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Cross beta-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory-Denk Bodies Compared With Other Hepatocyte Inclusions
Medical University of Graz.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Organic Chemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-5582-140X
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2011 (English)In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 141, no 3, 1080-U428 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND andamp; AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steato-hepatitis 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 beta-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 beta-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, alpha(1)-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5diethoxycarbonyl-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 beta-sheet conformation than K18, whereas p62 never had cross beta-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 alpha-helical to cross beta-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.

Place, publisher, year, edition, pages
WB Saunders , 2011. Vol. 141, no 3, 1080-U428 p.
Keyword [en]
Liver Disease, Protein Aggregation, Keratin, p62, NASH
National Category
Engineering and Technology
URN: urn:nbn:se:liu:diva-70739DOI: 10.1053/j.gastro.2011.05.039ISI: 000294281200047OAI: diva2:441420

Funding Agencies|Austrian Genome Program (GEN-AU)||Medical University of Graz||Swedish Foundation for Strategic Research||Knut and Alice Wallenberg Foundation||

Available from: 2011-09-16 Created: 2011-09-16 Last updated: 2015-05-28
In thesis
1. Fluorescent thiophene-based ligands for detection and characterization of disease-associated protein aggregates
Open this publication in new window or tab >>Fluorescent thiophene-based ligands for detection and characterization of disease-associated protein aggregates
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis the unique optical properties of fluorescent ligands termed luminescent conjugated oligothiophenes (LCOs) have been used to study a variety of protein aggregates associated with human protein misfolding disease. This heterogeneous group of diseases contains well known and fatal members such as Alzheimer´s and Huntington´s disease and the development of sensitive tools for the detection and characterization of protein aggregates is crucial for unravelling the complexity of these pathologies. Conventionally, the molecular dyes Congo red and thioflavin T (ThT) have been the primary choices for detecting and monitoring protein misfolding events. However, the rigid scaffold of both Congo red and ThT only offers an on or off mode and limits their ability to make fine distinctions at the molecular level. In contrast, LCOs have a flexible conjugated backbone and in addition to detect a broader subset of misfolded proteins, LCO can be used to visualize the heterogeneity of protein aggregates.

The work presented in this thesis has given novel insights regarding the close connection between LCO design and optical performance. By altering the backbone length and the arrangement of substituents as well as replacing thiophene units with moieties affecting conjugation length and conformational freedom, the structural requirements of an optimal LCO for a certain application have been revealed. LCOs having a pentameric thiophene backbone with carboxyl end-groups were able to i) cross the blood-brain barrier and selectively stain cerebral amyloid β (Aβ) plaques, ii) detect non-thioflavinophilic Aβ aggregates and non-congophilic prion aggregates, iii) spectrally discriminate Aβ from tau aggregates and iiii) strongly label protein inclusion bodies. However, in some applications this design was outdone by others and in general, the conjugation length and the level of conformational freedom of the backbone were important determinants of the performance of the LCO.

Overall, the findings in this thesis illustrate how small alterations in the LCO molecular scaffold may have large impact on the ligand properties. The results highlight the importance of having a toolbox of diverse ligands in order to increase our knowledge regarding the complex nature of protein aggregates.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. 81 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1518
National Category
Natural Sciences
urn:nbn:se:liu:diva-92772 (URN)978-91-7519-623-7 (ISBN)
Public defence
2013-05-31, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (Swedish)
Available from: 2013-05-21 Created: 2013-05-21 Last updated: 2014-04-08Bibliographically approved

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