ABSTRACT
All living organisms require exogenous foods/nutrients for producing energy in the form of Adenosine
Triphosphate (ATP), which is needed for numerous cellular functions and is efficiently produced in mitochondria
that are intimately involved in the generation of and defense against reactive oxygen species (ROS). An early
event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes
to the mitochondrial proteome, respiration defects and mitochondrial DNA (mtDNA) damage. These effects of
ethanol are prominent in the liver, the major site of ethanol metabolism in the body, promoting both apoptotic
and necrotic cell death and contribute to the onset or progression of alcohol-induced liver diseases (ALD), leading
to hepatic fibrosis/cirrhosis and cancer. SUMOylation is a posttranslational modification that involves addition of
SUMOs (small ubiquitin-like modifiers) modulating protein stability, activity and localization. Several studies have
intimated a close relationship between SUMOylation and ROS. We recently demonstrated that Ubiquitin
Conjugating Enzyme 9 (UBC9), the sole E2 protein required by SUMOylation machinery, is upregulated in
Intragastric fed-mice liver (IE) and cirrhotic tissues. We also found that UBC9 is phosphorylated and this is
correlated with high level of SUMOylation activity in lipopolysaccharides-activated Kupffer cells that leads to
inflammation development. In addition, we elucidated the key function of SUMOylated microsomal Cytochrome
P450 2E1 (CYP2E1) in ALD that sustains its enzymatic activity and protein stability. Much of the detailed
investigation of the role of SUMOylation in ALD has been started in our laboratory. However, several important
mechanistic pathways that are altered in ALD have not been investigated yet. In 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.
This proposal tests the novel hypothesis that ethanol induces mitochondrial dysfunction in ALD
regulating the SUMOylation status of key respiratory chain proteins and explore the molecular
mechanisms. Two specific aims are proposed: 1) Explore the role of SUMOylation in ethanol-modulated
ETC and ATP production, 2) Examine the role of SUMOylation in ethanol-induced CYP2E1 for
maintenance of mitochondrial oxygen homeostasis. If successfully completed, these studies should provide
highly novel information on the role of SUMOylation in the development of ALD and may provide novel
therapeutic strategies, which is of high