factor dimer has been suggested to hold exciting potential for inhibitory targeting in a wide range of muscle wastage diseases [28,29]. Less focus had been placed on the involvement of MstnPP or the propeptide region in disease until a role for MstnPP in the pathogenesis of sporadic inclusion body myositis (sIBM) was proposed [30,31]. sIBM is the most common progressive muscle wastage disease associated with aging where progressive muscle loss leads to severe atrophy and weakness. Although the pathogenesis is unknown, it is likely that oxidative damage contributes to aging of the muscle fibers [30]. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been demonstrated in sIBM muscle fibers [30,32]. Inflam- mation and amyloid formation appear to be predominant features but whether these are causally related and which is the primary cause of sIBM, remain matters of debate [30,33]. The presence of fibrillar inclusions in some diseased tissue suggests that sIBM may be an amyloid disease, where a prominent feature is protein aggregation and the subsequent formation and deposition of large amyloid fibrils analogous to those observed for neurodegenerative disorders such as Alzheimer’s, Parkinson’s and Huntington’s diseases, the spongiform encephalopathies, the systemic amyloid- oses and type II diabetes [34,35]. Despite extensive research, the mechanisms behind amyloid formation and how they contribute to disease remain poorly understood. It is well-documented that fibril formation occurs via one or more oligomeric intermediate stages [36–40] and it appears that an intermediate oligomeric species, rather than the mature fibril, is the mediator of cyto- and neurotoxicity [41–43]. Amyloid fibrils, characterised by extensive b-sheet secondary structure, are deposited in tissues and are resistant to degradation but may actually be chemically inert [35]. The filamentous inclusions of sIBM tissue contain a number of molecules normally alien to muscle fibers, predominantly the amyloid beta (Ab) protein of Alzheimer’s disease [44]. Biopsies from sIBM muscle fibers revealed a close association of the myostatin precursor protein (MstnPP) with Ab aggregates and increased levels of both MstnPP and the processed myostatin growth factor dimer [31]. Furthermore, when muscle cells are placed in conditions that result in endoplasmic reticulum (ER) stress, MstnPP is upregulated and an sIBM-like pathology is observed [45]. ER stress may lead to the misfolding and aggregation of MstnPP. Cultured human muscle fibers (CHMFs) overexpressing Ab precursor protein showed an increase in MstnPP protein levels and subsequent experimental inhibition of the proteasome caused co-accumulation of MstnPP and AbPP/Ab within aggresomes [46]. CHMFs subjected to tunicamycin or thapsigargin, compounds that inhibit protein glycosylation and disrupt ER calcium levels respectively inducing ER stress, also showed an increase in MstnPP expression (in addition to ER stress marker proteins GRP78 and Herp), possibly due to the activation of NF-kB [45]. In contrast to proteasome inhibition, where MstnPP mRNA levels were decreased, NF-kB activation increases transcription of MstnPP mRNA in a stressed situation. These differences may be explained by the observation that the nature and duration of ER stress may be important in determining whether a cell activates a protective pathway or enters apoptosis [47,48]. An additional mechanism for how myostatin/ MstnPP contributes to sIBM is focused on increased activity by the myostatin growth factor. As myostatin is a procachectic growth factor postnatally, increased production and processing of MstnPP may result in increased levels of circulating myostatin growth factor and signalling that leads to atrophy [45,46,49], though there is little evidence supporting this hypothesis. A number of peptide hormones, such as those in secretory granules of the endocrine system, have been found to aggregate into amyloids in secretory Myostatin Amyloid Formation pathways in a regulated, functional manner [50,51], so there is a possibility that myostatin aggregates are protective in the sIBM diseased state. The role of MstnPP in sIBM has not been firmly established. Our research shows for the first time that MstnPP is able to form amyloid fibrils independently in vitro via a pathway that produces cytotoxic intermediates. When MstnPP is refolded and purified from E. coli inclusion bodies, a population of MstnPP spontane- ously misfolds into amyloid protofibril-like aggregates that can be visualised using electron microscopy and bind the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the protofibril-like aggregates form straight and unbranched amyloid fibrils that are 15 nm in diameter and exhibit higher order amyloid structures such as twists and bundles. The fibrils are resistant to trypsin digest after incubation overnight at 37uC. Circular dichroism spectroscopy reveals that MstnPP undergoes a number of structural transitions through aggregation to amyloid protofibril and fibril formation that initiate at a physiologically relevant temperature. Significantly, MstnPP aggre- gates and protofibrils have a negative effect on the viability of C2C12 mouse myoblasts when added to the culture medium. These results support a role for MstnPP in the pathogenesis of sIBM and present the hypothesis that this role may be within a degenerative context. Results MstnPP Is Predicted to Be Prone to Aggregation and Disorder In Silico In silico programs have been shown to accurately predict the regions of a polypeptide chain that are prone to b-sheet aggregation and amyloid formation. The MstnPP amino acid sequence was analysed for b-aggregation propensity (Fig. 1A) using Tango [52] (Fig. S1A) and PASTA [53,54] (Fig. S1B) and for amyloidogenic regions using Waltz [55] (Fig. S1C). Both Tango and PASTA predicted a high propensity for b-aggregation for residues within the pro-peptide region, 154–177 (Fig. 1A); residues 139–153 were also predicted by PASTA to have b-aggregation tendencies, albeit at a comparatively lower level. Tango also identified growth factor domain sequences, 314–321 and 347–355. Waltz predicted the same propeptide amyloidogenic regions, 153–162 and 168–177 as well as the very C-terminus of the protein, 348–364. Mapping of aggregation-prone regions of the myostatin growth factor onto the published crystal structure [11] shows that residues 348–364 localise to the b-sheet ‘fingers’ of the growth factor (Fig. 1B). In Vitro Refolding of MstnPP Results in the Production of Large Soluble Aggregates Human MstnPP is found in inclusion bodies when overex- pressed in E. coli like other TGF-b family members [56,57] and can be refolded in vitro using a modification of methods described for zebra-fish myostatin [8]. After refolding, in addition to the native disulfide-bonded MstnPP dimer, two other species were apparent by non-reducing SDS-PAGE; misfolded monomer and a very high molecular weight species that has been characterised previously as soluble aggregates [8,9] (Fig. 2A), which are frequently observed during protein refolding [58]. Native MstnPP was purified by heparin affinity chromatography that separated the correctly folded MstnPP dimer from the majority of the aggregates followed by the removal of remaining aggregates by gel filtration. During heparin chromatography, the correctly folded dimer eluted at 200 mM NaCl (H1) and the majority of aggregates as a large peak at 600 mM NaCl (H2) (Fig. 2B and Fig. S2A). PLoS ONE | www.plosone.org 2 February 2010 | Volume 5 | Issue 2 | e9170