Sensitivity and specificity of in situ proximity ligation for protein interaction analysis in a model of steatohepatitis with Mallory-Denk bodies.

The in situ proximity ligation assay (isPLA) is an increasingly used technology for in situ detection of protein interactions, post-translational modifications, and spatial relationships of antigens in cells and tissues, in general. In order to test its performance we compared isPLA with immunofluorescence microscopy by analyzing protein interactions in cytoplasmic protein aggregates, so-called Mallory Denk bodies (MDBs). These structures represent protein inclusions in hepatocytes typically found in human steatohepatitis and they can be generated in mice by feeding of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). We investigated the colocalization of all three key MDB components, namely keratin 8 (K8), keratin 18 (K18), and p62 (sequestosome 1) by isPLA and immunofluorescence microscopy. Sensitivity and specificity of isPLA was assessed by using Krt8-/- and Krt18-/- mice as biological controls, along with a series of technical controls. isPLA signal visualization is a robust technology with excellent sensitivity and specificity. The biological relevance of signals generated critically depends on the performance of antibodies used, which requires careful testing of antibodies like in immunofluorescence microscopy. There is a clear advantage of isPLA in visualizing protein co-localization, particularly when antigens are present at markedly different concentrations. Furthermore, isPLA is superior to confocal microscopy with respect to spatial resolution of colocalizing antigens. Disadvantages compared to immunofluorescence are increased costs and longer duration of the laboratory protocol.

CD73 (ecto-5'-nucleotidase) hepatocyte levels differ across mouse strains and contribute to mallory-denk body formation.

UNLABELLED: Formation of hepatocyte Mallory-Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein, p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of MDB-susceptible C57BL and MDB-resistant C3H mouse strains can illuminate MDB-associated pathways. Using both targeted and unbiased metabolomic analyses, we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in messenger RNA (mRNA) level, protein expression, and enzymatic activity of the adenosine-generating enzyme, ecto-5'-nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and nonalcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB-inducing agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild-type (WT) and CD73(-/-) mice a DDC-containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73(-/-) , compared to WT livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was nearly absent in CD73(-/-) mice. CONCLUSION: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. Expression of the adenosine-generating enzyme, CD73, contributes to experimental MDB induction and is highly regulated in MDB-associated liver injury in mice and in chronic human liver disease.

Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory-Denk body formation.

Mallory-Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein keratin 8 (K8) and its cross-linking by transglutaminase-2 (TG2) are essential for MDB formation. K8 hyperphosphorylation occurs in association with liver injury and MDB formation, but the link between keratin phosphorylation and MDB formation is unknown. We used a mutational approach to identify K8 Q70 as a residue that is important for K8 cross-linking to itself and other liver proteins. K8 cross-linking is markedly enhanced on treating cells with a phosphatase inhibitor and decreases dramatically on K8 S74A or Q70N mutation in the presence of phosphatase inhibition. K8 Q70 cross-linking, in the context of synthetic peptides or intact proteins transfected into cells, is promoted by phosphorylation at K8 S74 or by an S74D substitution and is inhibited by S74A mutation. Transgenic mice that express K8 S74A or a K8 G62C liver disease variant that inhibits K8 S74 phosphorylation have a markedly reduced ability to form MDBs. Our findings support a model in which the stress-triggered phosphorylation of K8 S74 induces K8 cross-linking by TG2, leading to MDB formation. These findings may extend to neuropathies and myopathies that are characterized by intermediate filament-containing inclusions.

[Autophagy in liver diseases].

Two major degradation systems exist in cells: the lysosome and proteasome. In the lysosome system, extracellular materials are degraded via endocytosis. Intracellular materials are degraded by autophagy, a cellular pathway crucial for various intracellular events. It has recently been demonstrated that autophagy is involved in the pathogenesis of various liver diseases. In hepatitis B or hepatitis C virus infection, autophagy is enhanced in hepatocytes. In hepatic steatosis, hepatocyte autophagy is inhibited. The expression of the autophagy protein is disrupted in hepatocellular carcinoma. I summarize recent advances in the study of the involvement of autophagy in various liver diseases. The regulation of autophagy in the liver may be a useful therapeutic strategy for various liver diseases.