Abstract
Mallory-Denk bodies (MDBs) are hepatocytic protein aggregates found in steatohepatitis and several other chronic liver diseases as well as hepatocellular carcinoma. MDBs are mainly composed of phosphorylated keratins and stress protein p62/Sequestosome-1 (p62), which is a common component of cytoplasmic aggregates in a variety of protein aggregation diseases. In contrast to the well-established role of keratins, the role of p62 in MDB pathogenesis is still elusive. We have generated total and hepatocyte-specific p62 knockout mice, fed them with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce MDBs and allowed the mice to recover from DDC intoxication on a standard diet to investigate the role of p62 in MDB formation and elimination. In the absence of p62, smaller, granular and less distinct MDBs appeared, which failed to mature to larger and compact inclusions. Moreover, p62 deficiency impaired the binding of other proteins such as NBR1 and Hsp25 to MDBs and altered the cellular defense mechanism by downregulation of Nrf2 target genes. Upon recovery from DDC intoxication on a standard diet, there was an enhanced reduction of p62-deficient MDBs, which was accompanied by a pronounced decrease in ubiquitinated proteins. Our data provide strong evidence that keratin aggregation is the initial step in MDB formation in steatohepatitis-related mouse models. Interaction of p62 with keratin aggregates then leads to maturation i.e., enlargement and stabilization of the MDBs as well as recruitment of other MDB-associated proteins.
Highlights
The fate of misfolded proteins gained significant relevance in a variety of diseases characterized by the accumulation of abnormal proteins, collectively known as “protein aggregation diseases” [1, 2]
Double immunofluorescence staining with antibodies against known Mallory-Denk bodies (MDBs) components was correlated with the results of MM120-1immunostaining, which served as common reference, and colocalization efficiency was quantified for various proteins present in MDBs. doi:10.1371/journal.pone.0161083.t001
MDB size was observed after recovery in the different genotypes, which confirmed the observations of immunofluorescence staining (n = 5). (D) Immunoblotting of insoluble liver protein extracts with antibodies against keratin 8 (K8) and ubiquitin and corresponding densitometric quantification (n = 5) showed that loss of p62 did not affect the accumulation of insoluble ubiquitinated proteins or formation of cross-linked high molecular mass K8 species during DDC intoxication
Summary
The fate of misfolded proteins gained significant relevance in a variety of diseases characterized by the accumulation of abnormal proteins, collectively known as “protein aggregation diseases” [1, 2]. The folding of proteins into their functionally active three-dimensional structures is among the most fundamental roles of a living cell [2]. Misfolding on genetic or toxic basis may lead to aggregation-prone proteins, being deposited in the cytoplasm, nucleus or extracellular compartments [1, 3]. These inclusion bodies can serve as morphological hallmarks of a variety.
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