Limits...
Increased nitroxidative stress promotes mitochondrial dysfunction in alcoholic and nonalcoholic fatty liver disease.

Song BJ, Abdelmegeed MA, Henderson LE, Yoo SH, Wan J, Purohit V, Hardwick JP, Moon KH - Oxid Med Cell Longev (2013)

Bottom Line: Increased nitroxidative stress causes mitochondrial dysfunctions through oxidative modifications of mitochondrial DNA, lipids, and proteins.Many mitochondrial proteins including the enzymes involved in fat oxidation and energy supply could be oxidatively modified (including S-nitrosylation/nitration) under increased nitroxidative stress and thus inactivated, leading to increased fat accumulation and ATP depletion.To demonstrate the underlying mechanism(s) of mitochondrial dysfunction, we employed a redox proteomics approach using biotin-N-maleimide (biotin-NM) as a sensitive biotin-switch probe to identify oxidized Cys residues of mitochondrial proteins in the experimental models of alcoholic and acute liver disease.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pharmacology and Toxicology, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, 9000 Rockville Pike, Bethesda, MD 20892, USA. bj.song@nih.gov

ABSTRACT
Increased nitroxidative stress causes mitochondrial dysfunctions through oxidative modifications of mitochondrial DNA, lipids, and proteins. Persistent mitochondrial dysfunction sensitizes the target cells/organs to other pathological risk factors and thus ultimately contributes to the development of more severe disease states in alcoholic and nonalcoholic fatty liver disease. The incidences of nonalcoholic fatty liver disease continuously increase due to high prevalence of metabolic syndrome including hyperlipidemia, hypercholesterolemia, obesity, insulin resistance, and diabetes. Many mitochondrial proteins including the enzymes involved in fat oxidation and energy supply could be oxidatively modified (including S-nitrosylation/nitration) under increased nitroxidative stress and thus inactivated, leading to increased fat accumulation and ATP depletion. To demonstrate the underlying mechanism(s) of mitochondrial dysfunction, we employed a redox proteomics approach using biotin-N-maleimide (biotin-NM) as a sensitive biotin-switch probe to identify oxidized Cys residues of mitochondrial proteins in the experimental models of alcoholic and acute liver disease. The aims of this paper are to briefly describe the mechanisms, functional consequences, and detection methods of mitochondrial dysfunction. We also describe advantages and limitations of the Cys-targeted redox proteomics method with alternative approaches. Finally, we discuss various applications of this method in studying oxidatively modified mitochondrial proteins in extrahepatic tissues or different subcellular organelles and translational research.

Show MeSH

Related in: MedlinePlus

Translational research application of a redox proteomics approach by analyzing the oxidatively modified proteins in control and alcohol-exposed rats in the absence or presence of PUFA. Oxidatively modified mitochondrial proteins from each group were identified with a redox proteomics method using biotin-NM as a probe [106, 107], purified with streptavidin-agarose beads, then displayed on 2D gel, and stained with silver. The images of all gels were adjusted by optimizing the similar density of an endogenous, internal protein (*) in each gel. Protein spots in encircled areas in indicated samples reflect the appearance or disappearance of oxidized proteins depending on the treatment in each group, as described in and adapted from [137].
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3649774&req=5

fig4: Translational research application of a redox proteomics approach by analyzing the oxidatively modified proteins in control and alcohol-exposed rats in the absence or presence of PUFA. Oxidatively modified mitochondrial proteins from each group were identified with a redox proteomics method using biotin-NM as a probe [106, 107], purified with streptavidin-agarose beads, then displayed on 2D gel, and stained with silver. The images of all gels were adjusted by optimizing the similar density of an endogenous, internal protein (*) in each gel. Protein spots in encircled areas in indicated samples reflect the appearance or disappearance of oxidized proteins depending on the treatment in each group, as described in and adapted from [137].

Mentions: As shown in Figure 4, the number and levels of oxidatively modified mitochondrial proteins were increased in alcohol-fed control rats (Base ethanol) compared to pair-fed control rats (Base control). Our results [137] showed that increased production of hydrogen peroxide and peroxynitrite in alcohol-exposed rats (Base ethanol) compared to pair-fed control group (Base control). These results are consistent with elevated levels of CYP2E1 and iNOS in ethanol-fed rats. Immunoblot analyses of oxidized proteins from each group revealed the presence of oxidatively modified thiolase and α-ATP synthase only in the Base-ethanol group. However, the increased levels of oxidized proteins in the Base-ethanol group were markedly decreased in rats fed the same amounts of alcohol in the presence of PUFA (PUFA-ethanol). Addition of PUFA to ethanol-fed rats (PUFA-ethanol) improved histological data (i.e., disappearance of fat vacuoles) with the absence of the oxidized protein bands of both thiolase and α-ATP synthase detected only in the Base-ethanol group. Furthermore, the respective activities of thiolase, ATP synthase, and ALDH2, all suppressed in the Base-ethanol group, were restored in the PUFA-ethanol group. Further mechanistic studies revealed that the PUFA diet significantly prevented activation/induction of CYP2E1 and iNOS, which produce ROS and RNS, respectively, observed in alcohol-exposed tissues (Base ethanol). Consequently, the elevated levels of a potently toxic peroxynitrite, which can S-nitrosylate Cys residues and/or nitrate Tyr residues of various proteins [48], were significantly decreased in the PUFA-ethanol group compared to those in alcohol-fed control rats (Base ethanol).


Increased nitroxidative stress promotes mitochondrial dysfunction in alcoholic and nonalcoholic fatty liver disease.

Song BJ, Abdelmegeed MA, Henderson LE, Yoo SH, Wan J, Purohit V, Hardwick JP, Moon KH - Oxid Med Cell Longev (2013)

Translational research application of a redox proteomics approach by analyzing the oxidatively modified proteins in control and alcohol-exposed rats in the absence or presence of PUFA. Oxidatively modified mitochondrial proteins from each group were identified with a redox proteomics method using biotin-NM as a probe [106, 107], purified with streptavidin-agarose beads, then displayed on 2D gel, and stained with silver. The images of all gels were adjusted by optimizing the similar density of an endogenous, internal protein (*) in each gel. Protein spots in encircled areas in indicated samples reflect the appearance or disappearance of oxidized proteins depending on the treatment in each group, as described in and adapted from [137].
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649774&req=5

fig4: Translational research application of a redox proteomics approach by analyzing the oxidatively modified proteins in control and alcohol-exposed rats in the absence or presence of PUFA. Oxidatively modified mitochondrial proteins from each group were identified with a redox proteomics method using biotin-NM as a probe [106, 107], purified with streptavidin-agarose beads, then displayed on 2D gel, and stained with silver. The images of all gels were adjusted by optimizing the similar density of an endogenous, internal protein (*) in each gel. Protein spots in encircled areas in indicated samples reflect the appearance or disappearance of oxidized proteins depending on the treatment in each group, as described in and adapted from [137].
Mentions: As shown in Figure 4, the number and levels of oxidatively modified mitochondrial proteins were increased in alcohol-fed control rats (Base ethanol) compared to pair-fed control rats (Base control). Our results [137] showed that increased production of hydrogen peroxide and peroxynitrite in alcohol-exposed rats (Base ethanol) compared to pair-fed control group (Base control). These results are consistent with elevated levels of CYP2E1 and iNOS in ethanol-fed rats. Immunoblot analyses of oxidized proteins from each group revealed the presence of oxidatively modified thiolase and α-ATP synthase only in the Base-ethanol group. However, the increased levels of oxidized proteins in the Base-ethanol group were markedly decreased in rats fed the same amounts of alcohol in the presence of PUFA (PUFA-ethanol). Addition of PUFA to ethanol-fed rats (PUFA-ethanol) improved histological data (i.e., disappearance of fat vacuoles) with the absence of the oxidized protein bands of both thiolase and α-ATP synthase detected only in the Base-ethanol group. Furthermore, the respective activities of thiolase, ATP synthase, and ALDH2, all suppressed in the Base-ethanol group, were restored in the PUFA-ethanol group. Further mechanistic studies revealed that the PUFA diet significantly prevented activation/induction of CYP2E1 and iNOS, which produce ROS and RNS, respectively, observed in alcohol-exposed tissues (Base ethanol). Consequently, the elevated levels of a potently toxic peroxynitrite, which can S-nitrosylate Cys residues and/or nitrate Tyr residues of various proteins [48], were significantly decreased in the PUFA-ethanol group compared to those in alcohol-fed control rats (Base ethanol).

Bottom Line: Increased nitroxidative stress causes mitochondrial dysfunctions through oxidative modifications of mitochondrial DNA, lipids, and proteins.Many mitochondrial proteins including the enzymes involved in fat oxidation and energy supply could be oxidatively modified (including S-nitrosylation/nitration) under increased nitroxidative stress and thus inactivated, leading to increased fat accumulation and ATP depletion.To demonstrate the underlying mechanism(s) of mitochondrial dysfunction, we employed a redox proteomics approach using biotin-N-maleimide (biotin-NM) as a sensitive biotin-switch probe to identify oxidized Cys residues of mitochondrial proteins in the experimental models of alcoholic and acute liver disease.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Pharmacology and Toxicology, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, 9000 Rockville Pike, Bethesda, MD 20892, USA. bj.song@nih.gov

ABSTRACT
Increased nitroxidative stress causes mitochondrial dysfunctions through oxidative modifications of mitochondrial DNA, lipids, and proteins. Persistent mitochondrial dysfunction sensitizes the target cells/organs to other pathological risk factors and thus ultimately contributes to the development of more severe disease states in alcoholic and nonalcoholic fatty liver disease. The incidences of nonalcoholic fatty liver disease continuously increase due to high prevalence of metabolic syndrome including hyperlipidemia, hypercholesterolemia, obesity, insulin resistance, and diabetes. Many mitochondrial proteins including the enzymes involved in fat oxidation and energy supply could be oxidatively modified (including S-nitrosylation/nitration) under increased nitroxidative stress and thus inactivated, leading to increased fat accumulation and ATP depletion. To demonstrate the underlying mechanism(s) of mitochondrial dysfunction, we employed a redox proteomics approach using biotin-N-maleimide (biotin-NM) as a sensitive biotin-switch probe to identify oxidized Cys residues of mitochondrial proteins in the experimental models of alcoholic and acute liver disease. The aims of this paper are to briefly describe the mechanisms, functional consequences, and detection methods of mitochondrial dysfunction. We also describe advantages and limitations of the Cys-targeted redox proteomics method with alternative approaches. Finally, we discuss various applications of this method in studying oxidatively modified mitochondrial proteins in extrahepatic tissues or different subcellular organelles and translational research.

Show MeSH
Related in: MedlinePlus