Role of SUMOylation in Ethanol‐Modulated Mitochondria Homeostasis

Abstract

ProposeAlcohol‐induced liver disease (ALD) is a major health concern of alcohol abuse and a leading cause of liver‐related morbidity and mortality. Alterations in mitochondrial morphology and function are a hallmark of ALD. Mitochondria synthesize most of the adenosine triphosphate (ATP) needed by mammalian cells and are intimately involved in the generation of and defense against reactive oxygen species (ROS). Evidence support the importance of ROS production and oxidative stress in the development of ALD. SUMOylation is a dynamic and reversible posttranscriptional modification involved in many cellular pathways including transcription, intracellular transport, DNA repair, replication, and cell signaling. We recently demonstrated that UBC9, the sole E2 protein required by SUMOylation machinery, is upregulated in Intragastric ethanol‐fed mouse model (IE) and cirrhotic tissues. We also found that UBC9 is phosphorylated and this is correlated with high level of SUMOylation in lipopolysaccharides‐activated Kupffer cells that lead inflammation development. In addition, we described a novel key role of SUMOylation inducing the enzymatic activity and protein stability of Cytochrome P450 2E1 (CYP2E1), that is the major component of the microsomal ethanol‐oxidizing system (MEOS), in NIAAA mouse model. This study aims to investigate whether ethanol induces mitochondrial dysfunction in ALD regulating the SUMOylation status of key respiratory chain proteins and explore the molecular mechanisms.MethodsPrimary mouse hepatocytes and livers from NIAAA mouse model were used. mRNA and protein levels were analyzed by Real‐Time PCR and Western Blotting, respectively. OCR (oxygen consumption rate) and Δψm (mitochondrial membrane potential) were measured by Seahorse mito stress test and JC‐1 staining by confocal microscopy, respectively.ResultsIn order to explore the role of SUMOylation in ALD, Mass Spectrometry (MS) was performed to identify SUMOylated proteins in 10‐day ethanol‐feeding+1 Binge ethanol (NIAAA) model, where SUMOs binding columns were used to purify SUMOylated proteins from total livers. Interestingly, we found that ethanol induces changes in SUMOylation state of several mitochondrial proteins involved in ATP synthesis (ATP5B, SOD2, CLC25A5, HSPD1, FBP1, CYCS), ATP metabolism (ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5J2, ASPA8, AK3), and mitochondria disorder (UQCRC2, PC, HMGCS2, ECHS1, COX6B1, NDUFV3, COX6C). These finding could suggest a potential role of SUMOylation in oxidative phosphorylation, electron transport chain (ETC) and respiratory control ratio may be modulating the ability of these proteins to form the complex above and/or regulating their activity. Specifically, ethanol treatment decreases ATP production and maximal respiration in primary mouse hepatocytes, while UBC9 knockdown prevents the loss of respiration. Furthermore, UBC9 silencing prevents the drop of Δψm when ethanol is added. This finding clearly indicates that SUMOylation is involved in mitochondrial physiology.Conclusionsthese novel finding provides highly novel information on the role of SUMOylation in the development of ALD and may provide new therapeutic strategies, which is of high public health relevance.Support or Funding InformationNIH/NIAAA R21AA027325‐01A1