bromochloroacetic-acid and acetylleucyl-leucyl-norleucinal

The major pathways for protein degradation are the proteasomal and lysosomal systems. Derangement of protein degradation causes the formation of intracellular inclusions, and apoptosis and is associated with several diseases. We utilized hepatocyte-derived cell lines to examine the consequences of the cytoplasmic hepatocyte Mallory-Denk body-like inclusions on organelle organization, autophagy and apoptosis, and tested the hypothesis that autophagy affects inclusion turnover. Proteasome inhibitors (PIs) generate keratin-containing Mallory-Denk body-like inclusions in cultured cells and cause reorganization of mitochondria and other organelles, autophagy and apoptosis. In cultured hepatoma cells, caspase inhibition blocks PI-induced apoptosis but not inclusion formation or autophagy activation. Autophagy induction by rapamycin decreases the extent of PI-induced inclusions and apoptosis in Huh7 and OUMS29 cells. Surprisingly, blocking of autophagy sequestration by 3 methyl adenine or beclin 1 siRNA, but not bafilomycin A1 inhibition of autophagic degradation, also inhibits inclusion formation in the tested cells. Therefore, autophagy can be upstream of apoptosis and may promote or alleviate inclusion formation in cell culture in a context-dependent manner via putative autophagy-associated molecular triggers. Manipulation of autophagy may offer a strategy to address the importance of inclusion formation and its significance in inclusion-associated diseases.

Topics: Apoptosis; Autophagy; Cell Line; Cycloheximide; Cysteine Proteinase Inhibitors; Humans; Inclusion Bodies; Keratins; Leupeptins; Organelles; Proteasome Inhibitors; Protein Synthesis Inhibitors

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament (IF) proteins that are expressed in glandular epithelia. Although the mechanism of keratin turnover is poorly understood, caspase-mediated degradation of type I keratins occurs during apoptosis and the proteasome pathway has been indirectly implicated in keratin turnover based on colocalization of keratin-ubiquitin antibody staining. Here we show that K8 and K18 are ubiquitinated based on cotransfection of His-tagged ubiquitin and human K8 and/or K18 cDNAs, followed by purification of ubiquitinated proteins and immunoblotting with keratin antibodies. Transfection of K8 or K18 alone yields higher levels of keratin ubiquitination as compared with cotransfection of K8/18, likely due to stabilization of the keratin heteropolymer. Most of the ubiquitinated species partition with the noncytosolic keratin fraction. Proteasome inhibition stabilizes K8 and K18 turnover, and is associated with accumulation of phosphorylated keratins, which indicates that although keratins are stable they still turnover. Analysis of K8 and K18 ubiquitination and degradation showed that K8 phosphorylation contributes to its stabilization. Our results provide direct evidence for K8 and K18 ubiquitination, in a phosphorylation modulated fashion, as a mechanism for regulating their turnover and suggest that other IF proteins could undergo similar regulation. These and other data offer a model that links keratin ubiquitination and hyperphosphorylation that, in turn, are associated with Mallory body deposits in a variety of liver diseases.

Topics: Cell Line; Cysteine Endopeptidases; Enzyme Inhibitors; Humans; Intermediate Filament Proteins; Keratins; Leupeptins; Multienzyme Complexes; Mutation; Phosphorylation; Proteasome Endopeptidase Complex; Transfection; Ubiquitins