statistical significance was calculated using a paired Student's t-test where ** P<0.01 and * P<0.05.

Mentions: Oligomeric species in the amyloid formation pathway of a number of proteins are cytotoxic when added to the media of cultured cells [41], [42], [65]. The effect of MstnPP soluble aggregates, protofibrils and fibrils on the viability of C2C12 mouse myoblasts was investigated by monitoring the absorbance of formazan (Fig. 8) produced after addition of WST-1 (Roche). Only viable cells reduce WST-1 to produce formazan with the ratio of absorbances at 450 and 630 nm correlated to the number of viable cells in the culture. C2C12 mouse myoblasts are the standard model cell-line used for the analysis of myostatin activity [16], [18]. Although human MstnPP is used here, sequence identity between the mouse and human myostatin precursors is 96%; the murine cell-line is therefore suitable for initial studies. 25 µM of soluble aggregates and at least 10 µM of protofibrils decreased cell viability significantly compared to buffer only and correctly folded MstnPP dimer controls, at the same pH. A solution containing a mixture of MstnPP protofibrils and fibrils, as well as lower concentrations of soluble aggregates and protofibrils, had a reduced effect. These results indicate that oligomeric species in the amyloid formation pathway of MstnPP affect the normal functioning of C2C12 myoblasts.


Cytotoxic aggregation and amyloid formation by the myostatin precursor protein.

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

MstnPP aggregates and protofibrils (PF) are cytotoxic to C2C12 mouse myoblasts.C2C12 cytotoxicity assay using the WST-1 reagent where the difference in absorbance at 450 and 630 nm directly correlates to cell density after incubation with increasing concentrations of MstnPP soluble aggregates (SA, 5–25 µM), protofibrils (PF, 5–25 µM), 25 µM MstnPP dimer and 25 µM PF/F (fibrils). Concentrations are expressed as monomer equivalents. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Error bars represent the standard error of the mean for triplicate samples from two independent experiments. Statistical significance was calculated using a paired Student's t-test where ** P<0.01 and * P<0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820090&req=5

pone-0009170-g008: MstnPP aggregates and protofibrils (PF) are cytotoxic to C2C12 mouse myoblasts.C2C12 cytotoxicity assay using the WST-1 reagent where the difference in absorbance at 450 and 630 nm directly correlates to cell density after incubation with increasing concentrations of MstnPP soluble aggregates (SA, 5–25 µM), protofibrils (PF, 5–25 µM), 25 µM MstnPP dimer and 25 µM PF/F (fibrils). Concentrations are expressed as monomer equivalents. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Error bars represent the standard error of the mean for triplicate samples from two independent experiments. Statistical significance was calculated using a paired Student's t-test where ** P<0.01 and * P<0.05.
Mentions: Oligomeric species in the amyloid formation pathway of a number of proteins are cytotoxic when added to the media of cultured cells [41], [42], [65]. The effect of MstnPP soluble aggregates, protofibrils and fibrils on the viability of C2C12 mouse myoblasts was investigated by monitoring the absorbance of formazan (Fig. 8) produced after addition of WST-1 (Roche). Only viable cells reduce WST-1 to produce formazan with the ratio of absorbances at 450 and 630 nm correlated to the number of viable cells in the culture. C2C12 mouse myoblasts are the standard model cell-line used for the analysis of myostatin activity [16], [18]. Although human MstnPP is used here, sequence identity between the mouse and human myostatin precursors is 96%; the murine cell-line is therefore suitable for initial studies. 25 µM of soluble aggregates and at least 10 µM of protofibrils decreased cell viability significantly compared to buffer only and correctly folded MstnPP dimer controls, at the same pH. A solution containing a mixture of MstnPP protofibrils and fibrils, as well as lower concentrations of soluble aggregates and protofibrils, had a reduced effect. These results indicate that oligomeric species in the amyloid formation pathway of MstnPP affect the normal functioning of C2C12 myoblasts.

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