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Experimental & Molecular Medicine Mar 2023Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that...
Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that plasmalemmal phosphate (Pi) transporters, such as PiT-1/2, mediate depolarization, Ca influx, oxidative stress, and calcific changes in vascular smooth muscle cells (VSMCs). However, the pathogenic mechanism of mitochondrial Pi uptake in vascular calcification associated with hyperphosphatemia has not been elucidated. We demonstrated that the phosphate carrier (PiC) is the dominant mitochondrial Pi transporter responsible for high Pi-induced superoxide generation, osteogenic gene upregulation, and calcific changes in primary VSMCs isolated from rat aortas. Notably, acute incubation with high Pi markedly increased the protein abundance of PiC via ERK1/2- and mTOR-dependent translational upregulation. Genetic suppression of PiC prevented Pi-induced ERK1/2 activation, superoxide production, osteogenic differentiation, and vascular calcification of VSMCs in vitro and aortic rings ex vivo. Pharmacological inhibition of mitochondrial Pi transport using butyl malonate (BMA) or mersalyl abolished all pathologic changes involved in high Pi-induced vascular calcification. BMA or mersalyl also effectively prevented osteogenic gene upregulation and calcification of aortas from 5/6 subtotal nephrectomized mice fed a high-Pi diet. Our results suggest that mitochondrial Pi uptake via PiC is a critical molecular mechanism mediating mitochondrial superoxide generation and pathogenic calcific changes, which could be a novel therapeutic target for treating vascular calcification associated with hyperphosphatemia.
Topics: Rats; Mice; Animals; Hyperphosphatemia; Cells, Cultured; Superoxides; Osteogenesis; Mersalyl; Phosphates; Vascular Calcification; Phosphate Transport Proteins; Myocytes, Smooth Muscle
PubMed: 36854772
DOI: 10.1038/s12276-023-00950-0 -
International Journal of Molecular... Jan 2023Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most... (Comparative Study)
Comparative Study
A Comparative Study on the Effects of the Lysine Reagent Pyridoxal 5-Phosphate and Some Thiol Reagents in Opening the Tl-Induced Mitochondrial Permeability Transition Pore.
Lysine residues are essential in regulating enzymatic activity and the spatial structure maintenance of mitochondrial proteins and functional complexes. The most important parts of the mitochondrial permeability transition pore are F1F0 ATPase, the adenine nucleotide translocase (ANT), and the inorganic phosphate cotransporter. The ANT conformation play a significant role in the Tl-induced MPTP opening in the inner membrane of calcium-loaded rat liver mitochondria. The present study tests the effects of a lysine reagent, pyridoxal 5-phosphate (PLP), and thiol reagents (phenylarsine oxide, tert-butylhydroperoxide, eosin-5-maleimide, and mersalyl) to induce the MPTP opening that was accompanied by increased swelling, membrane potential decline, and decreased respiration in 3 and 3U (2,4-dinitrophenol uncoupled) states. This pore opening was more noticeable in increasing the concentration of PLP and thiol reagents. However, more significant concentrations of PLP were required to induce the above effects comparable to those of these thiol reagents. This study suggests that the Tl-induced MPTP opening can be associated not only with the state of functionally active cysteines of the pore parts, but may be due to a change in the state of the corresponding lysines forming the pore structure.
Topics: Animals; Rats; Calcium; Indicators and Reagents; Lysine; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Permeability; Pyridoxal Phosphate; Rats, Wistar; Sulfhydryl Reagents; Thallium
PubMed: 36768782
DOI: 10.3390/ijms24032460 -
BioMed Research International 2022One of the globally common cancers is colorectal cancer (CRC). At present, a surgical approach remains a good option for CRC patients; however, 20% of surgically treated...
One of the globally common cancers is colorectal cancer (CRC). At present, a surgical approach remains a good option for CRC patients; however, 20% of surgically treated CRC patients experience metastasis. Currently, even the first-line used drug, oxaliplatin, remains inadequate for treating metastatic CRC, and its side effect of neurotoxicity is a major problem when treating CRC. The Gene Omnibus GSE42387 database contains gene expression profiles of parental and oxaliplatin-resistant LoVo cell lines. Differentially expressed genes (DEGs) between parental and oxaliplatin-resistance LoVo cells, protein-protein interactions (PPIs), and a pathway analysis were determined to identify overall biological changes by an online DAVID bioinformatics analysis. The ability of DEGs to predict overall survival (OS) and disease-free survival (DFS) was validated by the SPSS 22.0, using liver metastasis CRC patient samples of GSE41258. The bioinformatics web tools of the GEPIA, the Human Protein Atlas, WebGestalt, and TIMER platforms were used. In total, 218 DEGs were identified, among which 105 were downregulated and 113 were upregulated. After mapping the PPI networks and pathways, 60 DEGs were identified as hub genes (with high degrees). Six genes (, , , , , and ) were involved with malaria, PPAR signaling, and the adipocytokine signaling pathway. High expressions of and were associated with the poor survival of CRC patients in the GSE41258 database. We predicted specific micro (mi)RNAs that targeted the 3' untranslated region (UTR) of by using miRWalk. It was found that three miRNAs, viz., miR-7-5p, miR-20a-3p, and miR-636, may be upstream targets of those genes. High expression levels of miR-7-5p, miR-20a-3p, and miR-636 were associated with poor OS of CRC patients, and the small-molecule compound, mersalyl, is a promising drug for treating oxaliplatin-resistant CRC. In conclusion, miR-7-5p miR-20a-3p, and miR-636 targeted the PCK1 biomarker in the PPAR signaling pathway, which is involved in oxaliplatin-resistant CRC. Meanwhile, mersalyl was identified as a potential drug for overcoming oxaliplatin resistance in CRC. Our findings may provide novel directions and strategies for CRC therapies.
Topics: 3' Untranslated Regions; Adipokines; Biomarkers; Colonic Neoplasms; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mersalyl; MicroRNAs; Oxaliplatin; Peroxisome Proliferator-Activated Receptors; Signal Transduction
PubMed: 36193307
DOI: 10.1155/2022/3825760 -
Biometals : An International Journal on... Oct 2021The effects of both Tl and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane... (Review)
Review
The effects of both Tl and thiol reagents were studied on the content of the inner membrane free SH-groups, detected with Ellman reagent, and the inner membrane potential as well as swelling and respiration of succinate-energized rat liver mitochondria in medium containing TlNO and KNO. These effects resulted in a rise in swelling and a decrease in the content, the potential, and mitochondrial respiration in 3 and 2,4-dinitrophenol-uncoupled states. A maximal effect was seen when phenylarsine oxide reacting with thiol groups recessed into the hydrophobic regions of the membrane. Compared with phenylarsine oxide, the effective concentrations of other reagents were approximately one order of magnitude higher in experiments with mersalyl and 4,4'-diisothiocyanostilbene-2,2'-disulfonate, and two orders of magnitude higher in experiments with tert-butyl hydroperoxide and diamide. The above effects of Tl and the thiol reagents became even more pronounced with calcium overload of mitochondria. However, the effects were suppressed by inhibitors of the mitochondrial permeability transition pore (cyclosporine A, ADP, and n-ethylmaleimide). These findings suggest that opening of the pore induced by Tl in the inner membrane can be dependent on the conformation state of the adenine nucleotide translocase, which depends on the activity of its thiol groups.
Topics: Animals; Calcium; Membrane Potential, Mitochondrial; Mitochondria; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Permeability; Rats; Rats, Wistar; Respiration; Succinic Acid; Sulfhydryl Compounds; Sulfhydryl Reagents; Thallium
PubMed: 34236558
DOI: 10.1007/s10534-021-00329-6 -
Pharmacological Research Nov 2020The mitochondrial citrate carrier (CIC) is a nuclear-encoded protein located in the inner mitochondrial membrane. By mediating efflux of citrate from the mitochondria to... (Review)
Review
The mitochondrial citrate carrier (CIC) is a nuclear-encoded protein located in the inner mitochondrial membrane. By mediating efflux of citrate from the mitochondria to the cytosol, CIC links mitochondrial central carbon metabolism and cytosolic lipogenesis together. Abnormal activity or expression of CIC was found in cancers, developmental disorders and many other diseases. Recently, the specific inhibitors of CIC were proved to modify basic cellular metabolism, which in turn led to changes in disease course such as reverted steatohepatitis and cancer cell death. CIC is believed to be a key player and may serve as a novel therapeutic target in types of human metabolic diseases. Therefore, in this paper, we integrally described the structure and function of CIC. Then, we gave an overview of CIC related diseases including cancers, congenital diseases, pro-inflammatory effects and some other diseases. At the same time, the potential molecular mechanisms of CIC in the above diseases were illuminated. Finally, we illuminated some emerging areas for future investigation.
Topics: Carrier Proteins; Energy Metabolism; Genetic Predisposition to Disease; Humans; Male; Metabolic Diseases; Mitochondria; Mutation; Phenotype; Protein Conformation; Structure-Activity Relationship
PubMed: 32814170
DOI: 10.1016/j.phrs.2020.105132 -
International Journal of Molecular... Jul 2018The idea of using metabolic aberrations as targets for diagnosis or therapeutic intervention has recently gained increasing interest. In a previous study, our group...
The idea of using metabolic aberrations as targets for diagnosis or therapeutic intervention has recently gained increasing interest. In a previous study, our group discovered intriguing differences in the oxidative mitochondrial respiration capacity of benign and prostate cancer (PCa) cells. In particular, we found that PCa cells had a higher total respiratory activity than benign cells. Moreover, PCa cells showed a substantial shift towards succinate-supported mitochondrial respiration compared to benign cells, indicating a re-programming of respiratory control. This study aimed to investigate the role of succinate and its main plasma membrane transporter NaDC3 (sodium-dependent dicarboxylate transporter member 3) in PCa cells and to determine whether targeting succinate metabolism can be potentially used to inhibit PCa cell growth. Using high-resolution respirometry analysis, we observed that ROUTINE respiration in viable cells and succinate-supported respiration in permeabilized cells was higher in cells lacking the tumor suppressor phosphatase and tensin-homolog deleted on chromosome 10 (PTEN), which is frequently lost in PCa. In addition, loss of PTEN was associated with increased intracellular succinate accumulation and higher expression of NaDC3. However, siRNA-mediated knockdown of NaDC3 only moderately influenced succinate metabolism and did not affect PCa cell growth. By contrast, mersalyl acid-a broad acting inhibitor of dicarboxylic acid carriers-strongly interfered with intracellular succinate levels and resulted in reduced numbers of PCa cells. These findings suggest that blocking NaDC3 alone is insufficient to intervene with altered succinate metabolism associated with PCa. In conclusion, our data provide evidence that loss of PTEN is associated with increased succinate accumulation and enhanced succinate-supported respiration, which cannot be overcome by inhibiting the succinate transporter NaDC3 alone.
Topics: Cell Line, Tumor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mitochondria; Oxidative Phosphorylation; PTEN Phosphohydrolase; Prostatic Neoplasms; Respiration; Succinic Acid
PubMed: 30037119
DOI: 10.3390/ijms19072129 -
Biochemical and Biophysical Research... Jan 2018It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO and KNO resulted in the Tl-induced mitochondrial permeability transition...
It was earlier shown that the calcium load of rat liver mitochondria in medium containing TlNO and KNO resulted in the Tl-induced mitochondrial permeability transition pore (MPTP) opening in the inner membrane. This opening was accompanied by an increase in swelling and membrane potential dissipation and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration. This respiratory decrease was markedly leveled by mersalyl (MSL), the phosphate symporter (PiC) inhibitor which poorly stimulated the calcium-induced swelling, but further increased the potential dissipation. All of these effects of Ca and MSL were visibly reduced in the presence of the MPTP inhibitors (ADP, N-ethylmaleimide, and cyclosporine A). High MSL concentrations attenuated the ability of ADP to inhibit the MPTP. Our data suggest that the PiC can participate in the Tl-induced MPTP opening in the inner membrane of Ca-loaded rat liver mitochondria.
Topics: Animals; Calcium; In Vitro Techniques; Ion Transport; Membrane Potential, Mitochondrial; Mersalyl; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oxygen Consumption; Rats; Rats, Wistar; Thallium
PubMed: 29223393
DOI: 10.1016/j.bbrc.2017.12.023 -
Biochemical and Biophysical Research... Sep 2015FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in...
FAD synthase (FMN:ATP adenylyl transferase, FMNAT or FADS, EC 2.7.7.2) is involved in the biochemical pathway for converting riboflavin into FAD. Human FADS exists in different isoforms. Two of these have been characterized and are localized in different subcellular compartments. hFADS2 containing 490 amino acids shows a two domain organization: the 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase domain, that is the FAD-forming catalytic domain, and a resembling molybdopterin-binding (MPTb) domain. By a multialignment of hFADS2 with other MPTb containing proteins of various organisms from bacteria to plants, the critical residues for hydrolytic function were identified. A homology model of the MPTb domain of hFADS2 was built, using as template the solved structure of a T. acidophilum enzyme. The capacity of hFADS2 to catalyse FAD hydrolysis was revealed. The recombinant hFADS2 was able to hydrolyse added FAD in a Co(2+) and mersalyl dependent reaction. The recombinant PAPS reductase domain is not able to perform the same function. The mutant C440A catalyses the same hydrolytic function of WT with no essential requirement for mersalyl, thus indicating the involvement of C440 in the control of hydrolysis switch. The enzyme C440A is also able to catalyse hydrolysis of FAD bound to the PAPS reductase domain, which is quantitatively converted into FMN.
Topics: Amino Acid Sequence; Binding Sites; Coenzymes; Computer Simulation; Enzyme Activation; Flavin-Adenine Dinucleotide; Humans; Hydrolases; Metalloproteins; Models, Chemical; Models, Molecular; Molecular Sequence Data; Molybdenum Cofactors; Multienzyme Complexes; Nucleotidyltransferases; Protein Binding; Protein Conformation; Protein Structure, Tertiary; Pteridines; Structure-Activity Relationship; Substrate Specificity
PubMed: 26277395
DOI: 10.1016/j.bbrc.2015.08.035 -
Free Radical Biology & Medicine Sep 2014It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In...
It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane.
Topics: Animals; Cytochrome-B(5) Reductase; Cytochromes c; Electron Transport; Male; Mitochondria, Liver; Mitochondrial Membranes; NAD; Osmolar Concentration; Oxidation-Reduction; Paraquat; Rats; Rats, Wistar; Substrate Cycling; Voltage-Dependent Anion Channel 1; Xenobiotics
PubMed: 24945955
DOI: 10.1016/j.freeradbiomed.2014.06.005