Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Lahiri, P.
Functional analysis of the role of p62/Sequestosome-1 in
protein aggregation diseases
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2016. pp.
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- Autor*innen der Med Uni Graz:
- Lahiri Pooja
- Betreuer*innen:
- Zatloukal Kurt
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- Abstract:
- How cells deal with misfolded proteins gained significant relevance in a variety of diseases characterized by accumulation of abnormal protein aggregates collectively known as “protein aggregation diseases”. They include among others, neuronal Lewy bodies in Parkinson’s disease, neurofibrillary tangles in Alzheimer’s disease, TDP-43 aggregates in amyotrophic lateral sclerosis, and Mallory-Denk bodies (MDBs) in steatohepatitis as well as other chronic liver disorders. As a reflection of a common pathogenesis, these inclusions often display, in addition to their disease-specific protein “backbone”, an analogous molecular composition, namely they all contain the multifunctional stress adaptor protein p62/Sequestosome-1, and ubiquitin as constant constituents. Moreover, these pathological aggregates share the common structural property of cross ¿-sheet (amyloid-like) conformation. Therefore, the investigation of these common structural and molecular composition are of paramount importance as they may provide new evidence on how misfolded proteins and the factors involved in aggregation machinery regulates protein aggregate formation, its clearance, and stress-induced cellular toxicity. In this study, we focused on the function of p62 in the formation of MDBs. MDBs are the best studied hepatocytic protein inclusions found in several chronic liver diseases, especially in steatohepatitis, chronic cholestasis, and hepatocellular carcinoma. To delve into the role of p62 in MDB formation, we have generated p62 total (p62-/-) and hepatocyte-specific (p62hep-/-) knockout mice and intoxicated them with DDC to induce MDBs. Key questions which are investigated are whether p62 initiates, enhances or inhibits MDB formation and influences their stability and degradation. Another aspect of this study was to comprehend how p62 modulates liver injury in the DDC model similar to that observed in autophagy deficiency. Furthermore, how p62-domains regulate the binding of various components for the formation of aggregates were also evaluated. In the absence of p62, small and granular MDBs appeared, which failed to mature to larger and compact MDBs. The lack of p62 impaired the binding of other proteins such as NBR1, Hsp25 and K17 to MDBs. Moreover, loss of p62 altered the cellular defense mechanism by downregulation of Nrf2 target genes during chronic injury. Upon recovery, there was a rapid numerical reduction of MDBs lacking p62 with a parallel decrease of cross-linked ubiquitinated proteins. Additionally, we identified a functionally relevant second isoform of human p62 lacking the PB1 domain. The absence of SH2 region of PB1 domain in the second isoform led to the acquisition of cross ß-sheet structure that enabled p62 to progressively form aggregates with primary components of protein inclusions without ubiquitin. The results indicate that p62 is essential for the maturation and stabilization of MDBs. The data highlight that keratins are the central proteins in MDB formation that proposes two different pathways of p62 containing protein aggregate formation in the liver. The first pathway is linked to human steatohepatitis and drug-induced hepatotoxicity models where keratin aggregation initiates MDB formation, and attachment of p62 then leads to maturation and stabilization of the aggregates. The second pathway involves a transition of p62-containing intracellular hyaline bodies (IHBs) into MDBs by incorporation of abnormal keratins in a pre-existing matrix of aggregated p62 as observed in idiopathic copper toxicosis and a few cases of hepatocellular carcinomas. Furthermore, the interesting new finding that a natural variant of p62 can acquire cross ß-sheet conformation (amyloid-like), and can progressively form aggregates in the absence of ubiquitin provide a new approach to study the role of p62 in various protein aggregation diseases.