Increased oxidation and degradation of cytosolic proteins in alcohol-exposed mouse liver and hepatoma cells

Abstract

We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 wk elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by MS. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.

Figure 1

Increased levels of CYP2E1 and 3-nitrotyrosine in alcohol-fed mouse liver. (A) Equal amounts of microsomal proteins (10 μg/well) from the livers of control mice or alcohol-fed mice for 6 weeks (n = 2 per lane) were separated on 10% SDS-PAGE, transferred to PVDF-Immobilon membranes, and stained with Coomassie blue (top) or subjected to immunoblot analysis by using anti-CYP2E1 antibody (bottom). The CYP2E1 protein band is indicated with an arrow. (B) Equal amounts (15 μg/lane) of freshly isolated cytosolic proteins from the same set of mouse livers were separated on 10% SDS-PAGE and subjected to immunoblot analyses using specific antibodies against 3-nitrotyrosine (3-NT) (top) or β-actin (bottom). Specific protein bands recognized by the antibodies against 3-NT are designated with arrows. This figure represents a typical result from two separate experiments.

Figure 2

Increased oxidation of cytosolic proteins in alcohol-fed mouse liver and E47 HepG2 cells. (A) Purified biotin-NM labeled cytosolic proteins (20 μg/well, n = 2 per lane) from pair-fed control and ethanol-fed mice for 6 weeks were separated on 10% SDS-PAGE, transferred to PVDF-Immobilon membranes, and subjected to immunoblot analysis using streptavidin-HRP. (B) Biotin-NM labeled cytosolic proteins from untreated and E47 HepG2 cells treated with 100 mM ethanol for 8 h were analyzed by immunoblot analysis using MAb-biotin-HRP. (B) This figure represents a typical result from two separate experiments.

Figure 3

Separation of biotin-NM labeled cytosolic proteins from mouse livers by 2-DE. Biotin-NM labeled cytosolic proteins from pair-fed control (A) and alcohol-fed mouse livers (B) were purified with streptavidin-agarose, resolved by 2-DE gels, and silver stained. Individual protein spots (spot 1-55) with differential intensities were marked with different numbers, excised out of this particular gel (pH range 3 - 10), and subjected to MS analysis following in-gel trypsin digestion. Spot 21, designated with a square, was used as an internal standard for comparison purpose between the two different gels for pair-fed control and alcohol-fed mouse livers. Protein spots with similar or decreased intensities after alcohol exposure are designated with circles in Fig. 3C.

Figure 4

Separation of biotin-NM labeled cytosolic proteins from mouse livers on 2-DE. Another batch of biotin-NM labeled cytosolic proteins was purified streptavidin-agarose, resolved on 2-DE, and silver stained. Clearly separated protein spots (spot 56 - 92) were excised out of this particular gel (pH range 4 - 7) and subjected to MS analysis following in-gel trypsin digestion. Spot 88, designated with a square, was used as an internal standard for comparison purpose between the two different gels.

Figure 5

Inactivation of peroxiredoxin through oxidation after alcohol exposure. (A) Equal amounts (20 μg/well) of cytosolic proteins from pair-fed control and alcohol-fed mice were separated on 12% SDS-polyacrylamide gel and subjected to immunoblot analysis using a specific antibody which recognizes either oxidized Prx-SO₃ (inactive form) or Prx (total). (B) Equal amounts (20 μg/well) of cytosolic proteins from E47 HepG2 cells untreated or treated with 100 mM ethanol for indicated times were subjected to immunoblot analysis using specific antibodies to Prx-SO₃ or Prx. This figure represents a typical result from three separate experiments.