The role of the ubiquitin-proteasome pathway in the formation of mallory bodies.
The dynamics of Mallory body (MB) formation are difficult to follow in vivo. Because of the lack of an in vitro mouse hepatocyte culture model, a cellular extract approach was developed. In this model an immunoprecipitate was obtained using an antibody to cytokeratin-8 (CK-8). The isolate contained a large number of compounds: CK-8, ubiquitin, a frameshift mutation of ubiquitin (UBB(+1)), proteasomal subunits beta5 (a catalytic subunit of the 20S proteasome) and Tbp7 (an ATPase subunit of the 26S proteasome), transglutaminase, tubulin, heat shock proteins 90 and 70, and MBs. In Western blots, CK-8 immunoprecipitates showed colocalization of these components in a complex of proteins colocalized in a high-molecular-weight smear. When the CK-8 immunoprecipitate was incubated with the isolate of proteasomes and an energy generating source (ATP), the components of the ubiquitinated protein smear increased. These observations taken together with the in vivo observation that these proteins colocalized at the edge of the MB shown in the present study suggest that these proteins form aggregates through covalent binding of CK-8, ubiquitin, and the proteasomes. Covalent aggregation is suggested by the fact that the protein complex found in the high-molecular-weight smear that forms in vitro fails to dissociate in SDS. This protein complex is present in the CK-8 immunoprecipitates of livers forming MBs but not in control livers. In conclusion, the results support the concept that Mallory bodies are aggresomes which form as the result of the failure of the ubiquitin-proteasome complex to adequately eliminate cytokeratins destined for proteolysis.
Bardag-Gorce
Fawzia
F
Department of Pathology and Medicine, Harbor- UCLA Medical Center, Torrance, California 90509, USA.
van Leeuwen
Frederik Willen
FW
Nguyen
Victoria
V
French
Barbara Alan
BA
Li
Jun
J
Riley
Nora
N
McPhaul
Laron Wilson
LW
Lue
Yan He
YH
French
Samuel William
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000284:Administration, Oral; D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System / Q000378:metabolism; D002719:Chlormethiazole / Q000008:administration & dosage / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000502:physiology*; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004095:Dihydropyridines / Q000008:administration & dosage / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000378:metabolism / Q000648:ultrastructure; D002479:Inclusion Bodies / Q000378:metabolism*; D007633:Keratins / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008815:Mice, Inbred Strains; D009097:Multienzyme Complexes / Q000502:physiology*; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D014452:Ubiquitins / Q000502:physiology*
D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004095:Dihydropyridines; D004791:Enzyme Inhibitors; C488922:Mallory body protein, mouse; D009097:Multienzyme Complexes; D011506:Proteins; D014452:Ubiquitins; D002719:Chlormethiazole; C061879:diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate; D007633:Keratins; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases