Fig. 4: Both ethanol and alcoholic steatohepatitis liver tissue extracts promote the thioflavin T-monitored aggregation of KRT8.
a,b, Structures of wild-type and mutant KRT858–64 segments co-crystallized with alcohol. The wild-type SGMGGIT structure (a) shows two ethanol molecules (gray) per asymmetric unit coordinated to the N terminus of the peptide, while mutant SGMGCIT (b) displays isopropanol binding alongside the middle of the peptide along the fibril axis, occupying the space between the beta-sheets (in gray) in the steric zipper. Both structures illustrate that alcohols can directly associate with KRT8 amyloid fibrils. Aggregated KRT8 is the primary component of MDBs, a common pathological feature observed in hepatocytes during alcoholic liver disease. c, ThT aggregation kinetics assay of wild-type KRT8 with increasing concentrations of ethanol (0, 1, and 2.5%). d,e, Aggregation of alpha-synuclein (d) and tau microtubule-binding domains (e) in the presence of ethanol. For alpha-synuclein (‘aSyn’), significant protein precipitation was noted for the 2.5% ethanol sample, thus results from only the 1% ethanol sample are reported. f, Extracts of liver tissue with ASH or HCC were used to seed the aggregation of KRT8. g, The effects of demeclocycline HCl on KRT8 aggregation as measured by ThT fluorescence. We mixed 50 µM of KRT81–90 with various concentrations of demeclocycline dissolved in DMSO. The ‘KRT8’ control was treated with equal concentration DMSO vehicle. h, In a state of cellular stress, increased expression of KRT8 may contribute to a normal stress response. However, in the context of disease, particularly in the presence of ethanol and/or mutations which may occur in liver disease, KRT8 may become aggregated. This may result in the formation of MDBs or an insufficient stress response. n = 3 experimental replicates were used for c–f. Error bars represent ±s.d.
