미토콘드리아 기능 장애에서 mitoNEET의 역할과 작용 기전 연구

Abstract

MitoNEET (mitochondrial protein containing Asn-Glu-Glu-Thr (NEET) sequence) is a 2Fe-2S cluster-containing integral membrane protein that resides in the mitochondrial outer membrane (MOM) and participates in a redox-sensitive signaling and Fe-S (iron-sulfur) cluster transfer. Thus, mitoNEET is a key regulator of mitochondrial oxidative capacity and iron homeostasis. Moreover, mitochondrial dysfunction and oxidative stress play critical roles in inflammatory diseases such as sepsis. Increased iron levels mediated by mitochondrial dysfunction lead to oxidative damage and generation of reactive oxygen species (ROS). Increasing evidence suggests that targeting mitoNEET to reverse mitochondrial dysfunction deserves further investigation. However, the role of mitoNEET in inflammatory diseases is unknown. Here, we investigated the mechanism of action and function of mitoNEET during lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. Levels of mitoNEET protein increased during microbial or LPS-induced sepsis. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) decreased the levels of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in animal models of sepsis, as well as LPS-induced inflammatory responses by macrophages in vitro. Inhibition of mitoNEET using NL-1 or mitoNEET shRNA abrogated LPS-induced ROS formation and mitochondrial dysfunction. Furthermore, mitochondrial iron accumulation led to generation of LPS-induced ROS, a process blocked by NL-1 or shRNA. Taken together, these data suggest that mitoNEET could be a key therapeutic molecule that targets mitochondrial dysfunction during inflammatory diseases and sepsis.|Inflammatory disease, such as sepsis is associated with both oxidative stress mediated by mitochondrial dysfunction and glycolysis inhibiting antioxidant capacity. Glycolysis as a source of energy is activated in inflammatory response. Glycolytic ATP (adenosine triphosphate) production inhibit AMPK (adenosine monophosphate-activated protein kinase) activity and expression of PGC-1α (Peroxisome proliferator-activated receptor-gamma coactivator-1alpha). PGC-1α is tightly linked to the anti-ROS system through the production of antioxidant enzymes and plays the role of the induction of mitochondrial biogenesis. Therefore, the activation of PGC-1α through inhibition of glycolysis is an interesting therapeutic target that improved survival rates and protected septic mice from organ oxidative injury and maintained healthy mitochondria. Glycolysis requires NAD+ for use in the production of glycolytic ATP and if NAD+ is not regenerated, glycolysis will stop. Earlier studies demonstrated that in the presence of oxygen, NADH is oxidized to NAD+ through mitochondrial NADH shuttle systems and it is supplied to glycolysis. Recently, increasing evidences suggest that mitoNEET (mitochondrial protein containing NEET sequence) as a novel redox enzyme oxidizing cytosolic NADH to NAD+ may indirectly activate glycolysis and glycolytic ATP production. MitoNEET as a ligand of a thiazolidinedione (TZD) regulate mitochondrial ROS and iron homeostasis. Therefore, mitoNEET has been suggested as a possible molecule of mitochondrial therapeutic target for oxidative stress during damaged mitochondrial disease. However, under oxidizing environment, that inhibition of the electron transfer activity of mitoNEET as a generator of NAD+ activates PGC-1α through regulation of glycolytic ATP has not been studied yet. In this study, we investigated mechanisms of PGC-1α in antioxidant effect mediated by inhibiting mitoNEET during LPS-induced oxidative stress. Levels of PGC-1α protein was increased by inhibition of mitoNEET using inhibitor, or shRNA. Pharmacological inhibition of mitoNEET with inhibitor, mitoNEET Ligand-1 (NL-1), suppressed LPS-induced ROS and increased antioxidant enzyme when compared with LPS alone in control shRNA cells, but not in PGC-1α shRNA expressing cells. Increase of PGC-1α expression by inhibition of mitoNEET is mediated though AMPK activity increased by prevention of ATP production under inflammatory stimuli. Taken together, these data demonstrated that PGC-1α is a key regulator in antioxidant effect mediated by inhibiting mitoNEET during inflammatory diseases. Therefore, we suggest that targeting mitoNEET is a possible therapeutic strategy bring the effect of energy metabolism control and antioxidant effect through activating PGC-1α in mitochondrial dysfunctional disease.|MitoNEET, a mitochondrial outer membrane protein containing the Asn-Glu-Glu-Thr (NEET) sequence, controls the formation of intermitochondrial junctions and confers autophagy resistance. Moreover, mitoNEET as a mitochondrial substrate undergoes ubiquitination by activated Parkin in the initiation of mitophagy. Therefore, mitoNEET is linked to the regulation of autophagy and mitophagy. Mitophagy is a selective removal process of damaged or unnecessary mitochondria, which is crucial to sustaining mitochondrial quality control. In numerous human diseases, the accumulation of damaged mitochondria by impaired mitophagy has been observed. The therapeutic strategy involving the targeting of mitoNEET as a mitophagy-enhancing mediator requires further research. Herein, we confirmed that mitophagy is indeed activated by mitoNEET inhibition. CCCP (carbonyl cyanide m-chlorophenyl hydrazone), which leads to mitochondrial depolarization, induces mitochondrial dysfunction and superoxide production. This, in turn, contributes to the induction of mitophagy. mitoNEET protein levels were initially increased before the increase in LC3-Ⅱ protein level following CCCP treatment. Pharmacological inhibition of mitoNEET using mitoNEET Ligand-1 (NL-1) promoted accumulation of PTEN-induced putative kinase 1 (Pink1) and Parkin, which are mitophagy-associated proteins, as well as activation of mitochondria–lysosome crosstalk, in comparison to CCCP alone. Inhibition of mitoNEET using NL-1, or mitoNEET shRNA transfected into RAW264.7 cells, abrogated CCCP-induced ROS and mitochondrial cell death, which demonstrated by confocal microscopy and flow cytometry or lactate dehydrogenase (LDH)-release assay; additionally, it activated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and superoxide dismutase (SOD2), regulators of oxidative metabolism, detected using western blotting. In particular, the increase in PGC-1α, which is a major regulator of mitochondrial biogenesis, promotes mitochondrial quality control. These data indicated that mitoNEET may be employed as a therapeutic target in numerous human diseases to enhance mitophagy and protect cells by maintaining a network of healthy mitochondria.