Myostatin Amyloid Formation Figure 7. ThT binding, CD spectroscopy and TEM show the formation of prefibrillar structures and protofibrils by MstnPP aggregates at 37uC and pH 5.3. A. ThT binding assays before incubation (blue), after overnight incubation (green) and after one week incubation (red). ***** P,0.001 by Student’s t-test using averaged values over peak. B. CD spectra of MstnPP soluble aggregates (blue), aggregates after overnight incubation at 37uC in pH 5.3 (green) and after one week incubation (red). C. TEM shows elongation and clustering after overnight incubation; and D. a granular to smooth transition and enhanced lateral association after one week. doi:10.1371/journal.pone.0009170.g007 pH 5.3 and 60uC, the aggregates form amyloid protofibrils and after one week are able to form long, linear and unbranching amyloid fibrils. At 37uC, protofibrils and mature fibrils form over an increased time period. These in vitro results show that MstnPP is capable of amyloid protofibril and fibril formation, supporting a role for misfolding of the myostatin precursor in the pathogenesis of sIBM. MstnPP aggregates and protofibrils have a cytotoxic effect on mouse myoblasts when added to the culture medium. CD spectroscopy shows there is a transition from the correctly folded protein structure to a structure consisting primarily of b- sheet. For a number of peptides and proteins this transition is from a loop or disordered region through an a-helical intermediate stage [36,75]. Analysis of the differently folded forms of MstnPP suggests a shift towards a predominance of a-helix on misfolding. Whether this a-helical shift represents the intermediate stage described for other amyloid species is not known but is an attractive possibility. TEM images show a number of different morphological forms present in the aggregate solution. These may represent different oligomeric intermediates at different stages of the fibril formation process, some of which may be the direct precursors of protofibrils. Overnight incubation of MstnPP aggregates in pH 5.3 at 60uC produces a transition from the a- helical structure to one dominated by b-sheet. This result is consistent with the major models proposed for amyloid formation, which describe an unfolded state in equilibrium with a partly unfolded a-helix-containing intermediate that accelerates fibril formation [36,37,76]. Aggregation of the intermediate into oligomers precedes structural rearrangement into b-sheet-rich protofibrils. A detailed structure of protofibrils has not been determined; they are defined primarily by morphology, the ability to bind dyes such as ThT and a structural shift to a predominance of b-sheet. Amyloid fibrils have been well-studied structurally by NMR and X-ray fiber diffraction [77]. For growth factors such as myostatin, and a number of other secreted proteins, perhaps the biggest obstacle to refolding is the formation of the correct disulfide-bonded cysteine pairs in the ER [78]. Myostatin has the characteristic TGF-b cysteine knot motif in its mature domain [2,10,11] and so likely requires both an appropriate redox environment and the presence of disulfide-bond chaperones for correct folding in the ER. The myostatin propeptide that lies N-terminal to the mature domain has been proposed to have a chaperone-like function in the correct folding of the mature domain [8,9]. During ER-stressed situations, such as an altered redox environment, misfolding can be extensive [48,79] with the unfolded protein response (UPR) resulting in the elimination of misfolded proteins and/or apoptosis if necessary [48,80,81]. If the PLoS ONE | www.plosone.org 8 February 2010 | Volume 5 | Issue 2 | e9170