Proteasome activation by hepatitis C core protein is reversed by ethanol-induced oxidative stress.
OBJECTIVE
The proteasome is a major cellular proteinase. Its activity is modulated by cellular oxidants. Hepatitis C core protein and ethanol exposure both cause enhanced oxidant generation. The aim was to investigate whether core protein, by its ability to generate oxidants, alters proteasome activity and whether these alterations are further affected by ethanol exposure.


METHODS
These interactions were examined in Huh-7 cell lines that expressed inducible HCV core protein and/or constitutive cytochrome P450 2E1 (CYP2E1) and as purified components in a cell-free system. Chymotrypsin-like proteasome activity was measured fluorometrically.


RESULTS
Proteasome activity in core-positive 191-20 cells was 20% higher than that in core-negative cells and was enhanced 3-fold in CYP2E1-expressing L14 cells. Exposure of core-positive cells to glutathione ethyl ester, catalase, or the CYP2E1 inhibitor diallyl sulfide partially reversed the elevation of proteasome activity in core-positive cells, whereas ethanol exposure suppressed proteasome activity. The results indicate that proteasome activity was up-regulated by low levels of core-induced oxidative stress but down-regulated by high levels of ethanol-elicited stress. These findings were partially mimicked in a cell-free system. Addition of core protein enhanced the peptidase activity of purified 20S proteasome containing the proteasome activator PA28 and was further potentiated by addition of liver mitochondrial and/or microsome fractions. However, proteasome activation was significantly attenuated when fractions were obtained from ethanol-fed animals.


CONCLUSIONS
HCV core protein interacts with PA28, mitochondrial, and endoplasmic reticulum proteins to cause low levels of oxidant stress and proteasome activation, which is dampened during ethanol metabolism when oxidant generation is higher.
Liver Study Unit, Omaha Veterans Affairs Medical Center, Omaha, Nebraska 68105, USA. nosna@unmc.edu
Natalia A
NA
Osna
Ronda L
RL
White
Viatcheslav M
VM
Krutik
Ting
T
Wang
Steven A
SA
Weinman
Terrence M
TM
Donohue
D000498:Allyl Compounds / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D045744:Cell Line, Tumor; D002918:Chymotrypsin / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000633:toxicity*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D016877:Oxidants / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013440:Sulfides / Q000494:pharmacology; D014162:Transfection; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D000498:Allyl Compounds; D065691:Cytochrome P-450 CYP2E1 Inhibitors; D004791:Enzyme Inhibitors; D016877:Oxidants; D011506:Proteins; D013440:Sulfides; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D000431:Ethanol; C038491:allyl sulfide; C042431:S-ethyl glutathione; D020122:tert-Butylhydroperoxide; D002374:Catalase; D019392:Cytochrome P-450 CYP2E1; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex; C486213:Psme1 protein, mouse; D005978:Glutathione