5F) Nearly identical results were obtained using SAM as a methyl

5F). Nearly identical results were obtained using SAM as a methyl donor instead of betaine (Fig. S6A-D). To further test the hypothesis that

the combined HCV-ethanol effect results from FOXO3 demethylation, we generated a FOXO3 R248K_R250K double mutant missing the arginines methylated in FOXO1.[17] This mutant demonstrated less total protein methylation than the WT-FOXO3 (Fig. 6A) and its half-life was significantly reduced (Fig. 6B). The methylation-defective mutant was similar to the WT protein in that it was still transcriptionally active (Fig. 6C), it was primarily CHIR-99021 price cytosolic in uninfected cells (Fig. 6D,E), and it translocated to the nucleus in response to HCV infection. The FOXO3 R248K_R250K double mutant differed from the WT protein in two respects. Its nuclear translocation was not inhibited by the combination of HCV and ethanol (Fig. 6D,E), and formation of the HCV-specific pI 5.85 peak was not inhibited by ethanol (Fig. 6F, compare to Fig. 2A or S7B). Finally, we used small interfering RNA (siRNA) to knock down expression of PRMT1, the methyltransferase responsible

for the majority of arginine methylation.[17] This knock down reduced nuclear levels and decreased transcriptional activity of WT but not mutant FOXO3 (Figs. 6G, S7C). We compared the pattern of FOXO3 species present in human liver nuclear extracts from normal donor livers, HCV cirrhosis, and nonalcoholic BMN 673 mouse steatohepatitis (NASH) cirrhosis. We observed the presence of a peak in the region of pI 6.0 for majority of the samples and a peak in the region of pI 5.85 in all HCV-positive and some HCV-negative samples (Fig. 7A). We quantitated the presence of an HCV-like effect as the 5.85 peak area divided by the sum of the areas of the 5.85 + 6.0 peaks. As shown in Fig. 7B, the 5.85 peak accounted for ∼60%-90%

of total in HCV versus only ∼10% in NASH or normal liver (P < 0.01). To determine if this peak was related to JNK activation we simultaneously analyzed the samples for the phosphorylated JNK species (pI 5.3 and 5.6) Urease in our samples by cIEF (Fig. 7C). Figure 7D shows that regardless of disease condition, there was a strong correlation between detection of pJNK species and the proportion of the pI 5.85 form of FOXO3. FOXO transcription factors regulate hepatic growth and metabolism and respond to stress conditions.[20-22] FOXO1 is activated by HCV infection, contributing to insulin resistance,[23, 24] and FOXO3 activity was noted to be increased in HCV infection where it modulated innate immune signaling.[25] The molecular mechanisms of FOXO3 regulation by HCV and how alcohol modifies HCV’s FOXO3 effects in the liver has not been determined. In the present study we have shown that HCV and ethanol induce specific and interactive patterns of FOXO3 posttranslational modifications that alter the function of this transcription factor.

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