Our current understanding of how ethanol regulates proteasome activity is based on studies conducted in ethanol-metabolizing liver cells, which revealed that the ethanol-induced decrease in proteasome activity is due to inhibition by ethanol metabolites, some of which directly forms adducts with proteasomal subunits [5
]. In addition, some studies have indicated a link between ethanol-elicited impaired methylation of histones and nuclear proteasome function [26
]. However, it was not clear from these studies whether hypomethylation of DNA and/or aberrant histone methylation induces the suppression of proteasome activity or altered methionine metabolism directly regulates proteasome function.
McClain et al
postulated a correlation between a decrease in proteasome activity and lower SAM levels in alcoholic liver disease (ALD) [27
], but the exact link between these events was not established. Here, we provide evidence that ethanol-induced alteration in methionine metabolism that results in decreased intracellular SAM:SAH ratios directly suppressed 20S proteasome in liver cells. To our knowledge, this is the first indication for a role of impaired methylation reactions as an important factor in the regulation of liver proteasome activity. Here, we report that cultured mouse hepatocytes exposed to a SAM:SAH ratio of 2.5 (as previously seen in hepatocytes and livers of ethanol-fed animals) exhibited decreased proteasome activity compared with cells exposed to a SAM:SAH ratio of 5 (as seen in controls). Further, ethanol-elicited inhibition of proteasome activity was blocked by overnight exposure of hepatocytes to ethanol and 0.1 mM SAM, indicating that ethanol-elicited suppression of proteasome activity can be prevented by this methyl donor. Furthermore, in hepatocyte-like Huh7CYP cells, proteasome was suppressed by exposure to the specific methylation reaction inhibitor, tubercidin, and this effect was also blocked by co-incubation with SAM. Interestingly, proteasome inhibition under hypomethylation conditions seems to be a liver-specific as other investigators reported lower proteasome activity in vascular smooth muscle cells exposed to SAM [28
The importance of a “normal” SAM:SAH ratio for proteasome function was further validated by experiments, in which we observed an inhibitory effect of tubercidin on proteasome activity in Huh7CYP cells. In addition, it is important to note that because CYP2E1 is a proteasome substrate [29
], the tubercidin-mediated increase in CYP2E1 activity, possibly due to CYP2E1 stabilization, could potentially induce oxidative stress. Nevertheless, despite the increase in CYP2E1 activity, tubercidin treatment evidently suppressed oxidative stress indices examined in this study.
Further, we found that tubercidin treatment suppressed proteasome activity in both nuclear and cytosolic fractions of Huh7CYP cells. However, we cannot exclude a possibility that cytosolic proteasome activity was reduced due to lower expression of proteasome components because of impaired gene methylation. To ascertain this, crude cytosolic proteasome from mouse hepatocytes was exposed to varying SAM:SAH ratios. Consistent with the effects observed on proteasome activity in cells exposed to tubercidin, we found that proteasome activity in cytosol was also suppressed when the SAM:SAH ratio was less than 2.5. The latter data suggests that some cytosolic proteins possess SAM-dependent methyltransferase activity, are regulated by altered SAM:SAH ratios and may directly modulate proteasome activity.
The observation that purified 20S proteasome when exposed to SAM:SAH of 2.5 or less possesses significant reduced activity is also noteworthy. In addition, we found that a 25 kDa subunit of purified 20S proteasome preparation exhibited immunoreactivity to antibody against methyl lysine. This is consistent with a recent proteome study that revealed the hepatic 20S proteasome subunits are arginine and lysine methylated [31
]. Further, we observed that differential SAM:SAH ratios regulated methylation of lysine residues on the 25 kDa proteasome subunit. This suggests that either the 20S proteasomal subunit(s) and/or other proteins that form a tight complex and are co-purified with commercially obtained 20S proteasome have (i) a SAM-dependent methyltransferase-like activity and; (ii) are sensitive to SAH inhibition.
Although SAM:SAH ratio-induced changes in proteasome activity were modest, this level of proteasome inhibition does result in significant alterations of downstream cell functions including peptide hydrolysis for antigen presentation [32
]. Further, the modest decreases in proteasome activity ensure that the viability of treated cells is maintained and that the results are not due to cell death since a profound inhibition of proteasome would likely cause apoptosis/necrosis. Thus, our data indicate that in addition to previously reported methylation-mediated regulation of proteasome activity in cells at the epigenetic level [19
], proteasome function is also regulated at the level of SAM:SAH-dependent methylation reactions.
Further, we observed greater inhibition of activity of IPr-enriched proteasome preparation with decreased SAM:SAH compared with 20S constitutive proteasome. This observation is consistent with higher sensitivity of IFNγ-treated hepatocytes to tubercidin compared with untreated cells (). These findings suggest that there may be an increased association of a lysine methyltransferase (and probably some others) with immunoproteasome subunits. However, establishment of the identity of such methyltransferases that regulate proteasome function is currently under investigation.
Thus, ethanol exposure to liver cells dually regulates proteasome activity: by ethanol-induced oxidative stress [1
] and by ethanol-induced defects in protein methylation reactions. The reduction in proteasome activity has serious implications in many important physiologically liver cell functions, such as protein aggregation (Mallory body formation) and antigen presentation. The fact that methylation defects can be corrected by treatment with regulators of methylation, like SAM or betaine, provides another therapeutic target of maintaining proteasome function and protecting against the development of liver injury from alcohol abuse.
We conclude that in liver cells, impaired methylation reactions directly suppress proteasome function, providing a novel mechanism for regulation of 20S proteasome activity.