title: Cross β-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.
abstract: OBJECTIVE
  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.
authors.affiliation: Institute of Pathology, Medical University of Graz, Graz, Austria.
authors.forename: Vineet
authors.initials: V
authors.lastname: Mahajan
authors.forename: Therése
authors.initials: T
authors.lastname: Klingstedt
authors.forename: Rozalyn
authors.initials: R
authors.lastname: Simon
authors.forename: K Peter R
authors.initials: KP
authors.lastname: Nilsson
authors.forename: Andrea
authors.initials: A
authors.lastname: Thueringer
authors.forename: Karl
authors.initials: K
authors.lastname: Kashofer
authors.forename: Johannes
authors.initials: J
authors.lastname: Haybaeck
authors.forename: Helmut
authors.initials: H
authors.lastname: Denk
authors.forename: Peter M
authors.initials: PM
authors.lastname: Abuja
authors.forename: Kurt
authors.initials: K
authors.lastname: Zatloukal
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D006224:Cricetinae; D003412:Cricetulus; D005234:Fatty Liver / Q000378:metabolism / Q000473:pathology; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism* / Q000648:ultrastructure*; D053538:Keratin-18 / Q000737:chemistry / Q000378:metabolism; D053533:Keratin-8 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D017433:Protein Structure, Secondary; D000071456:Sequestosome-1 Protein; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D006360:Heat-Shock Proteins; D053538:Keratin-18; D053533:Keratin-8; C486595:Krt8 protein, mouse; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein; C101343:Sqstm1 protein, mouse