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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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SDS-PAGE and TEM analysis showing MstnPP resistance to trypsin digestion.A. dimer; and B. soluble aggregates. Major bands are indicated: 1. MstnPP monomer; 2. MstnPP dimer; 3. MstnPP soluble aggregates. C. MstnPP fibrils: 4. prefibrillar aggregates plus fibrils. Samples were analysed in sample buffer +/− βME where βME concentration is 2 M. D. TEM shows resistance of (i) protofibrils and (ii) amyloid fibrils to trypsin. Trypsin digests were performed with a MstnPP∶trypsin ratio of 100∶1 (A and B) or 20∶1 (C and D) for 18 hours at 37°C.
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pone-0009170-g006: SDS-PAGE and TEM analysis showing MstnPP resistance to trypsin digestion.A. dimer; and B. soluble aggregates. Major bands are indicated: 1. MstnPP monomer; 2. MstnPP dimer; 3. MstnPP soluble aggregates. C. MstnPP fibrils: 4. prefibrillar aggregates plus fibrils. Samples were analysed in sample buffer +/− βME where βME concentration is 2 M. D. TEM shows resistance of (i) protofibrils and (ii) amyloid fibrils to trypsin. Trypsin digests were performed with a MstnPP∶trypsin ratio of 100∶1 (A and B) or 20∶1 (C and D) for 18 hours at 37°C.

Mentions: Resistance to proteolytic digestion is another defining characteristic for the presence of amyloid fibrils [73], [74]. Comparative trypsin digestions were performed for the MstnPP fibril-containing sample, soluble aggregates and native dimer (Fig. 6). Digestion of the MstnPP dimer and aggregates was carried out with a MstnPP to trypsin ratio of 100∶1 (w/w) at 4°C, room temperature (approximately 22°C) and 37°C with samples taken after 0.5, 1, 2, 3, 4 and 18 hours. The precursor dimer and aggregates have comparable trypsin susceptibility at 37°C and are fully digested after an overnight incubation (18 hours, Fig. 6A and B respectively). The susceptibility of the soluble aggregates to trypsin digest suggests an open, flexible structure, consistent with CD results and supporting the conclusion that this species represents a prefibrillar intermediate rather than protofibrils. Prior to digestion the fibril-containing sample is not able to enter the top of the 4% stacking gel owing to its extremely large size. Following incubation with trypsin this property is maintained even after overnight incubation (18 h) at 37°C and a 5-fold increase in trypsin concentration (MstnPP∶trypsin 20∶1) (Fig. 6C). Inability to enter the stacking gel is not a definitive indication of proteolysis resistance since partial hydrolysis may result in products smaller than fibrils and/or protofibrils yet still large enough to be retained in the stacking gel. To address this possibility, TEM analysis revealed that both MstnPP protofibrils (Fig. 6Di) and mature fibrils (Fig. 6Dii) had unchanged morphology after trypsin incubation.


Cytotoxic aggregation and amyloid formation by the myostatin precursor protein.

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

SDS-PAGE and TEM analysis showing MstnPP resistance to trypsin digestion.A. dimer; and B. soluble aggregates. Major bands are indicated: 1. MstnPP monomer; 2. MstnPP dimer; 3. MstnPP soluble aggregates. C. MstnPP fibrils: 4. prefibrillar aggregates plus fibrils. Samples were analysed in sample buffer +/− βME where βME concentration is 2 M. D. TEM shows resistance of (i) protofibrils and (ii) amyloid fibrils to trypsin. Trypsin digests were performed with a MstnPP∶trypsin ratio of 100∶1 (A and B) or 20∶1 (C and D) for 18 hours at 37°C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2820090&req=5

pone-0009170-g006: SDS-PAGE and TEM analysis showing MstnPP resistance to trypsin digestion.A. dimer; and B. soluble aggregates. Major bands are indicated: 1. MstnPP monomer; 2. MstnPP dimer; 3. MstnPP soluble aggregates. C. MstnPP fibrils: 4. prefibrillar aggregates plus fibrils. Samples were analysed in sample buffer +/− βME where βME concentration is 2 M. D. TEM shows resistance of (i) protofibrils and (ii) amyloid fibrils to trypsin. Trypsin digests were performed with a MstnPP∶trypsin ratio of 100∶1 (A and B) or 20∶1 (C and D) for 18 hours at 37°C.
Mentions: Resistance to proteolytic digestion is another defining characteristic for the presence of amyloid fibrils [73], [74]. Comparative trypsin digestions were performed for the MstnPP fibril-containing sample, soluble aggregates and native dimer (Fig. 6). Digestion of the MstnPP dimer and aggregates was carried out with a MstnPP to trypsin ratio of 100∶1 (w/w) at 4°C, room temperature (approximately 22°C) and 37°C with samples taken after 0.5, 1, 2, 3, 4 and 18 hours. The precursor dimer and aggregates have comparable trypsin susceptibility at 37°C and are fully digested after an overnight incubation (18 hours, Fig. 6A and B respectively). The susceptibility of the soluble aggregates to trypsin digest suggests an open, flexible structure, consistent with CD results and supporting the conclusion that this species represents a prefibrillar intermediate rather than protofibrils. Prior to digestion the fibril-containing sample is not able to enter the top of the 4% stacking gel owing to its extremely large size. Following incubation with trypsin this property is maintained even after overnight incubation (18 h) at 37°C and a 5-fold increase in trypsin concentration (MstnPP∶trypsin 20∶1) (Fig. 6C). Inability to enter the stacking gel is not a definitive indication of proteolysis resistance since partial hydrolysis may result in products smaller than fibrils and/or protofibrils yet still large enough to be retained in the stacking gel. To address this possibility, TEM analysis revealed that both MstnPP protofibrils (Fig. 6Di) and mature fibrils (Fig. 6Dii) had unchanged morphology after trypsin incubation.

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