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 β-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.
Highlights
Myostatin is a member of the transforming growth factor-beta (TGF-b) superfamily of growth and differentiation factors and is a primary regulator of muscle growth both pre- and postnatally, primarily via inhibition of myoblast proliferation and differentiation [1,2,3,4]
Like other members of the family, myostatin is translated as a precursor protein (MstnPP) that consists of an Nterminal signal sequence, a regulatory propeptide domain and a growth factor domain which dimerises at the C-terminus via an inter-molecular disulfide bond [5,6,7]
A role for MstnPP in sporadic inclusion body myositis (sIBM) was proposed after colocalisation and direct association with amyloid beta (Ab) was observed in diseased cells [31], and because endoplasmic reticulum (ER) stress causes an upregulation of myostatin expression [45]
Summary
Myostatin is a member of the transforming growth factor-beta (TGF-b) superfamily of growth and differentiation factors and is a primary regulator of muscle growth both pre- and postnatally, primarily via inhibition of myoblast proliferation and differentiation [1,2,3,4]. The mature growth factor dimer is cleaved from the propeptide region by furin convertase proteolysis in the endoplasmic reticulum (ER) at a conserved RSRR sequence [2,7]. The N-terminal propeptide plays a regulatory role after cleavage, remaining noncovalently associated with the mature dimer to form a latent complex which is exported from the cell [7]. Myostatin remains latent until a second cleavage event immediately N-terminal to aspartate 76 of the propeptide region, most probably by metalloproteinases, that disrupts the association [12,13]. The mature growth factor dimer is structurally similar to other members of the TGF-b family [11]; the structural characteristics of MstnPP or the propeptide region remain undetermined
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