Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared.
In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.
Bardag-Gorce
Fawzia
F
Department of Pathology, Harbor UCLA Medical Center, 1000 W. Carson Street, Torrance, CA 90502, USA.
Venkatesh
Ravi
R
Li
Jun
J
French
Barbara Alan
BA
French
Samuel William
SW
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004032:Diet; D004359:Drug Therapy, Combination; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D007274:Injections, Intraperitoneal; D008099:Liver / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D009097:Multienzyme Complexes / Q000302:isolation & purification / Q000378:metabolism*; D019319:Okadaic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D051381:Rats; D017208:Rats, Wistar
D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D019319:Okadaic Acid; D000431:Ethanol; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases