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Increasing evidences indicate the vital role of exosomes-mediated intercellular communication in the pathogenesis of liver fibrosis. However, the underlying mechanisms are still not clearly defined. In this study, we found that citreoviridin (CIT), a mycotoxin and ectopic ATP synthase (e-ATPS) inhibitor, induced liver fibrosis in mice. The exosomes derived from CIT-treated L-02 hepatocytes activated hepatic stellate cells (HSC) LX-2. With exosomal small RNA sequencing, we found 156 differentially expressed miRNAs in the exosomes from CIT-treated L-02 cells, and the predicted target genes of exosomal miRNAs were enriched in calcium signaling pathway. The exosomes from CIT-treated L-02 cells induced mitochondrial calcium accumulation in LX-2 cells. And pharmacological inhibition of mitochondrial calcium uptake relieved exosomes-activated fibrogenic response in LX-2 cells. The miR-181a-2-3p that was predicted to target-regulate mitochondrial calcium uptake 1 (MICU1) was significantly increased in the exosomes from CIT-treated L-02 cells. Exosomes-induced reduction of MICU1, mitochondrial calcium overload and activation of LX-2 cells were reversed by AntagomiR-181a-2-3p. In this study, we pointed out that exosomal miR-181a-2-3p from CIT-treated hepatocytes induced mitochondrial calcium accumulation and activated HSC subsequently through inhibiting the expression of MICU1, shedding new light on the mechanism underlying liver fibrosis and CIT hepatotoxicity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Citreoviridin (CIT), a mycotoxin and ATP synthase inhibitor, is regarded as one of aetiology factors of cardiac beriberi and Keshan disease. Thiamine (VB1) and selenium (Se) improve the recovery of these two diseases respectively. The underlying mechanisms of cardiotoxic effect of CIT and cardioprotective effect of VB1 and Se have not been fully elucidated. In this study, we found that ectopic ATP synthase was more sensitive to CIT treatment than mitochondrial ATP synthase in H9c2 cardiomyocytes. CIT inhibited the transcriptional activity of peroxisome proliferator activated receptor gamma (PPAR-γ) in mice hearts and H9c2 cells. PPAR-γ agonist attenuated the inhibitory effect of CIT on mechanistic target of rapamycin complex 2 (mTORC2) and stimulatory effect of CIT on autophagy in cardiomyocytes. CIT induced apoptosis through lysosomal-mitochondrial axis in cardiomyocytes. PPAR-γ agonist and autophagy inhibitor alleviated CIT-induced apoptosis and accelerated cardiac biomarker. VB1 and Se accelerated the basal transcriptional activity of PPAR-γ in mice hearts and H9c2 cells. Furthermore, VB1 and Se reversed the effect of CIT on PPAR-γ, autophagy and apoptosis. Our findings defined PPAR-γ-mTORC2-autophagy pathway as the key link between CIT cardiotoxicity and cardioprotective effect of VB1 and Se. The present study would shed new light on the pathogenesis of cardiomyopathy and the cardioprotective mechanism of micronutrients., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The natural killer group 2D (NKG2D) receptor on natural killer (NK) cells play an important role in immunosurveillance to cancer cells, which could mediate the eradication of tumor cells through specific interactions with NKG2D ligands on tumor cells. Here we report one natural compound aurovertin B from basidiomycete Albatrellus confluens significantly stimulates the expression of NKG2D ligands on tumor cells, which greatly sensitizes its recognition and lysis by NK cell. It is completely a novel role for aurovertin B to target tumor cells to death mediated by NK cells and our findings indicate aurovertin B may deserve further development as sensitizing agent in NK cell mediated cancer immunotherapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Citreoviridin (CIT) is a mycotoxin produced by Penicillum citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. CIT occurs naturally in moldy rice and corn. CIT is associated with the development of atherosclerosis in the general population. Alteration in hepatic lipid metabolism is a pathogenic factor in atherosclerosis. However the effect and the underlying mechanism of CIT on hepatic lipid metabolism are largely unknown. In this study, we reported that CIT induced triglyceride accumulation in mice liver and human liver HepG2 cells as shown in oil red O staining. CIT (0.1 mg/kg-0.3 mg/kg) for 6 weeks elevated liver triglyceride contents in mice. CIT inhibited the transactivation activity of peroxisome proliferator-activated receptor-α (PPAR-α) in hepatocyte in vivo and in vitro, as shown by the reduced mRNA levels of PPAR-α target genes which play key roles in lipid metabolism in various aspects. PPAR-α agonist fenofibrate attenuated CIT-induced triglyceride accumulation in HepG2 cells. Furthermore, CIT increased serum total cholesterol/high-density lipoprotein cholesterol ratio, a strong risk factor for cardiovascular disease. In summary, we reported that CIT induced PPAR-α-dependent hepatic triglyceride accumulation and dyslipidemia. Our data will provide new mechanistic insights into CIT-induced lipid alterations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The F 1 sector of ATP synthase (F O F 1 ) synthesizes or hydrolyses ATP via a rotational catalysis mechanism that couples chemical reaction with subunit rotation. Phytopolyphenols such as curcumin can inhibit bulk phase F 1 ATPase activity by extending the catalytic dwell time during subunit rotation (Sekiya, M., Hisasaka, R., Iwamoto-Kihara, A., Futai, M., Nakanishi-Matsui, M., Biochem. Biophys. Res. Commun. 452 (2014) 940-944). Citreoviridin, a polyene α-pyrone mycotoxin isolated from Penicillium sp, also inhibits ATPase activity. Molecular docking and mutational analysis indicated that these compounds interact with a region near the β-subunit Arg398 residue that lies at the interface with the α-subunit. Binding of these inhibitors lowered the rotation rate and increased the duration of the catalytic dwell synergistically with substitution of β-subunit Ser174 to Phe (βS174F), which rendered the enzyme defective for conformational transmission between β-subunits of different catalytic stages. Furthermore, substitution of α-subunit Glu402 to Ala (αE402A) in the α/β-interface also decreased the rotation rate by increasing the duration of the catalytic dwell. Interestingly, this mutation restored the catalytic dwell of the βS174F variant to that of the wild-type enzyme. These results suggest that the α/β-interface is involved in conformational changes of the β-subunit during rotational catalysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The mitochondrial F1F0 ATP synthase is a critical enzyme that works by coupling the proton motive force generated by the electron transport chain via proton transfer through the F0 or proton-pore forming domain of this enzyme to release ATP from the catalytic F1 domain. This enzyme is regulated by calcium, ADP, and inorganic phosphate as well as increased transcription through several pathways. This enzyme is also an ATP hydrolase under ischemic conditions. This "inefficient" hydrolysis of ATP consumes 90% of ATP consumed during ischemia as shown with non-selective ATPase inhibitors oligomycin and Aurovertin B. A benzopyran analog, BMS-199264, selectively inhibits F1F0 ATP hydrolase activity with no effect on ATP synthase activity. BMS-199264 had no effect on ATP before ischemia, but reduced the decline in ATP during ischemia. Selective hydrolase inhibition seen with the small molecule BMS-199264 suggests a conformational change in the F1F0 ATPase enzyme when switching from synthase to hydrolase activity.

A triazine-based combinatorial library of small molecules was screened in albino murine melanocytes to identify compounds that induce pigmentation. Six compounds (of 1536 screened) produced at least 3-fold increases in pigmentation. Immunohistochemical studies demonstrated that the compounds conferred correct routing of the mistrafficked enzyme tyrosinase, which is critical to normal melanogenesis. Affinity matrices of the immobilized compounds allowed the cellular target to be identified as the mitochondrial F1F0-ATP synthase. Oligomycin and aurovertin B, small molecules known to inhibit the mitochondrial ATP synthase, were shown to compete with the triazine-based compounds for their cellular target in albino melanocytes and confer similar effects on pigmentation and tyrosinase rerouting. This is the first demonstration of the mitochondrial ATP synthase as a potential therapeutic target for restoring pigmentation in albino melanocytes.

We have engineered a mutant form of Escherichia coli F1-ATPase which is tryptophan-free and contains five mutations, namely delta W28L/alpha W513F/gamma W108Y/gamma W206Y/beta W107F. A strain carrying all five mutations grew normally by oxidative phosphorylation. Purified mutant F1-ATPase showed Vmax and Km both 65% higher than wild-type, resulting in kcat/Km the same as wild-type. The pH dependence of ATPase activity in mutant enzyme was very similar to that in wild-type. Catalytic-site nucleotide-binding characteristics were measured using the analog lin-benzo-ADP and sensitivity to inhibitors was tested using dicyclohexylcarbodiimide, azide and aurovertin. The mutant enzyme was very similar to wild-type in each of these characteristics. The fluorescence spectrum of mutant enzyme confirmed the absence of tryptophan. We have therefore established that it is possible to generate a tryptophan-free enzyme which retains normal catalytic function, oligomeric stability and in vivo assembly.

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.

It is shown that F1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F1 was accompanied by recovery of alpha in equilibrium beta intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F1. The specific ATPase activities of the partial revertant F1 preparations were in the range 1-29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F1 preparation (uncA498) which has approximately half of normal catalytic rate are also briefly described.