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1.
J Phys Chem B ; 128(11): 2697-2706, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38447081

RESUMO

CLCF fluoride/proton antiporters move fluoride ions out of bacterial cells, leading to fluoride resistance in these bacteria. However, many details about their operating mechanisms remain unclear. Here, we report a combined quantum-mechanical/molecular-mechanical (QM/MM) study of a CLCF homologue from Enterococci casseliflavus (Eca), in accord with the previously proposed windmill mechanism. Our multiscale modeling sheds light on two critical steps in the transport cycle: (i) the external gating residue E118 pushing a fluoride in the external binding site into the extracellular vestibule and (ii) an incoming fluoride reconquering the external binding site by forcing out E118. Both steps feature competitions for the external binding site between the negatively charged carboxylate of E118 and the fluoride. Remarkably, the displaced E118 by fluoride accepts a proton from the nearby R117, initiating the next transport cycle. We also demonstrate the importance of accurate quantum descriptions of fluoride solvation. Our results provide clues to the mysterious E318 residue near the central binding site, suggesting that the transport activities are unlikely to be disrupted by the glutamate interacting with a well-solvated fluoride at the central binding site. This differs significantly from the structurally similar CLC chloride/proton antiporters, where a fluoride trapped deep in the hydrophobic pore causes the transporter to be locked down. A free-energy barrier of 10-15 kcal/mol was estimated via umbrella sampling for a fluoride ion traveling through the pore to repopulate the external binding site.


Assuntos
Antiporters , Prótons , Antiporters/química , Antiporters/metabolismo , Fluoretos/química , Modelos Moleculares , Proteínas de Membrana Transportadoras/metabolismo , Cloretos/química , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Transporte de Íons
2.
Sci Rep ; 14(1): 5915, 2024 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-38467695

RESUMO

Cell pH and Na+ homeostasis requires Na+/H+ antiporters. The crystal structure of NhaA, the main Escherichia coli Na+/H+ antiporter, revealed a unique NhaA structural fold shared by prokaryotic and eukaryotic membrane proteins. Out of the 12 NhaA transmembrane segments (TMs), TMs III-V and X-XII are topologically inverted repeats with unwound TMs IV and XI forming the X shape characterizing the NhaA fold. We show that intramolecular cross-linking under oxidizing conditions of a NhaA mutant with two Cys replacements across the crossing (D133C-T340C) inhibits antiporter activity and impairs NhaA-dependent cell growth in high-salts. The affinity purified D133C-T340C protein binds Li+ (the Na+ surrogate substrate of NhaA) under reducing conditions. The cross-linking traps the antiporter in an outward-facing conformation, blocking the antiport cycle. As many secondary transporters are found to share the NhaA fold, including some involved in human diseases, our data have importance for both basic and clinical research.


Assuntos
Proteínas de Escherichia coli , Humanos , Proteínas de Escherichia coli/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Escherichia coli/metabolismo , Antiporters/metabolismo , Transporte de Íons , Íons/metabolismo , Concentração de Íons de Hidrogênio
3.
BMC Genomics ; 25(1): 144, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38317113

RESUMO

BACKGROUND: The cation/proton antiporter (CPA) superfamily plays a crucial role in regulating ion homeostasis and pH in plant cells, contributing to stress resistance. However, in potato (Solanum tuberosum L.), systematic identification and analysis of CPA genes are lacking. RESULTS: A total of 33 StCPA members were identified and classified into StNHX (n = 7), StKEA (n = 6), and StCHX (n = 20) subfamilies. StCHX owned the highest number of conserved motifs, followed by StKEA and StNHX. The StNHX and StKEA subfamilies owned more exons than StCHX. NaCl stress induced the differentially expression of 19 genes in roots or leaves, among which StCHX14 and StCHX16 were specifically induced in leaves, while StCHX2 and StCHX19 were specifically expressed in the roots. A total of 11 strongly responded genes were further verified by qPCR. Six CPA family members, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19, were proved to transport Na+ through yeast complementation experiments. CONCLUSIONS: This study provides comprehensive insights into StCPAs and their response to NaCl stress, facilitating further functional characterization.


Assuntos
Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Prótons , Cloreto de Sódio/farmacologia , Antiporters/genética , Antiporters/metabolismo , Proteínas de Plantas/metabolismo , Filogenia , Regulação da Expressão Gênica de Plantas , Cátions/metabolismo , Estresse Fisiológico/genética
4.
Exp Eye Res ; 240: 109815, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38316204

RESUMO

Corneal endothelial dysfunction is a major indication for corneal transplantation. However, a global shortage of donor corneal tissues and risks associated with corneal surgeries have prompted exploration of alternative options, including tissue-engineered grafts or cell injection therapy. Nonetheless, these approaches require a controlled culture of primary human corneal endothelial cells (HCEnCs). Although HCEnCs established from young donors are generally more proliferative and maintain a better phenotype, corneas from old donors are more frequently accessible from eye banks due to a lower corneal endothelial cell count than the necessary threshold required for transplantation. In this study, we investigated various culture media to evaluate which one is the most appropriate for stimulating the proliferation while maintaining cell morphology and function of HCEnCs derived from old donors (age >65 years). All experiments were performed on paired research-grade donor corneas, divided for the conditions under investigation in order to minimize the inter-donor variability. Cell morphology as well as expression of specific markers were assessed at both mRNA (CD166, SLC4A11, ATP1A1, COL8A1, α-SMA, CD44, COL1A1, CDKN2A, LAP2A and LAP2B) and protein (ZO-1, α-SMA, Ki67 and LAP2) levels. Results obtained showed how the Dual Media formulation maintained the hexagonal phenotype more efficiently than Single Medium, but cell size gradually increased with passages. In contrast, the Single Medium provided a higher proliferation rate and a prolonged in vitro expansion but acquired an elongated morphology. To summarize, Single medium and Dual media preserve morphology and functional phenotype of HCEnCs from old donor corneas at low passages while maintenance of the same cell features at high passages remains an active area of research. The new insights revealed within this work become particularly relevant considering that the elderly population a) is the main target of corneal endothelial therapy, b) represents the majority of corneal donors. Therefore, the proper expansion of HCEnCs from old donors is essential to develop novel personalised therapeutic strategies and reduce requirement of human corneal tissues globally.


Assuntos
Células Endoteliais , Endotélio Corneano , Humanos , Idoso , Células Cultivadas , Endotélio Corneano/metabolismo , Córnea , Doadores de Tecidos , Meios de Cultura , Antiporters/metabolismo , Proteínas de Transporte de Ânions/metabolismo
5.
Sci Adv ; 10(7): eadk2317, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38354239

RESUMO

Lysosomal calcium (Ca2+) release is critical to cell signaling and is mediated by well-known lysosomal Ca2+ channels. Yet, how lysosomes refill their Ca2+ remains hitherto undescribed. Here, from an RNA interference screen in Caenorhabditis elegans, we identify an evolutionarily conserved gene, lci-1, that facilitates lysosomal Ca2+ entry in C. elegans and mammalian cells. We found that its human homolog TMEM165, previously designated as a Ca2+/H+ exchanger, imports Ca2+ pH dependently into lysosomes. Using two-ion mapping and electrophysiology, we show that TMEM165, hereafter referred to as human LCI, acts as a proton-activated, lysosomal Ca2+ importer. Defects in lysosomal Ca2+ channels cause several neurodegenerative diseases, and knowledge of lysosomal Ca2+ importers may provide previously unidentified avenues to explore the physiology of Ca2+ channels.


Assuntos
Cálcio , Proteínas de Transporte de Cátions , Animais , Humanos , Cálcio/metabolismo , Caenorhabditis elegans/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Transdução de Sinais , Lisossomos/metabolismo , Sinalização do Cálcio , Mamíferos/metabolismo , Antiporters/metabolismo , Proteínas de Transporte de Cátions/metabolismo
6.
Int J Mol Sci ; 25(4)2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38396725

RESUMO

The transcription of glycine-rich RNA-binding protein 2 (PeGRP2) transiently increased in the roots and shoots of Populus euphratica (a salt-resistant poplar) upon initial salt exposure and tended to decrease after long-term NaCl stress (100 mM, 12 days). PeGRP2 overexpression in the hybrid Populus tremula × P. alba '717-1B4' (P. × canescens) increased its salt sensitivity, which was reflected in the plant's growth and photosynthesis. PeGRP2 contains a conserved RNA recognition motif domain at the N-terminus, and RNA affinity purification (RAP) sequencing was developed to enrich the target mRNAs that physically interacted with PeGRP2 in P. × canescens. RAP sequencing combined with RT-qPCR revealed that NaCl decreased the transcripts of PeGRP2-interacting mRNAs encoding photosynthetic proteins, antioxidative enzymes, ATPases, and Na+/H+ antiporters in this transgenic poplar. Specifically, PeGRP2 negatively affected the stability of the target mRNAs encoding the photosynthetic proteins PETC and RBCMT; antioxidant enzymes SOD[Mn], CDSP32, and CYB1-2; ATPases AHA11, ACA8, and ACA9; and the Na+/H+ antiporter NHA1. This resulted in (i) a greater reduction in Fv/Fm, YII, ETR, and Pn; (ii) less pronounced activation of antioxidative enzymes; and (iii) a reduced ability to maintain Na+ homeostasis in the transgenic poplars during long-term salt stress, leading to their lowered ability to tolerate salinity stress.


Assuntos
Populus , Tolerância ao Sal , Tolerância ao Sal/genética , Populus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Cloreto de Sódio/metabolismo , Íons/metabolismo , Sódio/metabolismo , Homeostase , Adenosina Trifosfatases/metabolismo , Antiporters/metabolismo , Fotossíntese/genética , Regulação da Expressão Gênica de Plantas
7.
Phytomedicine ; 126: 155283, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38422652

RESUMO

BACKGROUND: Portulacae Herba and Granati Pericarpium pair (PGP) is a traditional Chinese herbal medicine treatment for colitis, clinically demonstrating a relatively favorable effect on relieving diarrhea and abnormal stools. However, the underlying mechanism remain uncertain. PURPOSE: The present study intends to evaluate the efficacy of PGP in treating colitis in mice and investigate its underlying mechanism. METHODS: The protective effect of PGP against colitis was determined by monitoring body weight, colon length, colon weight, and survival rate in mice. Colonic inflammation was assessed by serum cytokine levels, colonic H&E staining, and local neutrophil infiltration. The reversal of intestinal epithelial barrier damage by PGP was subsequently analyzed with Western blot and histological staining. Furthermore, RNA-seq analysis and molecular docking were performed to identify potential pathways recruited by PGP. Following the hints of the transcriptomic results, the role of PGP through the IL-6/STAT3/SOCS3 pathway in DSS-induced colitis mice was verified by Western blot. RESULTS: DSS-induced colitis in mice was significantly curbed by PGP treatment. PGP treatment significantly mitigated DSS-induced colitis in mice, as evidenced by improvements in body weight, DAI severity, survival rate, and inflammatory cytokines levels in serum and colon. Moreover, PGP treatment up-regulated the level of Slc26a3, thereby increasing the expressions of the tight junction/adherens junction proteins ZO-1, occludin and E-cadherin in the colon. RNA-seq analysis revealed that PGP inhibits the IL-6/STAT3/SOCS3 pathway at the transcriptional level. Molecular docking indicated that the major components of PGP could bind tightly to the proteins of IL-6 and SOCS3. Meanwhile, the result of Western blot revealed that the IL-6/STAT3/SOCS3 pathway was inhibited at the protein level after PGP administration. CONCLUSION: PGP could alleviate colonic inflammation and reverse damage to the intestinal epithelial barrier in DSS-induced colitis mice. The underlying mechanism involves the inhibition of the IL-6/STAT3/SOCS3 pathway.


Assuntos
Colite Ulcerativa , Colite , Extratos Vegetais , Punica granatum , Animais , Camundongos , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/metabolismo , Inflamação/metabolismo , Colo/patologia , Citocinas/metabolismo , Peso Corporal , Sulfato de Dextrana/efeitos adversos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Colite Ulcerativa/tratamento farmacológico , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/farmacologia , Transportadores de Sulfato/uso terapêutico , Antiporters/efeitos adversos , Antiporters/metabolismo
8.
Nature ; 627(8003): 382-388, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38418878

RESUMO

Calcium (Ca2+) is an essential nutrient for plants and a cellular signal, but excessive levels can be toxic and inhibit growth1,2. To thrive in dynamic environments, plants must monitor and maintain cytosolic Ca2+ homeostasis by regulating numerous Ca2+ transporters3. Here we report two signalling pathways in Arabidopsis thaliana that converge on the activation of vacuolar Ca2+/H+ exchangers (CAXs) to scavenge excess cytosolic Ca2+ in plants. One mechanism, activated in response to an elevated external Ca2+ level, entails calcineurin B-like (CBL) Ca2+ sensors and CBL-interacting protein kinases (CIPKs), which activate CAXs by phosphorylating a serine (S) cluster in the auto-inhibitory domain. The second pathway, triggered by molecular patterns associated with microorganisms, engages the immune receptor complex FLS2-BAK1 and the associated cytoplasmic kinases BIK1 and PBL1, which phosphorylate the same S-cluster in CAXs to modulate Ca2+ signals in immunity. These Ca2+-dependent (CBL-CIPK) and Ca2+-independent (FLS2-BAK1-BIK1/PBL1) mechanisms combine to balance plant growth and immunity by regulating cytosolic Ca2+ homeostasis.


Assuntos
Arabidopsis , Cálcio , Homeostase , Imunidade Vegetal , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Citosol/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Antiporters/metabolismo
9.
PLoS One ; 19(1): e0296928, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38252645

RESUMO

Mutations in the solute linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED) and Fuchs corneal endothelial dystrophy type 4 (FECD4), both characterized by corneal endothelial cell (CEnC) dysfunction and/or cell loss leading to corneal edema and visual impairment. In this study, we characterize the impact of CHED-/FECD4-associated SLC4A11 mutations on CEnC function and SLC4A11 protein localization by generating and comparing human CEnC (hCEnC) lines expressing wild type SLC4A11 (SLC4A11WT) or mutant SLC4A11 harboring CHED-/FECD4-associated SLC4A11 mutations (SLC4A11MU). SLC4A11WT and SLC4A11MU hCEnC lines were generated to express either SLC4A11 variant 2 (V2WT and V2MU) or variant 3 (V3WT and V3MU), the two major variants expressed in ex vivo hCEnC. Functional assays were performed to assess cell barrier, proliferation, viability, migration, and NH3-induced membrane conductance. We demonstrate SLC4A11-/- and SLC4A11MU hCEnC lines exhibited increased migration rates, altered proliferation and decreased cell viability compared to SLC4A11WT hCEnC. Additionally, SLC4A11-/- hCEnC demonstrated decreased cell-substrate adhesion and membrane capacitances compared to SLC4A11WT hCEnC. Induction with 10mM NH4Cl led SLC4A11WT hCEnC to depolarize; conversely, SLC4A11-/- hCEnC hyperpolarized and the majority of SLC4A11MU hCEnC either hyperpolarized or had minimal membrane potential changes following NH4Cl induction. Immunostaining of primary hCEnC and SLC4A11WT hCEnC lines for SLC4A11 demonstrated predominately plasma membrane staining with poor or partial colocalization with mitochondrial marker COX4 within a subset of punctate subcellular structures. Overall, our findings suggest CHED-associated SLC4A11 mutations likely lead to hCEnC dysfunction, and ultimately CHED, by interfering with cell migration, proliferation, viability, membrane conductance, barrier function, and/or cell surface localization of the SLC4A11 protein in hCEnC. Additionally, based on their similar subcellular localization and exhibiting similar cell functional profiles, protein isoforms encoded by SLC4A11 variant 2 and variant 3 likely have highly overlapping functional roles in hCEnC.


Assuntos
Proteínas de Transporte de Ânions , Antiporters , Distrofias Hereditárias da Córnea , Distrofia Endotelial de Fuchs , Humanos , Proteínas de Transporte de Ânions/genética , Antiporters/genética , Transtornos Cromossômicos , Distrofias Hereditárias da Córnea/genética , Células Endoteliais , Distrofia Endotelial de Fuchs/genética , Mutação , Proteínas SLC4A
10.
Planta ; 259(2): 49, 2024 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-38285217

RESUMO

MAIN CONCLUSION: ZmCBL8-1 enhances salt stress tolerance in maize by improving the antioxidant system to neutralize ROS homeostasis and inducing Na+/H+ antiporter gene expressions of leaves. Calcineurin B-like proteins (CBLs) as plant-specific calcium sensors have been explored for their roles in the regulation of abiotic stress tolerance. Further, the functional variations in ZmCBL8, encoding a component of the salt overly sensitive pathway, conferred the salt stress tolerance in maize. ZmCBL8-1 is a transcript of ZmCBL8 found in maize, but its function in the salt stress response is still unclear. The present study aimed to characterize the protein ZmCBL8-1 that was determined to be composed of 194 amino acids (aa) with three conserved EF hands responsible for binding Ca2+. However, a 20-aa fragment was found to be missing from its C-terminus relative to another transcript of ZmCBL8. Results indicated that it harbored a dual-lipid modification motif MGCXXS at its N-terminus and was located on the cell membrane. The accumulation of ZmCBL8-1 transcripts was high in the roots but relatively lower in the leaves of maize under normal condition. In contrast, its expression was significantly decreased in the roots, while increased in the leaves under NaCl treatment. The overexpression of ZmCBL8-1 resulted in higher salt stress resistance of transgenic Arabidopsis in a Ca2+-dependent manner relative to that of the wild type (WT). In ZmCBL8-1-overexpressing plants exposed to NaCl, the contents of malondialdehyde and hydrogen peroxide were decreased in comparison with those in the WT, and the expression of key genes involved in the antioxidant defense system and Na+/H+ antiporter were upregulated. These results suggested that ZmCBL8-1 played a positive role in the response of leaves to salt stress by inducing the expression of Na+/H+ antiporter genes and enhancing the antioxidant system to neutralize the accumulation of reactive oxygen species. These observations further indicate that ZmCBL8-1 confers salt stress tolerance, suggesting that transcriptional regulation of the ZmCBL8 gene is important for salt tolerance.


Assuntos
Arabidopsis , Estresse Salino , Zea mays , Aminoácidos , Antioxidantes , Antiporters , Arabidopsis/fisiologia , Calcineurina/genética , Cloreto de Sódio/farmacologia , Zea mays/genética
11.
Mol Genet Metab ; 141(3): 108144, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38277989

RESUMO

Glycogen storage disease type Ib (GSD Ib, biallelic variants in SLC37A4) is a rare disorder of glycogen metabolism complicated by neutropenia/neutrophil dysfunction. Since 2019, the SGLT2-inhibitor empagliflozin has provided a mechanism-based treatment option for the symptoms caused by neutropenia/neutrophil dysfunction (e.g. mucosal lesions, inflammatory bowel disease). Because of the rarity of GSD Ib, the published evidence on safety and efficacy of empagliflozin is still limited and does not allow to develop evidence-based guidelines. Here, an international group of experts provides 14 best practice consensus treatment recommendations based on expert practice and review of the published evidence. We recommend to start empagliflozin in all GSD Ib individuals with clinical or laboratory signs related to neutropenia/neutrophil dysfunction with a dose of 0.3-0.4 mg/kg/d given as a single dose in the morning. Treatment can be started in an outpatient setting. The dose should be adapted to the weight and in case of inadequate clinical treatment response or side effects. We strongly recommend to pause empagliflozin immediately in case of threatening dehydration and before planned longer surgeries. Discontinuation of G-CSF therapy should be attempted in all individuals. If available, 1,5-AG should be monitored. Individuals who have previously not tolerated starches should be encouraged to make a new attempt to introduce starch in their diet after initiation of empagliflozin treatment. We advise to monitor certain safety and efficacy parameters and recommend continuous, alternatively frequent glucose measurements during the introduction of empagliflozin. We provide specific recommendations for special circumstances like pregnancy and liver transplantation.


Assuntos
Compostos Benzidrílicos , Glucosídeos , Doença de Depósito de Glicogênio Tipo I , Neutropenia , Humanos , Neutrófilos/metabolismo , Consenso , Doença de Depósito de Glicogênio Tipo I/complicações , Doença de Depósito de Glicogênio Tipo I/tratamento farmacológico , Doença de Depósito de Glicogênio Tipo I/genética , Neutropenia/tratamento farmacológico , Neutropenia/etiologia , Proteínas de Transporte de Monossacarídeos , Antiporters/metabolismo
12.
Int J Mol Sci ; 25(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38203758

RESUMO

Ovarian cancer is one of the most dangerous gynecologic cancers worldwide and has a high fatality rate due to diagnosis at an advanced stage of the disease as well as a high recurrence rate due to the occurrence of chemotherapy resistance. In fact, chemoresistance weakens the therapeutic effects, worsening the outcome of this pathology. Solute Carrier Family 7 Member 11 (SLC7A11, also known as xCT) is the functional subunit of the Xc- system, an anionic L-cystine/L-glutamate antiporter expressed on the cell surface. SLC7A11 expression is significantly upregulated in several types of cancers in which it can inhibit ferroptosis and favor cancer cell proliferation, invasion and chemoresistance. SLC7A11 expression is also increased in ovarian cancer tissues, suggesting a possible role of this protein as a therapeutic target. In this review, we provide an overview of the current literature regarding the role of SLC7A11 in ovarian cancer to provide new insights on SLC7A11 modulation and evaluate the potential role of SLC7A11 as a therapeutic target.


Assuntos
Neoplasias dos Genitais Femininos , Neoplasias Ovarianas , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Antiporters , Membrana Celular , Ácido Glutâmico , Sistema y+ de Transporte de Aminoácidos/genética
13.
Nat Commun ; 15(1): 759, 2024 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-38272905

RESUMO

Anion exchanger 2 (AE2) is an electroneutral Na+-independent Cl-/HCO3- exchanger belongs to the SLC4 transporter family. The widely expressed AE2 participates in a variety of physiological processes, including transepithelial acid-base secretion and osteoclastogenesis. Both the transmembrane domains (TMDs) and the N-terminal cytoplasmic domain (NTD) are involved in regulation of AE2 activity. However, the regulatory mechanism remains unclear. Here, we report a 3.2 Å cryo-EM structure of the AE2 TMDs in complex with PIP2 and a 3.3 Å full-length mutant AE2 structure in the resting state without PIP2. We demonstrate that PIP2 at the TMD dimer interface is involved in the substrate exchange process. Mutation in the PIP2 binding site leads to the displacement of TM7 and further stabilizes the interaction between the TMD and the NTD. Reduced substrate transport activity and conformation similar to AE2 in acidic pH indicating the central contribution of PIP2 to the function of AE2.


Assuntos
Antiporters , Lipídeos , Humanos , Antiportadores de Cloreto-Bicarbonato/genética , Antiporters/genética , Proteínas SLC4A , Mutação , Proteínas de Transporte de Ânions/metabolismo , Concentração de Íons de Hidrogênio
14.
J Phys Chem Lett ; 15(3): 725-732, 2024 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-38215403

RESUMO

Transporter proteins change their conformations to carry their substrate across the cell membrane. The conformational dynamics is vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed a Gaussian accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully predicted the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward-open conformation. The results were complemented by an AlphaFold2 structure prediction. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations.


Assuntos
Simulação de Dinâmica Molecular , Oxalatos , Oxalatos/química , Oxalatos/metabolismo , Proteínas de Membrana Transportadoras/química , Antiporters/metabolismo , Formiatos/metabolismo , Conformação Proteica
15.
Sci Rep ; 14(1): 246, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168913

RESUMO

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death worldwide. Cigarette smoke which has approximately 2-3 µg of Cadmium (Cd) per cigarette contributes to the environmental exposure and development and severity of COPD. With the lack of a cadmium elimination mechanism in humans, the contribution of cadmium induced stress to lung epithelial cells remains unclear. Studies on cadmium responsive miRNAs suggest regulation of target genes with an emphasis on the critical role of miRNA-mRNA interaction for cellular homeostasis. Mir-381, the target miRNA in this study is negatively regulated by cadmium in airway epithelial cells. miR-381 is reported to also regulate ANO1 (Anoctamin 1) expression negatively and in this study low dose cadmium exposure to airway epithelial cells was observed to upregulate ANO1 mRNA expression via mir-381 inhibition. ANO1 which is a Ca2+-activated chloride channel has multiple effects on cellular functions such as proliferation, mucus hypersecretion and fibroblast differentiation in inflamed airways in chronic respiratory diseases. In vitro studies with cadmium at a high concentration range of 100-500 µM is reported to activate chloride channel, ANO1. The secretory epithelial cells are regulated by chloride channels like CFTR, ANO1 and SLC26A9. We examined "ever" smokers with COPD (n = 13) lung tissue sections compared to "never" smoker without COPD (n = 9). We found that "ever" smokers with COPD had higher ANO1 expression. Using mir-381 mimic to inhibit ANO1, we demonstrate here that ANO1 expression is significantly (p < 0.001) downregulated in COPD derived airway epithelial cells exposed to cadmium. Exposure to environmental cadmium contributes significantly to ANO1 expression.


Assuntos
MicroRNAs , Doença Pulmonar Obstrutiva Crônica , Humanos , Cádmio/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Células Epiteliais/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/genética , Proteínas de Neoplasias/metabolismo , Transportadores de Sulfato/metabolismo , Antiporters/metabolismo
16.
Plant Cell Environ ; 47(2): 557-573, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37916653

RESUMO

Multiple Arabidopsis H+ /Cation exchangers (CAXs) participate in high-capacity transport into the vacuole. Previous studies have analysed single and double mutants that marginally reduced transport; however, assessing phenotypes caused by transport loss has proven enigmatic. Here, we generated quadruple mutants (cax1-4: qKO) that exhibited growth inhibition, an 85% reduction in tonoplast-localised H+ /Ca transport, and enhanced tolerance to anoxic conditions compared to CAX1 mutants. Leveraging inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron X-ray fluorescence (SXRF), we demonstrate CAX transporters work together to regulate leaf elemental content: ICP-MS analysis showed that the elemental concentrations in leaves strongly correlated with the number of CAX mutations; SXRF imaging showed changes in element partitioning not present in single CAX mutants and qKO had a 40% reduction in calcium (Ca) abundance. Reduced endogenous Ca may promote anoxia tolerance; wild-type plants grown in Ca-limited conditions were anoxia tolerant. Sequential reduction of CAXs increased mRNA expression and protein abundance changes associated with reactive oxygen species and stress signalling pathways. Multiple CAXs participate in postanoxia recovery as their concerted removal heightened changes in postanoxia Ca signalling. This work showcases the integrated and diverse function of H+ /Cation transporters and demonstrates the ability to improve anoxia tolerance through diminishing endogenous Ca levels.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cálcio/metabolismo , Antiporters/genética , Antiporters/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cátions/metabolismo , Plantas/metabolismo
17.
Br J Haematol ; 204(1): 45-55, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-38049194

RESUMO

Neutrophils are the shortest-lived blood cells, which requires a prodigious degree of proliferation and differentiation to sustain physiologically sufficient numbers and be poised to respond quickly to infectious emergencies. More than 107 neutrophils are produced every minute in an adult bone marrow-a process that is tightly regulated by a small group of cytokines and chemical mediators and dependent on nutrients and energy. Like granulocyte colony-stimulating factor, the primary growth factor for granulopoiesis, they stimulate signalling pathways, some affecting metabolism. Nutrient or energy deficiency stresses the survival, proliferation, and differentiation of neutrophils and their precursors. Thus, it is not surprising that monogenic disorders related to metabolism exist that result in neutropenia. Among these are pathogenic mutations in HAX1, G6PC3, SLC37A4, TAFAZZIN, SBDS, EFL1 and the mitochondrial disorders. These mutations perturb carbohydrate, lipid and/or protein metabolism. We hypothesize that metabolic disturbances may drive the pathogenesis of a subset of inherited neutropenias just as defects in DNA damage response do in Fanconi anaemia, telomere maintenance in dyskeratosis congenita and ribosome formation in Diamond-Blackfan anaemia. Greater understanding of metabolic pathways in granulopoiesis will identify points of vulnerability in production and may point to new strategies for the treatment of neutropenias.


Assuntos
Doenças da Medula Óssea , Anemia de Fanconi , Neutropenia , Adulto , Humanos , Doenças da Medula Óssea/genética , Anemia de Fanconi/genética , Medula Óssea/patologia , Transtornos da Insuficiência da Medula Óssea , Neutropenia/patologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte de Monossacarídeos , Antiporters
18.
Psychopharmacology (Berl) ; 241(2): 291-304, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38049617

RESUMO

RATIONALE: While morphine has important therapeutic value it is also one of the most widely abused drugs in the world. As a newly discovered style of cell death, ferroptosis is involved in the occurrence and development of many diseases, however, the current understanding of the relationship between ferroptosis and morphine is still limited. OBJECTIVE: To clarify the role of opioid receptors in morphine-induced ferroptosis and to investigate the role of NRF2 in morphine-induced ferroptosis. METHODS: We first used different doses of morphine (0, 0.5, 1, and 1.5 mM) to investigate morphine-induced ferroptosis in SH-SY5Y cells, and we choose 1.5 mM morphine for subsequent experiments. We next inhibited opioid receptors and NRF2 separately and examined their influence on morphine-induced ferroptosis. Finally, we tested morphine-induced insufficient autophagy. RESULTS: Morphine triggered ferroptosis in a dose-dependent manner, which could be significantly rescued by the ferroptosis-specific inhibitor DFO. Moreover, GPX4 rather than xCT antiporter might be involved in morphine-induced ferroptosis. We also found naloxone could inhibit morphine-induced ferroptosis. Interestingly, our results demonstrated that NRF2 could promote rather than defend morphine-induced ferroptosis; this may be due to the increased p62-related insufficient autophagy. CONCLUSION: Morphine-induced ferroptosis is regulated by the opioid receptor and GPX4 rather than the xCT antiporter. NRF2-mediated ferroptosis in morphine-exposed cells may stem from increased p62-related insufficient autophagy.


Assuntos
Ferroptose , Neuroblastoma , Humanos , Antiporters , Autofagia , Morfina/farmacologia , Fator 2 Relacionado a NF-E2 , Receptores Opioides
19.
Gene ; 894: 148025, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38007163

RESUMO

Rapeseed (Brassica napus L.) is susceptible to nutrient stresses during growth and development; however, the CPA (cation proton antiporter) family genes have not been identified in B. napus and their biological functions remain unclear. This study was aimed to identify the molecular characteristics of rapeseed CPAs and their transcriptional responses to multiple nutrient stresses. Through bioinformatics analysis, 117 BnaCPAs, consisting of three subfamilies: Na+/H+ antiporter (NHX), K+ efflux antiporter (KEA), and cation/H+ antiporter (CHX), were identified in the rapeseed genome. Transcriptomic profiling showed that BnaCPAs, particularly BnaNHXs, were transcriptionally responsive to diverse nutrient stresses, including Cd toxicity, K starvation, salt stress, NH4+ toxicity, and low Pi. We found that the salt tolerance of the transgenic rapeseed lines overexpressing BnaA05.NHX2 was significantly higher than that of wild type. Subcellular localization showed that BnaA05.NHX2 was localized on the tonoplast, and TEM combined with X-ray energy spectrum analysis revealed that the vacuolar Na+ concentrations of the BnaA05.NHX2-overexpressing rapeseed plants were significantly higher than those of wild type. The findings of this study will provide insights into the complexity of the BnaCPA family and a valuable resource to explore the in-depth functions of CPAs in B. napus.


Assuntos
Brassica napus , Brassica rapa , Brassica napus/genética , Antiporters/genética , Prótons , Brassica rapa/genética , Vacúolos , Regulação da Expressão Gênica de Plantas , Estresse Fisiológico
20.
J Neuroinflammation ; 20(1): 292, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38057869

RESUMO

Neuroinflammation appears to involve some degree of excitotoxicity promulgated by microglia, which release glutamate via the system xC- (SxC-) cystine-glutamate antiporter. With the aim of mitigating this source of neuronal stress and toxicity, we have developed a panel of inhibitors of the SxC- antiporter. The compounds were based on L-tyrosine, as elements of its structure align with those of glutamate, a primary physiological substrate of the SxC- antiporter. In addition to 3,5-dibromotyrosine, ten compounds were synthesized via amidation of that parent molecule with a selection of acyl halides. These agents were tested for the ability to inhibit release of glutamate from microglia activated with lipopolysaccharide (LPS), an activity exhibited by eight of the compounds. To confirm that the compounds were inhibitors of SxC-, two of them were further tested for the ability to inhibit cystine uptake. Finally, these agents were shown to protect primary cortical neurons from the toxicity exhibited by activated microglia. These agents may hold promise in reducing the neurodegenerative effects of neuroinflammation in conditions, such as encephalitis, traumatic brain injury, stroke, or neurodegenerative diseases.


Assuntos
Ácido Glutâmico , Microglia , Humanos , Ácido Glutâmico/toxicidade , Microglia/metabolismo , Cistina/metabolismo , Doenças Neuroinflamatórias , Antiporters
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