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1.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
2.
BMC Plant Biol ; 19(1): 250, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185911

RESUMO

BACKGROUND: Cadmium (Cd) is a widespread toxic heavy metal pollutant in agricultural soil, and Cd accumulation in rice grains is a major intake source of Cd for Asian populations that adversely affect human health. However, the molecular mechanism underlying Cd uptake, translocation and accumulation has not been fully understood in rice plants. RESULTS: In this study, a mutant displaying extremely low Cd accumulation (lcd1) in rice plant and grain was generated by EMS mutagenesis from indica rice cultivar 9311 seeds. The candidate SNPs associated with low Cd accumulation phenotype in the lcd1 mutant were identified by MutMap and the transcriptome changes between lcd1 and WT under Cd exposure were analyzed by RNA-seq. The lcd1 mutant had lower Cd uptake and accumulation in rice root and shoot, as well as less growth inhibition compared with WT in the presence of 5 µM Cd. Genetic analysis showed that lcd1 was a single locus recessive mutation. The SNP responsible for low Cd accumulation in the lcd1 mutant located at position 8,887,787 on chromosome 7, corresponding to the seventh exon of OsNRAMP5. This SNP led to a Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 in the lcd1 mutant. A total of 1208 genes were differentially expressed between lcd1 and WT roots under Cd exposure, and DEGs were enriched in transmembrane transport process GO term. Increased OsHMA3 expression probably adds to the effect of OsNRAMP5 mutation to account for the significant decreases in Cd accumulation in rice plant and grain of the lcd1 mutant. CONCLUSIONS: An extremely low Cd mutant lcd1 was isolated and identified using MutMap and RNA-seq. A Pro236Leu amino acid substitution in the highly conserved region of OsNRAMP5 is likely responsible for low Cd accumulation in the lcd1 mutant. This work provides more insight into the mechanism of Cd uptake and accumulation in rice, and will be helpful for developing low Cd accumulation rice by marker-assisted breeding.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Oryza/genética , Proteínas de Plantas/genética , Poluentes do Solo/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica , Oryza/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
3.
Gene ; 710: 399-405, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31200088

RESUMO

Iron-responsive elements (IREs) are ~35-nucleotide (nt) stem-loop RNA structures located in 5' or 3' untranslated regions (UTRs) of mRNAs that mediate post-transcriptional regulation by their association with IRE-binding proteins (IRPs). IREs are characterized by their apical 6-nt loop motif 5'-CAGWGH-3' (W = A or U and H = A, C or U), the so-called pseudotriloop, of which the loop nts C1 and G5 are paired, and the none-paired C between the two stem regions. In this study, the yeast three-hybrid (Y3H) system was used to investigate the relevance of the pseudotriloop structure of ferritin light chain (FTL) for the IRE-IRP interaction and the binding affinities between variant IRE(-like) structures and the two IRP isoforms, IRP1 and 2. Destabilization of the pseudotriloop structure by a G5-to-A mutation reduced binding of IRP1 and 2, while restoring the pseudotriloop conformation by the compensatory C1-to-U mutation, restored binding to both IRPs. In particular, IRP1 showed even stronger binding to the C1U-G5A mutant than to the wildtype FTL IRE. On the other hand, deletion of the bulged-out U6 of the pseudotriloop did not significantly affect its binding to either IRP1 or 2, but substitution with C particularly enhanced the binding to IRP1. In comparison to FTL IRE, IRE-like structures of 5'-aminolevulinate synthase 2 (ALAS2) and SLC40A1 (also known as ferroportin-1) showed similar or, in the case of endothelial PAS domain protein 1 (EPAS1) IRE, slightly weaker binding affinity to IRPs. SLC11A2 (a.k.a. divalent metal transporter-1) IRE exhibited relatively weak binding to IRP1 and medium binding to IRP2. Notably, the IRE-like structure of α-synuclein showed no detectable binding to either IRP under the conditions used in this Y3H assay. Our results indicate that Y3H can be used to characterize binding between IRPs and various IRE-like structures in vivo.


Assuntos
Apoferritinas/química , Apoferritinas/genética , Proteína 1 Reguladora do Ferro/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , 5-Aminolevulinato Sintetase/química , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Animais , Apoferritinas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Mutação , Conformação de Ácido Nucleico , Técnicas do Sistema de Duplo-Híbrido , Regiões não Traduzidas
4.
BMC Plant Biol ; 19(1): 283, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248369

RESUMO

BACKGROUND: Metal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown. RESULTS: In this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress. CONCLUSION: OsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.


Assuntos
Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Cobre/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/genética , Proteínas de Plantas/genética , Zinco/metabolismo , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estresse Fisiológico
5.
Comput Biol Chem ; 80: 498-511, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176140

RESUMO

Magnesium (Mg) is an important micronutrient for various physiological processes in plants. In this study, putative Magnesium Transporter (MGT) genes have been identified in Solanum lycopersicum, Solanum tuberosum, Brachypodium distachyon, Fagaria vesca, Brassica juncea and were classified into 5 distinct groups based on their sequence homology. MGT genes are very diverse and possess very low sequence identity within its family. However, the Gly-Met-Asn (GMN) signature motif is present in most of the genes which are believed to be essential for Mg2+ recognition. In S. lycopersicum, different physiological root growth pattern was observed in both Mg excess and deficient conditions. Quantitative RT-PCR gene expression study shows that most of the SlMGT genes were upregulated in response to Mg deficient condition.


Assuntos
Proteínas de Transporte de Cátions/genética , Genoma , Magnoliopsida/genética , Proteínas de Plantas/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Magnésio/metabolismo , Magnoliopsida/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Conformação Proteica em alfa-Hélice , Reação em Cadeia da Polimerase em Tempo Real , Sementes/genética , Homologia de Sequência , Estresse Fisiológico/genética , Regulação para Cima
6.
Inorg Chem ; 58(11): 7488-7498, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31083932

RESUMO

It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)-hCtr2 interaction in the recovery of copper from lysosomes.


Assuntos
Proteínas de Transporte de Cátions/química , Cobre/metabolismo , Espaço Extracelular/química , Lisossomos/química , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Espaço Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Modelos Moleculares , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica
7.
Inorg Chem ; 58(9): 5932-5942, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-30986048

RESUMO

Cluster 2 (288HDDDNAHAHTH298) from Neisseria meningitidis ZnuD is a flexible loop that captures zinc(II) ions, acting as a "fishing net". We describe its Zn(II) and Cu(II) binding capabilities, focusing on the thermodynamics of such interactions and comparing them with the complexes of the 1MAHHHHHHL9-NH2 region. Copper(II) complexes with the studied ZnuD regions are thermodynamically more stable than the zinc(II) ones-Cu(II) complexes dominate in solution even in close to physiological ratios of the studied metal ions (a 10-fold excess of Zn(II) over Cu(II)). While the binding of native Zn(II) has no significant impact on the structure of its transporter, Cu(II) binding induces a conformational change of cluster 2 to a polyproline II-like helix. To the best of our knowledge, this is the first evidence of a copper(II)-induced formation of a polyproline II-like structure in a sequence that does not contain proline residues. Cu(II) coordination also changes the structure of an intracellular, N-terminal, His-rich region, folding it to an α helix.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Neisseria meningitidis/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Cobre/química , Humanos , Infecções Meningocócicas/microbiologia , Modelos Moleculares , Neisseria meningitidis/química , Ligação Proteica , Termodinâmica , Zinco/química
8.
Acta Crystallogr D Struct Biol ; 75(Pt 4): 357-367, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30988253

RESUMO

The cation diffusion facilitators (CDFs) are a family of membrane-bound proteins that maintain cellular homeostasis of essential metal ions. In humans, the zinc-transporter CDF family members (ZnTs) play important roles in zinc homeostasis. They do this by facilitating zinc efflux from the cytoplasm to the extracellular space across the plasma membrane or into intracellular organelles. Several ZnTs have been implicated in human health owing to their association with type 2 diabetes and neurodegenerative diseases. Although the structure determination of CDF family members is not trivial, recent advances in membrane-protein structural biology have resulted in two structures of bacterial YiiPs and several structures of their soluble C-terminal domains. These data reveal new insights into the molecular mechanism of ZnT proteins, suggesting a unique rocking-bundle mechanism that provides alternating access to the metal-binding site.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Família Multigênica , Zinco/metabolismo , Difusão , Humanos
9.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823627

RESUMO

Abiotic stresses generally cause a series of morphological, biochemical and molecular changes that unfavorably affect plant growth and productivity. Among these stresses, soil salinity is a major threat that can seriously impair crop yield. To cope with the effects of high salinity on plants, it is important to understand the mechanisms that plants use to deal with it, including those activated in response to disturbed Na⁺ and K⁺ homeostasis at cellular and molecular levels. HKT1-type transporters are key determinants of Na⁺ and K⁺ homeostasis under salt stress and they contribute to reduce Na⁺-specific toxicity in plants. In this review, we provide a brief overview of the function of HKT1-type transporters and their importance in different plant species under salt stress. Comparison between HKT1 homologs in different plant species will shed light on different approaches plants may use to cope with salinity.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Plantas/genética , Estresse Salino , Plantas Tolerantes a Sal/genética , Simportadores/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Simportadores/química , Simportadores/metabolismo
10.
Int J Mol Sci ; 20(5)2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30845649

RESUMO

The cyclin and cystathionine ß-synthase (CBS) domain magnesium transport mediators, CNNMs, are key players in maintaining the homeostasis of magnesium in different organs. The human family includes four members, whose impaired activity causes diseases such as Jalili Syndrome or Familial Hypomagnesemia, but is also linked to neuropathologic disorders, altered blood pressure, and infertility. Recent findings demonstrated that CNNMs are associated with the highly oncogenic phosphatases of the regenerating liver to promote tumor growth and metastasis, which has attracted renewed focus on their potential exploitation as targets for cancer treatment. However, the exact function of CNNMs remains unclear and is subject to debate, proposed as either direct transporters, sensors, or homeostatic factors. This review gathers the current structural knowledge on the CNNM family, highlighting similarities and differences with the closely related structural partners such as the bacterial Mg2+/Co2+ efflux protein CorC and the Mg2+ channel MgtE.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Proteínas de Transporte de Cátions/genética , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica
11.
PLoS Comput Biol ; 15(3): e1006882, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30893306

RESUMO

Zinc is a vital trace element crucial for the proper function of some 3,000 cellular proteins. Specifically, zinc is essential for key physiological processes including nucleic acid metabolism, regulation of gene expression, signal transduction, cell division, immune- and nervous system functions, wound healing, and apoptosis. Consequently, impairment of zinc homeostasis disrupts key cellular functions resulting in various human pathologies. Mammalian zinc transport proceeds via two transporter families ZnT and ZIP. However, the detailed mechanism of action of ZnT2, which is responsible for vesicular zinc accumulation and zinc secretion into breast milk during lactation, is currently unknown. Moreover, although the putative coupling of zinc transport to the proton gradient in acidic vesicles has been suggested, it has not been conclusively established. Herein we modeled the mechanism of action of ZnT2 and demonstrated both computationally and experimentally, using functional zinc transport assays, that ZnT2 is indeed a proton-coupled zinc antiporter. Bafilomycin A1, a specific inhibitor of vacuolar-type proton ATPase (V-ATPase) which alkalizes acidic vesicles, abolished ZnT2-dependent zinc transport into intracellular vesicles. Moreover, using LysoTracker Red and Lyso-pHluorin, we further showed that upon transient ZnT2 overexpression in intracellular vesicles and addition of exogenous zinc, the vesicular pH underwent alkalization, presumably due to a proton-zinc antiport; this phenomenon was reversed in the presence of TPEN, a specific zinc chelator. Finally, based on computational energy calculations, we propose that ZnT2 functions as an antiporter with a stoichiometry of 2H+/Zn2+ ion. Hence, ZnT2 is a proton motive force-driven, electroneutral vesicular zinc exchanger, concentrating zinc in acidic vesicles on the expense of proton extrusion to the cytoplasm.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Zinco/metabolismo , Ácidos/metabolismo , Álcalis/metabolismo , Proteínas de Transporte de Cátions/química , Quelantes/metabolismo , Relação Dose-Resposta a Droga , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Macrolídeos/farmacologia , Modelos Moleculares , Prótons , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo
12.
J Am Soc Mass Spectrom ; 30(5): 886-892, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30887461

RESUMO

Recent advances in native mass spectrometry (MS) have enabled the elucidation of how small molecule binding to membrane proteins modulates their structure and function. The protein-stabilizing osmolyte, trimethylamine oxide (TMAO), exhibits attractive properties for native MS studies. Here, we report significant charge reduction, nearly threefold, for three membrane protein complexes in the presence of this osmolyte without compromising mass spectral resolution. TMAO improves the ability to resolve individual lipid-binding events to the ammonia channel (AmtB) by over 200% compared to typical native conditions. The generation of ions with compact structure and access to a larger number of lipid-binding events through the incorporation of TMAO increases the utility of IM-MS for structural biology studies. Graphical Abstract.


Assuntos
Aquaporinas/química , Proteínas de Bactérias/química , Proteínas de Transporte de Cátions/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Canais Iônicos/química , Espectrometria de Mobilidade Iônica/métodos , Metilaminas/química , Mycobacterium tuberculosis/química , Excipientes/química , Íons/química , Conformação Proteica
13.
Nat Plants ; 5(3): 308-315, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742036

RESUMO

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.


Assuntos
Proteínas de Transporte de Cátions/química , Óleo de Eucalipto/química , Proteínas de Transporte de Cátions/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Ferro/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Vacúolos/metabolismo
14.
Mol Carcinog ; 58(5): 794-807, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30614075

RESUMO

Core fucosylation catalyzed by core fucosyltransferase (Fut8) contributes to the progressions of epithelial ovarian cancer (EOC). Copper transporter 1 (CTR1), which contains one N-glycan on Asn15 , mediates cellular transport of cisplatin (cDDP), and plays an important role in the process of cDDP-resistance in EOC. In the present study, we found that the core fucosylation level elevated significantly in the sera of cDDP-treated EOC patients. The in vitro assays also indicate that core fucosylation of CTR1 was significantly upregulated in cDDP-resistant A2780CP cells compared to the cDDP-sensitive A2780S cells. Intriguingly, the hyper core fucosylation suppressed the CTR1-cDDP interactions and cDDP-uptake into A2780CP cells. Conversely, contrast to the Fut8+/+ mouse ovarian epithelial cells, the Fut8-deleted (Fut8-/- ) cells obviously showed higher cDDP-uptake. Furthermore, the recovered core fucosylation induced the suppression of cDDP-uptake in Fut8-restored ovarian epithelial cells. In addition, the core fucosylation could regulate the phosphorylation of cDDP-resistance-associated molecules, such as AKT, ERK, JNK, and mTOR. Our findings suggest that the core fucosylation of CTR1 plays an important role in the cellular cDDP-uptake and thus provide new strategies for improving the outcome of cDDP based chemotherapy of EOC.


Assuntos
Carcinoma Epitelial do Ovário/tratamento farmacológico , Proteínas de Transporte de Cátions/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Fucose/metabolismo , Fucosiltransferases/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Carcinoma Epitelial do Ovário/metabolismo , Carcinoma Epitelial do Ovário/patologia , Proteínas de Transporte de Cátions/química , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais Cultivadas
15.
Genetics ; 211(3): 893-911, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30647069

RESUMO

Spore germination is a process whereby spores exit dormancy to become competent for mitotic cell division. In Schizosaccharomyces pombe, one critical step of germination is the formation of a germ tube that hatches out the spore wall in a stage called outgrowth. Here, we show that iron deficiency blocks the outgrowth of germinating spores. The siderophore synthetase Sib1 and the ornithine N5-oxygenase Sib2 participate in ferrichrome biosynthesis, whereas Str1 functions as a ferrichrome transporter. Expression profiles of sib1+ , sib2+ , and str1+ transcripts reveal that they are induced shortly after induction of germination and their expression remains upregulated throughout the germination program under low-iron conditions. sib1Δ sib2Δ mutant spores are unable to form a germ tube under iron-poor conditions. Supplementation with exogenous ferrichrome suppresses this phenotype when str1+ is present. Str1 localizes at the contour of swollen spores 4 hr after induction of germination. At the onset of outgrowth, localization of Str1 changes and it moves away from the mother spore to primarily localize at the periphery of the new daughter cell. Two conserved Tyr residues (Tyr553 and Tyr567) are predicted to be located in the last extracellular loop region of Str1. Results show that these amino acid residues are critical to ensure timely completion of the outgrowth phase of spores in response to exogenous ferrichrome. Taken together, the results reveal the essential requirement of ferrichrome biosynthesis to promote outgrowth, as well as the necessity to take up ferrichrome from an external source via Str1 when ferrichrome biosynthesis is blocked.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Ferricromo/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Esporos Fúngicos/genética , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Ferro/metabolismo , Domínios Proteicos , Transporte Proteico , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética
16.
J Biol Chem ; 294(8): 2815-2826, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30593504

RESUMO

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Endocitose/fisiologia , Matriz Extracelular/metabolismo , Histidina/química , Proteínas Mutantes/metabolismo , Mutação , Zinco/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Células HEK293 , Histidina/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Transporte Proteico , Homologia de Sequência
17.
J Biol Chem ; 293(52): 20008-20009, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30593530

RESUMO

Magnesium homeostasis relies on transporters like the CNNM family, but little information on these proteins' structure and regulation limits our understanding of their biology and functions in disease. New characterization of a conserved cytoplasmic domain now confirms the presence of a self-liganded architecture that is indispensable for Mg2+ efflux and suggests a possible role for a dimeric assembly.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Magnésio/metabolismo , Animais , Cristalografia por Raios X , Ciclinas/química , Ciclinas/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos
18.
Proc Natl Acad Sci U S A ; 115(26): 6691-6696, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891712

RESUMO

Strong interactions between lipids and proteins occur primarily through association of charged headgroups and amino acid side chains, rendering the protonation status of both partners important. Here we use native mass spectrometry to explore lipid binding as a function of charge of the outer membrane porin F (OmpF). We find that binding of anionic phosphatidylglycerol (POPG) or zwitterionic phosphatidylcholine (POPC) to OmpF is sensitive to electrospray polarity while the effects of charge are less pronounced for other proteins in outer or mitochondrial membranes: the ferripyoverdine receptor (FpvA) or the voltage-dependent anion channel (VDAC). Only marginal charge-induced differences were observed for inner membrane proteins: the ammonia channel (AmtB) or the mechanosensitive channel. To understand these different sensitivities, we performed an extensive bioinformatics analysis of membrane protein structures and found that OmpF, and to a lesser extent FpvA and VDAC, have atypically high local densities of basic and acidic residues in their lipid headgroup-binding regions. Coarse-grained molecular dynamics simulations, in mixed lipid bilayers, further implicate changes in charge by demonstrating preferential binding of anionic POPG over zwitterionic POPC to protonated OmpF, an effect not observed to the same extent for AmtB. Moreover, electrophysiology and mass-spectrometry-based ligand-binding experiments, at low pH, show that POPG can maintain OmpF channels in open conformations for extended time periods. Since the outer membrane is composed almost entirely of anionic lipopolysaccharide, with similar headgroup properties to POPG, such anionic lipid binding could prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways.


Assuntos
Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , Porinas/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Modelos Químicos , Modelos Moleculares , Simulação de Dinâmica Molecular , Porinas/química , Ligação Proteica , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Canais de Ânion Dependentes de Voltagem/química , Canais de Ânion Dependentes de Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/química , Canais de Sódio Disparados por Voltagem/metabolismo
19.
Int J Mol Sci ; 19(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748478

RESUMO

Arabidopsis thaliana natural resistance-associated macrophage protein 3 (AtNRAMP3) is involved in the transport of cadmium (Cd), iron (Fe), and manganese (Mn). Here, we present a structure-function analysis of AtNRAMP3 based on site-directed mutagenesis and metal toxicity growth assays involving yeast mutants, combined with three-dimensional (3D) structure modeling based on the crystal structure of the Eremococcus coleocola NRAMP family transporter, EcoDMT. We demonstrated that two conservative sites, D72 and N75, are essential for the transport activity. The M248A mutation resulted in a decrease in Cd sensitivity, while maintaining Mn transport. The mutation involving G61 caused a significant impairment of Fe and Mn transport, thereby indicating the importance of the conserved residue for proper protein function. The mutation involving G171 disrupted Fe transport activity but not that of Mn and Cd, suggesting that G171 is essential to metal binding and selectivity. Two residues, E194 and R262, may play an important role in stabilizing outward-facing conformation, which is essential for transport activity. Deletion assays indicated that the N-terminus is necessary for the function of AtNRAMP3. The findings of the present study revealed the structure-function relationship of AtNRAMP3 and metal transport activity and selectivity, which may possibly be applied to other plant NRAMP proteins.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Proteínas de Transporte de Cátions/química , Metais/metabolismo , Relação Estrutura-Atividade , Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Cádmio/química , Cádmio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Ferro/química , Manganês/química , Manganês/metabolismo , Metais/química , Mutagênese Sítio-Dirigida , Mutação , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
20.
PLoS One ; 13(5): e0196230, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29723216

RESUMO

Studies have demonstrated that the solute carrier family 11 member 1 (SLC11A1) is heavily glycosylated and phosphorylated in macrophages. However, the mechanisms of SLC11A1 phosphorylation, and the effects of phosphorylation on SLC11A1 activity remain largely unknown. Here, the tyrosine phosphorylation of SLC11A1 is observed in SLC11A1-expressing U937 cells when differentiated into macrophages by phorbol myristate acetate (PMA). The phosphorylation of SLC11A1 is almost completely blocked by treatment with PP2, a selective inhibitor of Src family kinases. Furthermore, we found that SLC11A1 is a direct substrate for active c-Src kinase and siRNA-mediated knockdown of cellular Src (c-Src) expression results in a significant decrease in tyrosine phosphorylation. We found that PMA induces the interaction of SLC11A1 with c-Src kinase. We demonstrated that SLC11A1 is phosphorylated by Src family kinases at tyrosine 15 and this type of phosphorylation is required for SLC11A1-mediated modulation of NF-κB activation and nitric oxide (NO) production induced by LPS. Our results demonstrate important roles for c-Src tyrosine kinase in phosphorylation and activation of SLC11A1 in macrophages.


Assuntos
Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Diferenciação Celular , Macrófagos/citologia , Tirosina/metabolismo , Quinases da Família src/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , NF-kappa B/metabolismo , Óxido Nítrico/biossíntese , Fosforilação
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