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1.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674457

RESUMO

CLC family genes, comprising anion channels and anion/H+ antiporters, are widely represented in nearly all prokaryotes and eukaryotes. CLC proteins carry out a plethora of functions at the cellular level. Here the coding sequences of the SaCLCa2 and SaCLCc2 genes, homologous to Arabidopsis thaliana CLCa and CLCc, were cloned from the euhalophyte Suaeda altissima (L.) Pall. Both the genes cloned belong to the CLC family as supported by the presence of the key conserved motifs and glutamates inherent for CLC proteins. SaCLCa2 and SaCLCc2 were heterologously expressed in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encodes the only CLC family protein, the Cl- transporter Gef1p, in undisrupted strains of yeast. The Δgef1 strain is characterized by inability to grow on YPD yeast medium containing Mn2+ ions. Expression of SaCLCa2 in Δgef1 cells growing on this medium did not rescue the growth defect phenotype of the mutant. However, a partial growth restoration occurred when the Δgef1 strain was transformed by SaCLCa2(C544T), the gene encoding protein in which proline, specific for nitrate, was replaced with serine, specific for chloride, in the selectivity filter. Unlike SaCLCa2, expression of SaCLCc2 in Δgef1 resulted in a partial growth restoration under these conditions. Analysis of SaCLCa2 and SaCLCc2 expression in the euhalophyte Suaeda altissima (L.) Pall by quantitative real-time PCR (qRT-PCR) under different growth conditions demonstrated stimulation of SaCLCa2 expression by nitrate and stimulation of SaCLCc2 expression by chloride. The results of yeast complementation assay, the presence of both the "gating" and "proton" glutamates in aa sequences of both the proteins, as well results of the gene expression in euhalophyte Suaeda altissima (L.) Pall suggest that SaCLCa2 and SaCLCc2 function as anion/H+ antiporters with nitrate and chloride specificities, respectively. The general bioinformatic overview of seven CLC genes cloned from euhalophyte Suaeda altissima is given, together with results on their expression in roots and leaves under different levels of salinity.


Assuntos
Chenopodiaceae , Canais de Cloreto , Proteínas de Plantas , Sequência de Aminoácidos , Ânions/metabolismo , Antiporters/metabolismo , Arabidopsis/genética , Chenopodiaceae/genética , Chenopodiaceae/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Clonagem Molecular , Nitratos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
2.
Biopharm Drug Dispos ; 43(6): 255-264, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36494876

RESUMO

Two-thirds of patients with type 2 diabetes mellitus have hypertension, and thus the combination of two or more drugs to treat these diseases is common. It has been shown that the combination of metformin and enalapril has beneficial effects, but few studies have evaluated the interactions between these two drugs. This study investigated the effects of enalapril on the pharmacokinetics and urinary excretion of metformin in rats, with a focus on transporter-mediated drug interactions. Rats were dosed orally with metformin alone (100 mg/kg) or in combination with enalapril (4 mg/kg). The concentration of metformin was measured by high performance liquid chromatography and the level of organic cation transporters (rOCTs) and multidrug and toxin excretion protein 1 (rMATE1), which mediate the uptake and efflux of metformin, respectively, were evaluated by immunoblotting. After single and 7-day dosing, the plasma concentration of metformin in the co-administration group was significantly lower than that in the metformin-only group, and the CL/F and urinary excretion were increased in the co-administration group. Enalapril did not affect the Kp of metformin but reduced renal slice-uptake of metformin. The expression of rMATE1 was increased, whereas rOCT2 expression was decreased in rat kidney. Importantly, long-term co-administration of metformin and enalapril markedly decreased the level of lactic acid and uric acid in the blood. Enalapril increases the urinary excretion of metformin through the up-regulation of rMATE1. This reveals a new mechanism of drug interactions and provides a basis for drug dosage adjustment when these drugs are co-administered.


Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Ratos , Animais , Metformina/farmacocinética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Enalapril/farmacologia , Enalapril/metabolismo , Ratos Wistar , Antiporters/metabolismo , Rim/metabolismo
3.
FASEB J ; 36(12): e22659, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36394534

RESUMO

The arsenical resistance-3 (ACR3) family constitutes the most common pathway that confers high-level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter from Bacillus subtilis (Acr3Bs ) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X-shaped structure. As the structural information available for these important arsenite-efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site-directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X-shaped of NhaA fold.


Assuntos
Antiporters , Arsenitos , Antiporters/genética , Antiporters/metabolismo , Arsenitos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Secundária de Proteína
4.
Nat Commun ; 13(1): 6383, 2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36289233

RESUMO

The strict exchange of protons for sodium ions across cell membranes by Na+/H+ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na+/H+ exchange can be modelled as competition between H+ and Na+ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na+/H+ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 Å. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na+ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Prótons , Antiporters/metabolismo , Histidina/metabolismo , Concentração de Íons de Hidrogênio , Escherichia coli/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Íons/metabolismo , Sódio/metabolismo , Água/metabolismo
5.
Nat Commun ; 13(1): 6091, 2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36241630

RESUMO

Multiple resistance and pH adaptation (Mrp) cation/proton antiporters are essential for growth of a variety of halophilic and alkaliphilic bacteria under stress conditions. Mrp-type antiporters are closely related to the membrane domain of respiratory complex I. We determined the structure of the Mrp antiporter from Bacillus pseudofirmus by electron cryo-microscopy at 2.2 Å resolution. The structure resolves more than 99% of the sidechains of the seven membrane subunits MrpA to MrpG plus 360 water molecules, including ~70 in putative ion translocation pathways. Molecular dynamics simulations based on the high-resolution structure revealed details of the antiport mechanism. We find that switching the position of a histidine residue between three hydrated pathways in the MrpA subunit is critical for proton transfer that drives gated trans-membrane sodium translocation. Several lines of evidence indicate that the same histidine-switch mechanism operates in respiratory complex I.


Assuntos
Antiporters , Simulação de Dinâmica Molecular , Antiporters/metabolismo , Proteínas de Bactérias/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Histidina , Concentração de Íons de Hidrogênio , Prótons , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Água/metabolismo
6.
FASEB J ; 36(11): e22534, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36183361

RESUMO

The solute carrier 26 family member A9 (SLC26A9) is an epithelial anion transporter that is assumed to contribute to airway chloride secretion and surface hydration. Whether SLC26A9 or CFTR is responsible for airway Cl- transport under basal conditions is still unclear, due to the lack of a specific inhibitor for SLC26A9. In the present study, we report a novel potent and specific inhibitor for SLC26A9, identified by screening of a drug-like molecule library and subsequent chemical modifications. The most potent compound S9-A13 inhibited SLC26A9 with an IC50 of 90.9 ± 13.4 nM. S9-A13 did not inhibit other members of the SLC26 family and had no effects on Cl- channels such as CFTR, TMEM16A, or VRAC. S9-A13 inhibited SLC26A9 Cl- currents in cells that lack expression of CFTR. It also inhibited proton secretion by HGT-1 human gastric cells. In contrast, S9-A13 had minimal effects on ion transport in human airway epithelia and mouse trachea, despite clear expression of SLC26A9 in the apical membrane of ciliated cells. In both tissues, basal and stimulated Cl- secretion was due to CFTR, while acidification of airway surface liquid by S9-A13 suggests a role of SLC26A9 for airway bicarbonate secretion.


Assuntos
Cloretos , Regulador de Condutância Transmembrana em Fibrose Cística , Animais , Antiporters/metabolismo , Bicarbonatos/metabolismo , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Prótons , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
7.
Biol Pharm Bull ; 45(10): 1585-1589, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36184519

RESUMO

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.


Assuntos
Antiporters , Barreira Hematoencefálica , Cloreto de Amônio , Analgésicos Opioides/metabolismo , Antiporters/metabolismo , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Cátions/metabolismo , Humanos , Naltrexona/metabolismo , Naltrexona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Prótons , Azida Sódica/metabolismo
8.
Mol Biol Evol ; 39(10)2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36181435

RESUMO

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.


Assuntos
Archaea , Hidrogenase , Archaea/genética , Archaea/metabolismo , Hidrogenase/química , Hidrogenase/genética , Hidrogenase/metabolismo , Cloreto de Sódio/metabolismo , Filogenia , Sistema Respiratório/metabolismo , Aminoácidos/genética , Antiporters/genética , Antiporters/metabolismo
9.
Sci Rep ; 12(1): 15871, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36151227

RESUMO

An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate1-:alanine0 exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of L-aspartate (-Asp) and L-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of L-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of L-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of L-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (L-Ala or L-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of L-Ala differed from that in the presence of L-Asp. From these results, we concluded that L-Ala binding yields a conformation different from the apo or L-Asp binding conformations.


Assuntos
Antiporters , Ácido Aspártico , Alanina/metabolismo , Antiporters/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Cisteína , Corantes Fluorescentes , Ácido Láctico , Lipossomos , Maleimidas , Conformação Proteica
10.
Sci Rep ; 12(1): 15870, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36151270

RESUMO

Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.


Assuntos
Glutamina , Leucina , Alvo Mecanístico do Complexo 1 de Rapamicina , Aminoácidos/metabolismo , Animais , Antiporters/metabolismo , Ingestão de Alimentos , Fator de Iniciação 4E em Eucariotos/metabolismo , Glutamato Desidrogenase/metabolismo , Glutamina/administração & dosagem , Leucina/administração & dosagem , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Proteínas Musculares/metabolismo , Fosforilação , Puromicina , Triptofano/metabolismo
11.
Environ Sci Technol ; 56(19): 14146-14153, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36121644

RESUMO

Selenate enhances arsenic (As) accumulation in As-hyperaccumulator Pteris vittata, but the associated molecular mechanisms are unclear. Here, we investigated the mechanisms of selenate-induced arsenic accumulation by exposing P. vittata to 50 µM arsenate (AsV50) and 1.25 (Se1.25) or 5 µM (Se5) selenate in hydroponics. After 2 weeks, plant biomass, plant As and Se contents, As speciation in plant and growth media, and important genes related to As detoxification in P. vittata were determined. These genes included P transporters PvPht1;3 and PvPht1;4 (AsV uptake), arsenate reductases PvHAC1 and PvHAC2 (AsV reduction), and arsenite (AsIII) antiporters PvACR3 and PvACR3;2 (AsIII translocation) in the roots, and AsIII antiporters PvACR3;1 and PvACR3;3 (AsIII sequestration) in the fronds. The results show that Se1.25 was more effective than Se5 in increasing As accumulation in both P. vittata roots and fronds, which increased by 27 and 153% to 353 and 506 mg kg-1. The As speciation analyses show that selenate increased the AsIII levels in P. vittata, with 124-282% more AsIII being translocated into the fronds. The qPCR analyses indicate that Se1.25 upregulated the gene expression of PvHAC1 by 1.2-fold, and PvACR3 and PvACR3;2 by 1.0- to 2.5-fold in the roots, and PvACR3;1 and PvACR3;3 by 0.6- to 1.1-fold in the fronds under AsV50 treatment. Though arsenate enhanced gene expression of P transporters PvPht1;3 and PvPht1;4, selenate had little effect. Our results indicate that selenate effectively increased As accumulation in P. vittata, mostly by increasing reduction of AsV to AsIII in the roots, AsIII translocation from the roots to fronds, and AsIII sequestration into the vacuoles in the fronds. The results suggest that selenate may be used to enhance phytoremediation of As-contaminated soils using P. vittata.


Assuntos
Arsênio , Arsenitos , Pteris , Selênio , Poluentes do Solo , Antiporters/metabolismo , Antiporters/farmacologia , Arseniato Redutases/genética , Arseniato Redutases/metabolismo , Arseniatos , Arsênio/metabolismo , Arsenitos/metabolismo , Biodegradação Ambiental , Raízes de Plantas/metabolismo , Pteris/genética , Pteris/metabolismo , Ácido Selênico , Selênio/metabolismo , Solo , Poluentes do Solo/metabolismo
12.
Pharmacol Ther ; 239: 108283, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36162727

RESUMO

About 30% of all small molecular drugs are organic cations (OCs). If these are more or less hydrophilic, they require membrane transporters to pass through biological membranes. Here, the proton-organic cation (H+ OC) antiporter may play a physiologically most relevant role, particularly concerning passage through the blood-brain barrier. Membrane transport of about 70 OCs is significantly enhanced by this H+ OC antiporter. Surprisingly still today the gene coding for this antiporter was not yet identified. However, the H+ OC antiporter is characterized by concentration- and pH-dependent uptake, antiport with another OC, and susceptibility to inhibition by specific inhibitors. Moreover, in the studied tissues and cell types, transport is not mediated by already well-known organic cation transporters. The review explains the typically used assays to identify potential substrates of the H+ OC antiporter. Thus far, the gene encoding for this transporter has not yet been identified, but a better understanding of this protein may be most relevant because it may affect the pharmacokinetics of up to 10% of all low molecular substances. This review summarizes the known functional characteristics of the H+ OC antiporter, its cell and tissue expression, and its substrate spectrum. Summarizing the features of the substrates of the H+ OC antiporter may even suggest that for OCs, good penetration through the blood-brain barrier is almost synonymous with being a substrate of the H+ OC antiporter. In clinical studies, pharmacokinetics of typical substrates of the antiporter showed outstanding between-subject variability.


Assuntos
Antiporters , Prótons , Humanos , Antiporters/genética , Antiporters/metabolismo , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Barreira Hematoencefálica/metabolismo , Transporte Biológico , Cátions/metabolismo
13.
Orphanet J Rare Dis ; 17(1): 361, 2022 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-36115991

RESUMO

BACKGROUND: Congenital hereditary endothelial dystrophy (CHED) is a rare form of corneal dystrophy caused by SLC4A11 gene variations. This study aims to find the genetic alterations in SLC4A11, in two Indian familial CHED cases with affected members n = 3 and n = 2 respectively and five sporadic CHED cases using direct sequencing, followed by in silico analysis and characterization of the identified variants. RESULTS: All three affected members of the first CHED family were identified with a novel homozygous c.1514C > G (p.Ser489Trp) variation while second family showed presence of a compound heterozygous variation c.529A > C (p.Arg161Arg) + c.2461insT (p.Val805fs). Among five sporadic cases, two showed novel changes, homozygous c.1487G > T (p.Ser480Ile) and c.620-2A > G, while the other one had previously reported homozygous c.2653C > T (p.Arg869Cys) variation. The remaining two cases did not reveal the presence of SLC4A11-related pathogenic variations. The identified variations were excluded from the Indian control (n = 80). In silico analysis using homology-based protein modeling and pathogenicity prediction tools, which revealed these alterations as pathogenic, changing their protein stability, local flexibility, residue contact clashes, and the hydrogen bond interactions. CONCLUSIONS: This study contributed to the CHED mutational spectrum, adding four novel variations and confirming a previously reported one. It demonstrates different type of variations in CHED cases, including coding, non-coding, homozygous, synonymous, and compound heterozygous variations. The identified variations revealed different degrees of pathogenic effects in silico. Moreover, two sporadic cases could not be identified with pathogenic variation emphasizing the involvement of other genes or genetic mechanisms.


Assuntos
Proteínas de Transporte de Ânions , Antiporters , Distrofias Hereditárias da Córnea , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antiporters/genética , Antiporters/metabolismo , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/metabolismo , Homozigoto , Humanos , Índia , Mutação/genética
14.
Kidney Int ; 102(6): 1259-1275, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36063875

RESUMO

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD). Cell death such as apoptosis plays a critical role in vascular calcification. Ferroptosis is a type of iron-catalyzed and regulated cell death resulting from excessive iron-dependent reactive oxygen species and lipid peroxidation. However, it is unclear whether ferroptosis of vascular smooth muscle cells (VSMCs) regulates vascular calcification in CKD. Our results showed that high calcium and phosphate concentrations induced ferroptosis in rat VSMCs in vitro. Inhibition of ferroptosis by ferrostatin-1 dose-dependently reduced mineral deposition in rat VSMCs under pro-osteogenic conditions, as indicated by alizarin red staining and quantification of calcium content. In addition, gene expression analysis revealed that ferrostatin-1 inhibited osteogenic differentiation of rat VSMCs. Similarly, ferrostatin-1 remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in vitamin D3-overloaded mice in vivo. Moreover, inhibition of ferroptosis by either ferrostatin-1 or deferoxamine attenuated aortic calcification in rats with CKD. Mechanistically, high calcium and phosphate downregulated expression of SLC7A11 (a cystine-glutamate antiporter), and reduced glutathione (GSH) content in VSMCs. Additionally, GSH depletion induced by erastin (a small molecule initiating ferroptotic cell death) significantly promoted calcification of VSMCs under pro-osteogenic conditions, whereas GSH supplement by N-acetylcysteine reduced calcification of VSMCs. Consistently, knockdown of SLC7A11 by siRNA markedly promoted VSMC calcification. Furthermore, high calcium and phosphate downregulated glutathione peroxidase 4 (GPX4) expression, and reduced glutathione peroxidase activity. Inhibition of GPX4 by RSL3 promoted VSMC calcification. Thus, repression of the SLC7A11/GSH/GPX4 axis triggers ferroptosis of VSMCs to promote vascular calcification under CKD conditions, providing a novel targeting strategy for vascular calcification.


Assuntos
Ferroptose , Insuficiência Renal Crônica , Calcificação Vascular , Humanos , Ratos , Camundongos , Animais , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Músculo Liso Vascular , Osteogênese , Cálcio/metabolismo , Antiporters/metabolismo , Miócitos de Músculo Liso/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/prevenção & controle , Ferro/metabolismo , Glutationa/metabolismo , Insuficiência Renal Crônica/patologia , Fosfatos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo
15.
Proc Natl Acad Sci U S A ; 119(35): e2205810119, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-35994672

RESUMO

In recent years, it has become clear that many homo- and heterodimeric cytoplasmic proteins in both prokaryotic and eukaryotic cells start to dimerize cotranslationally (i.e., while at least one of the two chains is still attached to the ribosome). Whether this is also possible for integral membrane proteins is, however, unknown. Here, we apply force profile analysis (FPA)-a method where a translational arrest peptide (AP) engineered into the polypeptide chain is used to detect force generated on the nascent chain during membrane insertion-to demonstrate cotranslational interactions between a fully membrane-inserted monomer and a nascent, ribosome-tethered monomer of the Escherichia coli inner membrane protein EmrE. Similar cotranslational interactions are also seen when the two monomers are fused into a single polypeptide. Further, we uncover an apparent intrachain interaction between E14 in transmembrane helix 1 (TMH1) and S64 in TMH3 that forms at a precise nascent chain length during cotranslational membrane insertion of an EmrE monomer. Like soluble proteins, inner membrane proteins thus appear to be able to both start to fold and start to dimerize during the cotranslational membrane insertion process.


Assuntos
Antiporters , Proteínas de Escherichia coli , Escherichia coli , Antiporters/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Dobramento de Proteína
16.
Int J Mol Sci ; 23(15)2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35955563

RESUMO

Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood-brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute-1 × g protein-1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug-drug interactions and variations in pharmacokinetics.


Assuntos
Antiporters , Fator 1 de Transcrição de Octâmero/metabolismo , Transportador 1 de Cátions Orgânicos , Antiporters/genética , Antiporters/metabolismo , Transporte Biológico , Encéfalo/metabolismo , Cátions , Humanos , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Prótons
17.
Plant Physiol ; 190(4): 2617-2636, 2022 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-35972350

RESUMO

A plant's oxygen supply can vary from normal (normoxia) to total depletion (anoxia). Tolerance to anoxia is relevant to wetland species, rice (Oryza sativa) cultivation, and submergence tolerance of crops. Decoding and transmitting calcium (Ca) signals may be an important component to anoxia tolerance; however, the contribution of intracellular Ca transporters to this process is poorly understood. Four functional cation/proton exchangers (CAX1-4) in Arabidopsis (Arabidopsis thaliana) help regulate Ca homeostasis around the vacuole. Our results demonstrate that cax1 mutants are more tolerant to both anoxic conditions and submergence. Using phenotypic measurements, RNA-sequencing, and proteomic approaches, we identified cax1-mediated anoxia changes that phenocopy changes present in anoxia-tolerant crops: altered metabolic processes, diminished reactive oxygen species production post anoxia, and altered hormone signaling. Comparing wild-type and cax1 expressing genetically encoded Ca indicators demonstrated altered cytosolic Ca signals in cax1 during reoxygenation. Anoxia-induced Ca signals around the plant vacuole are involved in the control of numerous signaling events related to adaptation to low oxygen stress. This work suggests that cax1 anoxia response pathway could be engineered to circumvent the adverse effects of flooding that impair production agriculture.


Assuntos
Arabidopsis , Proteínas de Transporte de Cátions , Humanos , Vacúolos/metabolismo , Cálcio/metabolismo , Antiporters/metabolismo , Prótons , Proteômica , Proteínas de Transporte de Cátions/metabolismo , Arabidopsis/metabolismo , Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo
18.
Microbiology (Reading) ; 168(8)2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35920804

RESUMO

Polyamines bind to various cellular components, such as nucleic acids, phospholipids, proteins and nucleotides. They are involved in the virulence and protection against physiological stresses of several bacterial species. Streptococcus agalactiae is able to colonize the vaginal tract of asymptomatic pregnant women and to resist, by an as yet poorly characterized mechanism, pH 4.0, the low physiological pH of this environment. We identified a transporter of the amino acid/polyamine antiporter family (SAK_1604 in strain A909) that shares 39.8 % similar amino acids with CadB and 34.7 % with PotE, two transporters implicated in acid resistance in Escherichia coli. We found that sak_1604 is overexpressed in the presence of spermidine and during citric acid stress at the vaginal pH, but not during lactic acid or HCl stresses at the same pH or during a sodium citrate stress at pH 7.4. Dihydrogen citrate is the predominant form of citric acid at pH 4.0. Using a deletion mutant, we proved that SAK_1604 is involved in the survival of S. agalactiae during citric acid stress at pH 4.0 in the presence of spermidine, and we showed by TLC analysis that it is involved in spermidine transport in these conditions. Our data open new perspectives on the comprehension of the molecular mechanisms allowing S. agalactiae to survive at the physiological pH of the vagina and on the unsuspected role of an ionic form of citric acid.


Assuntos
Antiporters , Espermidina , Aminoácidos/metabolismo , Antiporters/genética , Antiporters/metabolismo , Ácido Cítrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/metabolismo , Poliaminas/metabolismo , Gravidez , Espermidina/metabolismo , Streptococcus agalactiae/genética , Streptococcus agalactiae/metabolismo
19.
PLoS One ; 17(8): e0273685, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36037197

RESUMO

AIM: To identify the molecular basis of Congenital Hereditary Endothelial Dystrophy CHED caused by mutations in SLC4A11, in the consanguineous Pakistani families. METHODS: A total of 7 consanguineous families affected with Congenital Hereditary Endothelial Dystrophy were diagnosed and registered with the help of ophthalmologists. Blood samples were collected from affected and unaffected members of the enrolled families. Mutational analysis was carried out by DNA sequencing using both Sanger and Whole Exome Sequencing (WES). Probands of each pedigree from the 7 families were used for WES. Results were analyzed with the help of different bioinformatics tools. RESULTS: The sequencing results demonstrated three known homozygous mutations in gene SLC4A11 in probands of 7 families. These mutations p.Glu675Ala, p.Val824Met, and p.Arg158fs include 2 missense and 1 frameshift mutation. The mutations result in amino acids that were highly conserved in SLC4A11 across different species. The mutations were segregated with the disease phenotype in the families. CONCLUSION: This study reports 3 mutations in 7 families. One of the pathogenic mutations (p.R158fs) was identified for the first time in the Pakistani population. However, two mutations (p.Glu675Ala, p.Val824Met) were previously reported in two and one Pakistani family respectively. As these mutations segregate with the disease phenotype and bioinformatics tool also liable them as pathogenic, they are deemed as probable cause of underlying disease.


Assuntos
Distrofias Hereditárias da Córnea , Simportadores , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Antiporters/metabolismo , Boratos/metabolismo , Distrofias Hereditárias da Córnea/genética , Análise Mutacional de DNA , Humanos , Mutação , Paquistão , Linhagem , Sódio/metabolismo , Simportadores/genética
20.
Plant Cell Physiol ; 63(9): 1242-1252, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35876035

RESUMO

NGATHA-Like 1 (NGAL1) transcription factor has been identified as a gene regulated through AUG-stop-mediated boron (B)-dependent translation stall; however, its function in B response remains unknown. Here, we show that NGAL1 plays an important role in the maintenance of B transport under both low- and high-B conditions in Arabidopsis thaliana. NGAL1 mRNA is accumulated predominantly in shoots in response to B stress. Independent ngal1 mutants carrying transferred DNA (T-DNA) and Ds-transposon insertions exhibit reduced B concentrations in aerial tissues and produce shortened and reduced number of siliques when B supply is limited. Furthermore, the expression of B transporter genes including nodulin 26-like intrinsic protein 6; 1 (NIP6;1), NIP5;1, NIP7;1 and borate exporter 1 (BOR1) is significantly decreased in ngal1 mutants under low-B condition, suggesting that NGAL1 is required for the transcript accumulation of B transporter genes to facilitate B transport and distribution under B limitation. Under high-B condition, ngal1 mutants exhibit reduced growth and increased B concentration in their shoots. The accumulation of BOR4 mRNA, a B transporter required for B efflux to soil, is significantly reduced in roots of ngal1 plants under high-B condition, suggesting that NGAL1 is involved in the upregulation of BOR4 in response to excess B. Together, our results indicate that NGAL1 is involved in the transcriptional regulation of B transporter genes to facilitate B transport and distribution under both low- and high-B conditions.


Assuntos
Aquaporinas , Proteínas de Arabidopsis , Arabidopsis , Antiporters/metabolismo , Aquaporinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Boro/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo
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