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NAD þ depletion increased acetylated α-tubulin levels in 16HBE and 3T3-L1 cells. (A) Intracellular pyridine nucleotide concentrations in 16HBE cells (n = 3) treated with DMSO or FK866 (500 nM) for the indicated times. DMSO controls were treated for 72 h. (B) Immunoblot analysis for acetyl α-tubulin in 16HBE and 3T3-L1 cells treated with DMSO or FK866 (500 nM) for the indicated times. (C) Immunostaining for acetyl α-tubulin in 16HBE and 3T3-L1 cells treated with DMSO or FK866 for the indicated times. (D) Immunostaining for SIRT2 and NQO1 in 16HBE cells demonstrating the perinuclear localization of SIRT2 and NQO1.
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The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD⁺-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K⁴⁰) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD⁺ and may supplement levels of NAD⁺ near microtubules to aid SIRT2 deacetylase activity. While H...
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... intracellular NAD + concentrations and acetyl α-tubulin levels we treated 16HBE cells or 3T3-L1 fibroblasts with the nicotinamide phosphoribosyl transferase inhibitor FK866. Treatment with FK866 gradually decreased NAD + levels in both cell lines over 72 h, while during the same time course the levels of acetyl α-tubulin gradually increased ( Fig. 1A and B). Immunostaining for acetyl α-tubulin confirmed the immunoblot data and showed that following treatment with FK866 there was a gradual increase in immunostaining for acetyl α-tubulin in both cell lines (Fig. 1C). These data also showed that the increase in immunostaining for acetyl α-tubulin initially appeared in perinuclear regions, ...
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... NAD + levels in both cell lines over 72 h, while during the same time course the levels of acetyl α-tubulin gradually increased ( Fig. 1A and B). Immunostaining for acetyl α-tubulin confirmed the immunoblot data and showed that following treatment with FK866 there was a gradual increase in immunostaining for acetyl α-tubulin in both cell lines (Fig. 1C). These data also showed that the increase in immunostaining for acetyl α-tubulin initially appeared in perinuclear regions, which is consistent with previous studies that have suggested that SIRT2 may be responsible for regulating acetylation of perinuclear microtubules [27]. We have confirmed the perinuclear localization of SIRT2 and ...
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... also showed that the increase in immunostaining for acetyl α-tubulin initially appeared in perinuclear regions, which is consistent with previous studies that have suggested that SIRT2 may be responsible for regulating acetylation of perinuclear microtubules [27]. We have confirmed the perinuclear localization of SIRT2 and NQO1 in 16HBE cells (Fig. 1D). Double immunostaining for NQO1 and α-tubulin is shown in Supplementary Material, Fig. S1 and colocalization of NQO1 with microtubules was shown in previous work using immunocytochemistry and proximate ligation assays ...
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... in perinuclear regions, which is consistent with previous studies that have suggested that SIRT2 may be responsible for regulating acetylation of perinuclear microtubules [27]. We have confirmed the perinuclear localization of SIRT2 and NQO1 in 16HBE cells (Fig. 1D). Double immunostaining for NQO1 and α-tubulin is shown in Supplementary Material, Fig. S1 and colocalization of NQO1 with microtubules was shown in previous work using immunocytochemistry and proximate ligation assays ...
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... h) to deplete cells of pyridine nucleotides via DNA damage and PARP-mediated NAD + consumption. Following treatments, NQO1 was immunoprecipitated from lysates using the redox-dependent anti-NQO1 antibody that targets the C-terminus. In the absence of hydrogen peroxide, the majority of NQO1 is not immunoprecipitated due to its reduced conformation (Fig. 7B, lane 1); however, in samples pretreated with MI2321 in the absence of hydrogen peroxide, moderate levels of NQO1 could be pulled down (Fig. 7B, lane 2) suggesting MI2321 induces a change in the conformation of NQO1. In samples treated with hydrogen peroxide alone, large amounts of NQO1 could be immunoprecipitated due to the conversion of NQO1 ...
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... could be immunoprecipitated due to the conversion of NQO1 to an oxidized conformation (Fig. 7B, lane 3). Pretreatment with MI2321 before hydrogen peroxide decreased the amount of NQO1 immunoprecipitated (Fig. 7B, lane 4, compare to lane 3). In contrast, M3190, a non-inhibitory analog of MI2321, had little effect on the amount of NQO1 pulled down (Fig. 7B, lanes 6 and 7, compare to lanes 1 and 3). β-lapachone was used as a positive control for oxidized NQO1 (Fig. 7B, lane 5 [15]). Taken together these data suggest that binding of MI2321 (but not the inactive analog of the inhibitor, M3190) alters the structure of NQO1 and locks the protein into an inactivated conformation which is non-responsive to pyridine nucleotides ( Fig. ...
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... with MI2321 before hydrogen peroxide decreased the amount of NQO1 immunoprecipitated (Fig. 7B, lane 4, compare to lane 3). In contrast, M3190, a non-inhibitory analog of MI2321, had little effect on the amount of NQO1 pulled down (Fig. 7B, lanes 6 and 7, compare to lanes 1 and 3). β-lapachone was used as a positive control for oxidized NQO1 (Fig. 7B, lane 5 [15]). Taken together these data suggest that binding of MI2321 (but not the inactive analog of the inhibitor, M3190) alters the structure of NQO1 and locks the protein into an inactivated conformation which is non-responsive to pyridine nucleotides ( Fig. 7A and B, lanes 2 and ...
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... of pyridine nucleotides. To demonstrate that inactivation by MI2321 alters the structure of NQO1 purified rhNQO1 was incubated with NADH and MI2321 then analyzed by nondenaturing polyacrylamide gel electrophoresis (Fig. 7D). Inactivation of NQO1 by MI2321 (Fig. 7D, lane 4) resulted in slower migration of NQO1 compared to non-inactivated proteins (Fig. 7D, lanes ...
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Citations
... GPX4 is another critical enzyme of the antioxidant pathway that metabolizes lipid peroxides and limits damage to the cellular membranes (Brigelius-Flohe and Maiorino 2013). Furthermore, NAD(P) H quinone oxidoreductase-1 (NQO1) is a highly inducible redox-dependent enzyme (Siegel et al. 2021). Our data showed that the expression levels of GCLM, GPX4, and NQO1 were downregulated in the cardiac tissues of the WT + DOX mice. ...
Doxorubicin (DOX) is one of the most frequently used chemotherapeutic drugs belonging to the class of anthracyclines. However, the cardiotoxic effects of anthracyclines limit their clinical use. Recent studies have suggested that ferroptosis is the main underlying pathogenetic mechanism of DOX-induced cardiomyopathy (DIC). BTB-and-CNC homology 1 (Bach1) acts as a key role in the regulation of ferroptosis. However, the mechanistic role of Bach1 in DIC remains unclear. Therefore, this study aimed to investigate the underlying mechanistic role of Bach1 in DOX-induced cardiotoxicity using the DIC mice in vivo (DOX at cumulative dose of 20 mg/kg) and the DOX-treated H9c2 cardiomyocytes in vitro (1 μM). Our results show a marked upregulation in the expression of Bach1 in the cardiac tissues of the DOX-treated mice and the DOX-treated cardiomyocytes. However, Bach1−/− mice exhibited reduced lipid peroxidation and less severe cardiomyopathy after DOX treatment. Bach1 knockdown protected against DOX-induced ferroptosis in both in vivo and in vitro models. Ferrostatin-1 (Fer-1), a potent inhibitor of ferroptosis, significantly alleviated DOX-induced cardiac damage. However, the cardioprotective effects of Bach1 knockdown were reversed by pre-treatment with Zinc Protoporphyrin (ZnPP), a selective inhibitor of heme oxygenase-1(HO-1). Taken together, these findings demonstrated that Bach1 promoted oxidative stress and ferroptosis through suppressing the expression of HO-1. Therefore, Bach1 may present as a promising new therapeutic target for the prevention and early intervention of DOX-induced cardiotoxicity.
... Posttranslational acetylation of tubulin plays an important role in regulating the structure and stability of microtubules and has been shown to affect intracellular signal transduction, cell migration, and neuropathy. 13 The histone deacetylase (HDAC) family contains many deacetylases that differ in structure and substrate specificity, enzymatic mechanisms, and subcellular localization. ...
Aims
To identify an effective strategy for promoting microvascular endothelial cells (MECs) to phagocytize myelin debris and reduce secretion of inflammatory factors following spinal cord injury (SCI).
Methods
We established a coculture model of myelin debris and vascular‐like structures. The efficiency with which MECs phagocytize myelin debris under different conditions was examined via ELISA, flow cytometry, and immunofluorescence. Tubastatin‐A was used to interfere with the coculture model. The anti‐inflammatory effects of Tubastatin‐A were observed by HE staining, flow cytometry, immunofluorescence, and ELISA.
Results
MECs phagocytized myelin debris via IgM opsonization, and phagocytosis promoted the secretion of inflammatory factors, whereas IgG‐opsonized myelin debris had no effect on inflammatory factors. Application of the HDAC6 inhibitor Tubastatin‐A increased the IgG levels and decreased the IgM levels by regulating the proliferation and differentiation of B cells. Tubastatin‐A exerted a regulatory effect on the HDAC6‐mediated autophagy‐lysosome pathway, promoting MECs to phagocytize myelin debris, reducing the secretion of inflammatory factors, and accelerating the repair of SCI.
Conclusions
Inhibition of HDAC6 to regulate the immune‐inflammatory response and promote MECs to phagocytize myelin debris may represent a novel strategy in the treatment of SCI.
... SIRT1 and 2 are present in all three places, with SIRT2 having particular rich expression in the plasma membrane and cytoskeleton-associated organelles. The localization of NAD(P)H:quinone oxidoreductase 1 (NQO1) near MTs leads to the hypothesis that the NQO1-catalyzed oxidation of NADH to NAD + may drive the deacetylase activity of SIRT2, according to the results of a novel study [177]. While HDAC6 is known to regulate the majority of K40 acetylation, SIRT2 modulates K40 acetylation in the perinuclear region. ...
The inter-neuronal communication occurring in extensively branched neuronal cells is achieved primarily through the microtubule (MT)-mediated axonal transport system. This mechanistically regulated system delivers cargos (proteins, mRNAs and organelles such as mitochondria) back and forth from the soma to the synapse. Motor proteins like kinesins and dynein mechanistically regulate polarized anterograde (from the soma to the synapse) and retrograde (from the synapse to the soma) commute of the cargos, respectively. Proficient axonal transport of such cargos is achieved by altering the microtubule stability via post-translational modifications (PTMs) of α- and β-tubulin heterodimers, core components constructing the MTs. Occurring within the lumen of MTs, K40 acetylation of α-tubulin via α-tubulin acetyl transferase and its subsequent deacetylation by HDAC6 and SIRT2 are widely scrutinized PTMs that make the MTs highly flexible, which in turn promotes their lifespan. The movement of various motor proteins, including kinesin-1 (responsible for axonal mitochondrial commute), is enhanced by this PTM, and dyshomeostasis of neuronal MT acetylation has been observed in a variety of neurodegenerative conditions, including Alzheimer’s disease and Parkinson’s disease (PD). PD is the second most common neurodegenerative condition and is closely associated with impaired MT dynamics and deregulated tubulin acetylation levels. Although the relationship between status of MT acetylation and progression of PD pathogenesis has become a chicken-and-egg question, our review aims to provide insights into the MT-mediated axonal commute of mitochondria and dyshomeostasis of MT acetylation in PD. The enzymatic regulators of MT acetylation along with their synthetic modulators have also been briefly explored. Moving towards a tubulin-based therapy that enhances MT acetylation could serve as a disease-modifying treatment in neurological conditions that lack it.
Graphical abstract
... Human NAD(P)H:quinone oxidoreductase 1 (UniProt ID: P15559) is a soluble, typically cytosolic, dimeric protein at the hub of the antioxidant defense and stabilization of up to 50 different proteins, including p53 and HIF-1α [1,2]. Although hNQO1 has historically been labeled as a cytosolic enzyme, it is likely found in multiple subcellular locations [2,3]. As an enzyme, it catalyzes the two-electron reduction of a wide range of quinones to hydroquinones using NAD(P)H as a coenzyme (see Table A1 in [2]), displaying negative cooperativity regarding catalysis and FAD binding [4][5][6][7] and also detoxifying superoxide radicals [8]. ...
... NQO1 is highly inducible upon stress through the Nrf2 or Ah pathways [1,8]. The Nrf2 regulatory pathway mediates the delicate balance between oxidative signaling and antioxidant defense, and it is likely that the NQO1 antioxidant properties and its modulation of reduced/oxidized forms of NAD + play important roles [3,8,[22][23][24][25]. The Nrf-2 pathway is associated with multiple human pathologies, including alcohol-induced liver disease, cigarette smoking, cancer and neurodegeneration [25]. ...
... Importantly, acetylation was highly susceptible to functional ligand binding (NADH mediated reduction of FAD largely prevented acetylation), and deacetylation at K262 and K271 was quickly catalyzed by different sirtuins in vitro [40]. This phenomenon is likely associated with the NADH-dependent localization of hNQO1 around microtubules [3], and also highlights the plasticity of hNQO1 functionality in different ligation states and different subcellular locations [2,3,13]. Studies from our laboratory using mimetic mutations (at K31 and K209) have shown that the effect of acetylation on hNQO1 activity may be mainly ascribed to the K31 site (see the mutant K31Q in Table 1). ...
Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is a multifunctional and antioxidant stress protein whose expression is controlled by the Nrf2 signaling pathway. hNQO1 dysregulation is associated with cancer and neurological disorders. Recent works have shown that its activity is also modulated by different post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, and these may synergize with naturally-occurring and inactivating polymorphisms and mutations. Herein, I describe recent advances in the study of the effect of PTMs and genetic variations on the structure and function of hNQO1 and their relationship with disease development in different genetic backgrounds, as well as the physiological roles of these modifications. I pay particular attention to the long-range allosteric effects exerted by PTMs and natural variation on the multiple functions of hNQO1.
... Frontiers in Pharmacology frontiersin.org 08 2016; Siegel et al., 2021). Models have been proposed where NQO1 could provide NAD + for sirtuin-dependent deacetylation of other target proteins (Kim et al., 2014;Kang et al., 2018;Khadka et al., 2018;Qiu et al., 2022), or alternatively, sirtuin enzymes may modulate the redox-conformation of NQO1 through the consumption of NAD + ultimately lowering NADH levels (Siegel et al., 2018). ...
The stress induced protein NQO1 can participate in a wide range of biological pathways which are dependent upon the interaction of NQO1 with protein targets. Many of the protein-protein interactions involving NQO1 have been shown to be regulated by the pyridine nucleotide redox balance. NQO1 can modify its conformation as a result of redox changes in pyridine nucleotides and sites on the C-terminal and helix seven regions of NQO1 have been identified as potential areas that may be involved in redox-dependent protein-protein interactions. Since post-translational modifications can modify the functionality of proteins, we examined whether redox-dependent conformational changes induced in NQO1 would alter lysine acetylation. Recombinant NQO1 was incubated with and without NADH then acetylated non-enzymatically by acetic anhydride or S-acetylglutathione (Ac-GSH). NQO1 acetylation was determined by immunoblot and site-specific lysine acetylation was quantified by mass spectrometry (MS). NQO1 was readily acetylated by acetic anhydride and Ac-GSH. Interestingly, despite a large number of lysine residues (9%) in NQO1 only a small subset of lysines were acetylated and the majority of these were located in or near the functional C-terminal or helix seven regions. Reduction of NQO1 by NADH prior to acetylation resulted in almost complete protection of NQO1 from lysine acetylation as confirmed by immunoblot analysis and MS. Lysines located within the redox-active C-terminus and helix seven regions were readily acetylated when NQO1 was in an oxidized conformation but were protected from acetylation when NQO1 was in the reduced conformation. To investigate regulatory mechanisms of enzymatic deacetylation, NQO1 was acetylated by Ac-GSH then exposed to purified sirtuins (SIRT 1-3) or histone deacetylase 6 (HDAC6). NQO1 could be deacetylated by all sirtuin isoforms and quantitative MS analysis performed using SIRT2 revealed very robust deacetylation of NQO1, specifically at K262 and K271 in the C-terminal region. No deacetylation of NQO1 by HDAC6 was detected. These data demonstrate that the same subset of key lysine residues in the C-terminal and helix seven regions of NQO1 undergo redox dependent acetylation and are regulated by sirtuin-mediated deacetylation.
... FAD binding renders a conformational state (NQO1 holo ), ready to initiate catalysis, intracellularly stable and with a different interactomic pattern than that of NQO1 apo [10,19]. Upon NAD(P)H binding, the FAD is reduced to FADH 2 constituting a catalytically relevant state that also shuttles the proteins to interact with microtubules and enhances PPI [6,16,20]. Binding of dicoumarol (Dic), a competitive inhibitor of both NAD(P)H and the substrate [16], leads to an inactive state likely reflecting the ternary complex (NQO1holo:NAD(P)H) or transition state analogue, also abrogating PPI [6]. It is interesting to note that we have recently characterized the effects of FAD and Dic binding to WT NQO1, revealing that ligand binding sequentially stabilizes the FAD (FBS) and Dic (DBS) binding sites, and these stabilizing effects propagate far beyond these sites affecting the entire structure of the enzyme [21]. ...
Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequence of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.
... NQO1 encodes NAD(P)H dehydrogenase, which is mainly responsible for protecting cellular membranes from peroxidative injury . Following hydride transfer from NAD(P)H to the FAD cofactor in NQO1, NQO1 assumes a reduced shape under typical settings where NAD(P)H levels are abundant (Siegel et al., 2021). Numerous studies have shown that NQO1 plays a crucial role in the antioxidant defense mechanisms (Onda et al., 2015;Zhao et al., 2017;Ziemba et al., 2018), however, how NQO1 regulates ferroptosis in preeclampsia still needs to be further studied. ...
Background: Preeclampsia (PE) is one of the leading causes of maternal and fetal morbidity and mortality worldwide. Placental oxidative stress has been identified as a major pathway to the development of PE. Ferroptosis is a new form of regulated cell death that is associated with iron metabolism and oxidative stress, and likely mediates PE pathogenesis. The aim of the study was to identify the key molecules involved in ferroptosis to further explore the mechanism of ferroptosis in PE.
Methods: Gene expression data and clinical information were downloaded from the GEO database. The limma R package was used to screen differentially expressed genes (DEGs) and intersected with ferroptosis genes. The GO and KEGG pathways were then analyzed. Next, hub genes were identified via weighted gene co-expression network analysis (WGCNA). Receiver operating curves (ROCs) were performed for diagnostic and Pearson’s correlation of hub genes and clinicopathological characteristics. Immunohistochemistry and Western blot analysis were used to verify the expression of hub genes.
Results: A total of 3,142 DEGs were identified and 30 ferroptosis-related DEGs were obtained. In addition, ferroptosis-related pathways were enriched by GO and KEGG using DEGs. Two critical modules and six hub genes that were highly related to diagnosis of PE were identified through WGCNA. The analysis of the clinicopathological features showed that NQO1 and SRXN1 were closely correlated with PE characteristics and diagnosis. Finally, Western blot and immunohistochemistry analysis confirmed that the expression of the SRXN1 protein in the placental tissue of patients with PE was significantly elevated, while the expression of NQO1 was significantly decreased.
Conclusions: SRXN1 and NQO1 may be key ferroptosis-related proteins in the pathogenesis of PE. The study may provide a theoretical and experimental basis for revealing the pathogenesis of PE and improving the diagnosis of PE.
... Indeed, and as we will further discuss in Sections 2.2 and 2.3, several of the ligation states populated by NQO1 during the catalytic cycle (NQO1 holo , either oxidized and reduced, or the deadend complex of the holo-enzyme with dicoumarol, NQO1 dic ) are very relevant to the discussion of PPIs developed by NQO1. Among them, the reduced form of NQO1 seems to be most critical for strengthening PPI as well as the change in the subcellular location of the enzyme [84][85][86]. From a (bio)chemical point of view, its intrinsically low kinetic stability (i.e., a very small half-life, in the order of a few ms for the reduced flavin state) in the presence of model substrates is intriguing [76]. ...
... From a (bio)chemical point of view, its intrinsically low kinetic stability (i.e., a very small half-life, in the order of a few ms for the reduced flavin state) in the presence of model substrates is intriguing [76]. However, the results from Ross and coworkers have additionally supported that from a cellular/physiological viewpoint; this NQO1 state with the flavin reduced could be of high relevance, locally controlling the NADH/NAD + ratio, the cellular location of NQO1 and α-tubulin organization [85,86]. NQO1 is known to undergo different post-translational modifications (PTMs), some of which have been characterized in a site-specific manner. ...
... For instance, recent studies have supported that NQO1 may provide an adequate supply of NAD + for the deacetylase activity of different sirtuins associated with microtubule dynamics [85,122,126]. These studies also highlight the potential plasticity of NQO1 subcellular location during different cellular conditions or stages (e.g., the recruitment of cytosolic NQO1 to cytoskeletal structures during cell division) [85,86]. (ODC, NDUSF7, NME1, ADK, GOT1 and AK4) could also interact with NQO1 in these compartments. ...
HIF-1α is a master regulator of oxygen homeostasis involved in different stages of cancer development. Thus, HIF-1α inhibition represents an interesting target for anti-cancer therapy. It was recently shown that the HIF-1α interaction with NQO1 inhibits proteasomal degradation of the former, thus suggesting that targeting the stability and/or function of NQO1 could lead to the destabilization of HIF-1α as a therapeutic approach. Since the molecular interactions of NQO1 with HIF-1α are beginning to be unraveled, in this review we discuss: (1) Structure–function relationships of HIF-1α; (2) our current knowledge on the intracellular functions and stability of NQO1; (3) the pharmacological modulation of NQO1 by small ligands regarding function and stability; (4) the potential effects of genetic variability of NQO1 in HIF-1α levels and function; (5) the molecular determinants of NQO1 as a chaperone of many different proteins including cancer-associated factors such as HIF-1α, p53 and p73α. This knowledge is then further discussed in the context of potentially targeting the intracellular stability of HIF-1α by acting on its chaperone, NQO1. This could result in novel anti-cancer therapies, always considering that the substantial genetic variability in NQO1 would likely result in different phenotypic responses among individuals.
... CoQ10 can be reduced by different enzymes in cancer cells, for example FSP1 in the plasma membrane [13] and DHODH in the mitochondrial inner membrane [14]. We hypothesized that in melanoma cells also NQO1, transcriptional target of NRF2, could be responsible for the regeneration of the reduced form of CoQ10, since NQO1 is part of a plasma membrane redox system and elevated levels of NQO1 are associated with poor melanoma patient outcome [15,41,53]. According to our hypothesis, we found that NQO1 KD induced ROS increase and lipid peroxidation and that NQO1 is required for survival in melanoma cells. ...
Cutaneous melanoma is the deadliest type of skin cancer, although it accounts for a minority of all skin cancers. Oxidative stress is involved in all stages of melanomagenesis and cutaneous melanoma can sustain a much higher load of Reactive Oxygen Species (ROS) than normal tissues. Melanoma cells exploit specific antioxidant machinery to support redox homeostasis. The enzyme UBIA prenyltransferase domain-containing protein 1 (UBIAD1) is responsible for the biosynthesis of non-mitochondrial CoQ10 and plays an important role as antioxidant enzyme. Whether UBIAD1 is involved in melanoma progression has not been addressed, yet. Here, we provide evidence that UBIAD1 expression is associated with poor overall survival (OS) in human melanoma patients. Furthermore, UBIAD1 and CoQ10 levels are upregulated in melanoma cells with respect to melanocytes. We show that UBIAD1 and plasma membrane CoQ10 sustain melanoma cell survival and proliferation by preventing lipid peroxidation and cell death. Additionally, we show that the NAD(P)H Quinone Dehydrogenase 1 (NQO1), responsible for the 2-electron reduction of CoQ10 on plasma membranes, acts downstream of UBIAD1 to support melanoma survival. By showing that the CoQ10-producing enzyme UBIAD1 counteracts oxidative stress and lipid peroxidation events in cutaneous melanoma, this work may open to new therapeutic investigations based on UBIAD1/CoQ10 loss to cure melanoma.
... Microtubules hold high levels of acetylated αtubulin K40 on their luminal sides, and double-immunostaining studies have demonstrated that NQO1 colocalizes with these acetylated structures [102]. Binding of NQO1 dimers to microtubules may occur via their exposed positively charged C-terminal tails when NQO1 is in the oxidized state ( Figure 3) [102,104]. Numerous NAD + -dependent enzymes, including PARP and sirtuin 2 (SIRT2), have also been shown to colocalize with these acetylated microtubule structures, indicating that NQO1 may provide NAD + for these enzymes [26,[105][106][107]. Recent reports have also demonstrated that the progression of mitosis is delayed when NQO1 is compromised [108]. ...
... Microtubules hold high levels of lated α-tubulin K40 on their luminal sides, and double-immunostaining studies demonstrated that NQO1 colocalizes with these acetylated structures [102]. Bind NQO1 dimers to microtubules may occur via their exposed positively charged C-ter tails when NQO1 is in the oxidized state ( Figure 3) [102,104]. Numerous NAD + -depe enzymes, including PARP and sirtuin 2 (SIRT2), have also been shown to colocalize these acetylated microtubule structures, indicating that NQO1 may provide NA these enzymes [26,[105][106][107]. Recent reports have also demonstrated that the progre of mitosis is delayed when NQO1 is compromised [108]. ...
... NQO1 then undergoes a conformational change, discharging the oxidized pyridine nucleotide and developing an environment for quinone binding [17]. Owing to this Ping-Pong Bi-Bi kinetic mechanism, NQO1 may exist in the form of either a reduced (FADH 2 ) or an oxidized (FAD) conformation, depending on the relative concentrations of the substrates and the reduced pyridine nucleotides (Figure 3) [6,104]. Subsequent studies have revealed that NQO1 exists in at least three conformational forms-i.e., oxidized, reduced, and inactivated forms-which have heterogeneous immunoreactivity to antibodies and, therefore, distinct implications for reacting with downstream proteins [26,104]. ...
NAD(P)H:quinone oxidoreductase (NQO) is an antioxidant flavoprotein that catalyzes the reduction of highly reactive quinone metabolites by employing NAD(P)H as an electron donor. There are two NQO enzymes—NQO1 and NQO2—in mammalian systems. In particular, NQO1 exerts many biological activities, including antioxidant activities, anti-inflammatory effects, and interactions with tumor suppressors. Moreover, several recent studies have revealed the promising roles of NQO1 in protecting against cardiovascular damage and related diseases, such as dyslipidemia, atherosclerosis, insulin resistance, and metabolic syndrome. In this review, we discuss recent developments in the molecular regulation and biochemical properties of NQO1, and describe the potential beneficial roles of NQO1 in diseases associated with oxidative stress.
