Cross β-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions

Abstract

Background & aims: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis.

Methods: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross β-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies.

Results: LCO analysis showed cross β-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α₁-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross β-sheet conformation than K18, whereas p62 never had cross β-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis.

Conclusions: K8 can undergo conformational changes from predominantly α-helical to cross β-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8⁻/⁻ mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.