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1.
J Med Chem ; 63(24): 15773-15784, 2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33314931

RESUMO

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by ∼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by ∼2-fold (with an EC50 of ∼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Peptídeos Cíclicos/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Estabilidade de Medicamentos , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Mutação , Domínios PDZ , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(41): 25517-25522, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32973095

RESUMO

Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.


Assuntos
Proteínas de Escherichia coli , Prótons , Trocadores de Sódio-Hidrogênio , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Lisina/química , Lisina/genética , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Eletricidade Estática
3.
Biochim Biophys Acta Biomembr ; 1862(6): 183272, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32169592

RESUMO

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas de Membrana/química , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Humanos , Proteínas de Membrana/biossíntese , Conformação Proteica , Receptores da Somatotropina/química , Proteínas Recombinantes/biossíntese , Trocadores de Sódio-Hidrogênio/química , Leveduras/genética
4.
Mol Cell Biochem ; 468(1-2): 13-20, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32130622

RESUMO

Mammalian Na+/H+ exchanger type I isoform (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH (pHi) by removing one intracellular proton in exchange for one extracellular sodium ion. Abnormal activity of the protein occurs in cardiovascular disease and breast cancer. The purpose of this study is to examine the role of negatively charged amino acids of extracellular loop 3 (EL3) in the activity of the NHE protein. We mutated glutamic acid 217 and aspartic acid 226 to alanine, and to glutamine and asparagine, respectively. We examined effects on expression levels, cell surface targeting and activity of NHE1, and also characterized affinity for extracellular sodium and lithium ions. Individual mutation of these amino acids had little effect on protein function. However, mutation of both these amino acids together impaired transport, decreasing the Vmax for both Na+ and Li+ ions. We suggested that amino acids E217 and D226 form part of a negatively charged coordination sphere, which facilitates cation transport in the NHE1 protein.


Assuntos
Aminoácidos Acídicos/química , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Aminoácidos Acídicos/genética , Animais , Proteínas de Transporte de Cátions/genética , Linhagem Celular , Membrana Celular/química , Membrana Celular/genética , Cricetulus , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Domínios Proteicos/genética , Trocador 1 de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética
5.
Biochim Biophys Acta Biomembr ; 1862(3): 183111, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678368

RESUMO

The plant plasma membrane Na+/H+ antiporter SOS1 (Salt Overlay Sensitive 1) of Arabidopsis thaliana is the major transporter extruding Na+ out of cells in exchange for an intracellular H+. The sodium extrusion process maintains a low intracellular Na+ concentration and thereby facilitates salt tolerance. A. thaliana SOS1 consists of 1146 amino acids, with the first 450 in a N-terminal membrane transport domain and the balance forming a cytosolic regulatory domain. For studies on characterization of the protein, two different constructs of SOS1 comprising of the residues 28 to 460 and 28 to 990 were cloned and overexpressed in methylotropic yeast strain of Pichia pastoris with a C-terminal histidine tag using the expression vector pPICZA. Styrene malic acid copolymers (SMA) were used as a cost-effective alternative to detergent for solubilization and isolation of this membrane protein. Immobilized Ni2+-ion affinity chromatography was used to purify the expressed protein resulting in a yield of ~0.6-2 mg of SOS1 per liter of Pichia pastoris culture. The SMA purified protein containing amino acids 28 to 990 was directly reconstituted into liposomes for determination of Na+ transport activity and was functionally active. However, similar reconstitution with amino acids 28-460 did not yield a functional protein. Other results have shown that the truncated SOS1 protein at amino acid 481 is active, which infers the presence of an element between residues 461-481 which is necessary for SOS1 activity. This region contains several conserved segments that may be important in SOS1 structure and function.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Citoplasma/metabolismo , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Pichia/metabolismo , Tolerância ao Sal/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
6.
Sci Rep ; 9(1): 17662, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776461

RESUMO

Cardiolipin (CL) was shown to bound to the dimer interface of NhaA Na+/H+ antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl ß-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein-lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.


Assuntos
Cardiolipinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Sítios de Ligação , Cardiolipinas/farmacologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Humanos , Fosfolipídeos/metabolismo , Trocadores de Sódio-Hidrogênio/química
7.
Sci Rep ; 9(1): 15390, 2019 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-31659210

RESUMO

Bacterial NhaB Na+/H+ exchangers belonging to the Ion Transporter superfamily are poorly characterized in contrast to Na+/H+ exchangers of the Cation Proton Antiporter superfamily which have NhaA from Escherichia coli as a prominent member. For a more detailed understanding of the intricacies of the exchanger's transport mechanism, mutational studies are essential. Therefore, we mutated two protonatable residues present in the putative transmembrane region of NhaB from Klebsiella pneumoniae (KpNhaB), which could serve as substrate binding sites, Asp146 and Asp404, to either glutamate or alanine and analyzed transport function and stability of the mutants using electrophysiological and fluorimetric techniques. While mutation of either Asp residue to Glu only had slight to moderate effects on the transport activity of the exchanger, the mutations D404A and D146A, in particular, had more profound effects on the transport function. Furthermore, a double mutant, D146A/D404A, exhibited a remarkable behavior at alkaline pH, where recorded electrical currents changed polarity, showing steady-state transport with a stoichiometry of H+:Na+ < 1, as opposed to the H+:Na+ > 1 stoichiometry of the WT. Thus, we showed that Asp146 and Asp404 are part of the substrate binding site(s) of KpNhaB and engineered a Na+/H+ exchanger with a variable stoichiometry.


Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Klebsiella pneumoniae/química , Trocadores de Sódio-Hidrogênio/química , Ácido Aspártico/química , Ácido Aspártico/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/fisiologia , Potenciais da Membrana , Domínios Proteicos , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo
8.
Chem Commun (Camb) ; 55(87): 13152-13155, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31617527

RESUMO

We designed a supported lipid bilayer (SLB) biomimetic membrane system that comprised polyaniline (PANI) to support a lipid bilayer membrane that incorporated Na+/H+ transporter proteins (NhaA) to give the system the capability of controllable electrogenic ion transport. The high turnover rate of NhaA (∼105 per min) provides the basis for this PANI-SLB-NhaA system to be a high-speed rechargeable biocapacitor that functions as a low-energy-consuming fast switch for biological engineering applications.


Assuntos
Compostos de Anilina/metabolismo , Materiais Biomiméticos/metabolismo , Técnicas Biossensoriais , Proteínas de Escherichia coli/metabolismo , Bicamadas Lipídicas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Compostos de Anilina/química , Materiais Biomiméticos/química , Espectroscopia Dielétrica , Eletrodos , Proteínas de Escherichia coli/química , Ouro/química , Ouro/metabolismo , Bicamadas Lipídicas/química , Trocadores de Sódio-Hidrogênio/química
9.
Physiol Rev ; 99(4): 2015-2113, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31507243

RESUMO

Na+/H+ exchangers play pivotal roles in the control of cell and tissue pH by mediating the electroneutral exchange of Na+ and H+ across cellular membranes. They belong to an ancient family of highly evolutionarily conserved proteins, and they play essential physiological roles in all phyla. In this review, we focus on the mammalian Na+/H+ exchangers (NHEs), the solute carrier (SLC) 9 family. This family of electroneutral transporters constitutes three branches: SLC9A, -B, and -C. Within these, each isoform exhibits distinct tissue expression profiles, regulation, and physiological roles. Some of these transporters are highly studied, with hundreds of original articles, and some are still only rudimentarily understood. In this review, we present and discuss the pioneering original work as well as the current state-of-the-art research on mammalian NHEs. We aim to provide the reader with a comprehensive view of core knowledge and recent insights into each family member, from gene organization over protein structure and regulation to physiological and pathophysiological roles. Particular attention is given to the integrated physiology of NHEs in the main organ systems. We provide several novel analyses and useful overviews, and we pinpoint main remaining enigmas, which we hope will inspire novel research on these highly versatile proteins.


Assuntos
Equilíbrio Ácido-Base , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Evolução Molecular , Regulação da Expressão Gênica , Humanos , Conformação Proteica , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Relação Estrutura-Atividade , Distribuição Tecidual
10.
J Biol Chem ; 294(29): 11297-11310, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31171716

RESUMO

The Na+/H+ exchange regulatory cofactor 1 (NHERF1) protein modulates the assembly and intracellular trafficking of several transmembrane G protein-coupled receptors (GPCRs) and ion transport proteins with the membrane-cytoskeleton adapter protein ezrin. Here, we applied solution NMR and small-angle neutron scattering (SANS) to structurally characterize full-length NHERF1 and disease-associated variants that are implicated in impaired phosphate homeostasis. Using NMR, we mapped the modular architecture of NHERF1, which is composed of two structurally-independent PDZ domains that are connected by a flexible, disordered linker. We observed that the ultra-long and disordered C-terminal tail of NHERF1 has a type 1 PDZ-binding motif that interacts weakly with the proximal, second PDZ domain to form a dynamically autoinhibited structure. Using ensemble-optimized analysis of SANS data, we extracted the molecular size distribution of structures from the extensive conformational space sampled by the flexible chain. Our results revealed that NHERF1 is a diffuse ensemble of variable PDZ domain configurations and a disordered C-terminal tail. The joint NMR/SANS data analyses of three disease variants (L110V, R153Q, and E225K) revealed significant differences in the local PDZ domain structures and in the global conformations compared with the WT protein. Furthermore, we show that the substitutions affect the affinity and kinetics of NHERF1 binding to ezrin and to a C-terminal peptide from G protein-coupled receptor kinase 6A (GRK6A). These findings provide important insight into the modulation of the intrinsic flexibility of NHERF1 by disease-associated point mutations that alter the dynamic assembly of signaling complexes.


Assuntos
Fosfoproteínas/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo , Humanos , Cinética , Mutação , Ressonância Magnética Nuclear Biomolecular , Domínios PDZ , Fosfoproteínas/química , Ligação Proteica , Estrutura Secundária de Proteína , Trocadores de Sódio-Hidrogênio/química , Ressonância de Plasmônio de Superfície
11.
Anal Chem ; 91(11): 6976-6980, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31082219

RESUMO

Hydrogen-deuterium exchange-mass spectrometry (HDXMS) is a powerful technology to characterize conformations and conformational dynamics of proteins and protein complexes. HDXMS has been widely used in the field of therapeutics for the development of protein drugs. Although sufficient sequence coverage is critical to the success of HDXMS, it is sometimes difficult to achieve. In this study, we developed a HDXMS data analysis strategy that includes parallel post-translational modification (PTM) scanning in HDXMS analysis. Using a membrane-delimited G protein-coupled receptor (vasopressin type 2 receptor; V2R) and a cytosolic protein (Na+/H+ exchanger regulatory factor-1; NHERF1) as examples, we demonstrate that this strategy substantially improves protein sequence coverage, especially in key structural regions likely including PTMs themselves that play important roles in protein conformational dynamics and function.


Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteínas/metabolismo , Glicosilação , Humanos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
12.
Nat Commun ; 10(1): 1742, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988359

RESUMO

Na+/H+ antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.


Assuntos
Pyrococcus abyssi/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Modelos Moleculares , Prótons , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
13.
Sci Rep ; 9(1): 6191, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996236

RESUMO

The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu74 and Arg77 of transmembrane segment 3. Glu74 and Arg77 are conserved in yeast species while Glu74 is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg341 and Arg342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp389, Glu390, Glu392, Glu397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na+/H+ exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function.


Assuntos
Membrana Celular/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Trocadores de Sódio-Hidrogênio/química , Sequência de Aminoácidos , Aminoácidos Acídicos/fisiologia , Sequência Conservada , Mutação , Tolerância ao Sal/genética , Schizosaccharomyces/ultraestrutura
14.
Gene ; 695: 101-112, 2019 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-30763666

RESUMO

The fluted giant clam, Tridacna squamosa, lives in symbiosis with photosynthetic zooxanthellae, and can engage in light-enhanced growth and shell formation. Light-enhanced shell formation necessitates the elimination of excess H+ from the extrapallial fluid adjacent to the shell. This study aimed to clone Na+/H+Exchanger (NHE) from the whitish inner mantle adjacent to the extrapallial fluid of T. squamosa, to determine its cellular and subcellular localization, and to evaluate the effect of light exposure on its mRNA expression level and protein abundance therein. The complete coding cDNA sequence of NHE obtained was identified as a homolog of beta NHE (ßNHE-like). It consisted of 2925 bp, encoding for a polypeptide of 974 amino acids and 107.1 kDa, and was expressed predominantly in the inner mantle. There, ßNHE-like was localized in the apical membrane of the seawater-facing epithelium by immunofluorescence microscopy. After exposure to light for 12 h, the seawater-facing epithelium of the inner mantle displayed consistently stronger immunostaining than that of the control exposed to 12 h of darkness. Western blotting confirmed that light exposure significantly enhanced the protein abundance of ßNHE-like in the inner mantle. These results denote that some of the excess H+ generated during light-enhanced shell formation can be excreted through the light-dependent ßNHE-like of the seawater-facing epithelium to minimize the impact on the whole-body pH. Importantly, the excreted H+ could dehydrate exogenous HCO3-, and facilitate the absorption of inorganic carbon through the seawater-facing epithelium dedicated for light-enhanced shell formation due to its close proximity with the shell-facing epithelium. NUCLEOTIDE SYMBOL COMBINATIONS: Pairs: R = A/G; W = A/T; Y = C/T. Triples: D = A/G/T.


Assuntos
Bivalves/genética , Trocadores de Sódio-Hidrogênio/genética , Simbiose/genética , Sequência de Aminoácidos/genética , Animais , Bivalves/fisiologia , Clonagem Molecular , Epitélio/química , Epitélio/metabolismo , Luz , Fases de Leitura Aberta/genética , Fotossíntese/genética , RNA Mensageiro/genética , Água do Mar/microbiologia , Trocadores de Sódio-Hidrogênio/química
15.
Biomed Res Int ; 2019: 1031839, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30729118

RESUMO

Plant NHX antiporters are responsible for monovalent cation/H+ exchange across cellular membranes and play therefore a critical role for cellular pH regulation, Na+ and K+ homeostasis, and salt tolerance. Six members of grapevine NHX family (VvNHX1-6) have been structurally characterized. Phylogenetic analysis revealed their organization in two groups: VvNHX1-5 belonging to group I (vacuolar) and VvNHX6 belonging to group II (endosomal). Conserved domain analysis of these VvNHXs indicates the presence of different kinds of domains. Out of these, two domains function as monovalent cation-proton antiporters and one as the aspartate-alanine exchange; the remaining are not yet with defined function. Overall, VvNHXs proteins are typically made of 11-13 putative transmembrane regions at their N-terminus which contain the consensus amiloride-binding domain in the 3rd TM domain and a cation-binding site in between the 5th and 6th TM domain, followed by a hydrophilic C-terminus that is the target of several and diverse regulatory posttranslational modifications. Using a combination of primary structure analysis, secondary structure alignments, and the tertiary structural models, the VvNHXs revealed mainly 18 α helices although without ß sheets. Homology modeling of the 3D structure showed that VvNHX antiporters are similar to the bacterial sodium proton antiporters MjNhaP1 (Methanocaldococcus jannaschii) and PaNhaP (Pyrococcus abyssi).


Assuntos
Antiporters/química , Homeostase/genética , Filogenia , Trocadores de Sódio-Hidrogênio/química , Antiporters/genética , Cátions Monovalentes/química , Concentração de Íons de Hidrogênio , Methanocaldococcus , Potássio/metabolismo , Domínios Proteicos , Pyrococcus abyssi , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Vitis/química , Vitis/genética
16.
J Biol Chem ; 294(12): 4546-4571, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30696771

RESUMO

Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.


Assuntos
Hormônio Paratireóideo/fisiologia , Fosfatos/metabolismo , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/fisiologia , Sequência de Aminoácidos , Cristalografia por Raios X , Furanos/farmacologia , Células HEK293 , Humanos , Transporte de Íons/fisiologia , Lipídeos/farmacologia , Fosfoproteínas/química , Fosforilação , Conformação Proteica , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismo , Serina/metabolismo , Trocadores de Sódio-Hidrogênio/química
17.
Neurobiol Dis ; 121: 187-204, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30296617

RESUMO

Loss-of-function mutations in the recycling endosomal (Na+,K+)/H+ exchanger gene SLC9A6/NHE6 result in overacidification and dysfunction of endosomal-lysosomal compartments, and cause a neurodevelopmental and degenerative form of X-linked intellectual disability called Christianson Syndrome (CS). However, knowledge of the disease heterogeneity of CS is limited. Here, we describe the clinical features and underlying molecular and cellular mechanisms associated with a CS patient carrying a de novo missense variant (p.Gly218Arg; G218R) of a conserved residue in its ion translocation domain that results in a potential gain-of-function. The patient manifested several core symptoms typical of CS, including pronounced cognitive impairment, mutism, epilepsy, ataxia and microcephaly; however, deterioration of motor function often observed after the first decade of life in CS children with total loss of SLC9A6/NHE6 function was not evident. In transfected non-neuronal cells, complex glycosylation and half-life of the G218R were significantly decreased compared to the wild-type transporter. This correlated with elevated ubiquitination and partial proteasomal-mediated proteolysis of G218R. However, a major fraction was delivered to the plasma membrane and endocytic pathways. Compared to wild-type, G218R-containing endosomes were atypically alkaline and showed impaired uptake of recycling endosomal cargo. Moreover, instead of accumulating in recycling endosomes, G218R was redirected to multivesicular bodies/late endosomes and ejected extracellularly in exosomes rather than progressing to lysosomes for degradation. Attenuated acidification and trafficking of G218R-containing endosomes were also observed in transfected hippocampal neurons, and correlated with diminished dendritic branching and density of mature mushroom-shaped spines and increased appearance of filopodia-like protrusions. Collectively, these findings expand our understanding of the genetic diversity of CS and further elucidate a critical role for SLC9A6/NHE6 in fine-tuning recycling endosomal pH and cargo trafficking, processes crucial for the maintenance of neuronal polarity and mature synaptic structures.


Assuntos
Ataxia/genética , Ataxia/patologia , Endossomos/metabolismo , Epilepsia/genética , Epilepsia/patologia , Mutação com Ganho de Função , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Microcefalia/genética , Microcefalia/patologia , Neurônios/patologia , Transtornos da Motilidade Ocular/genética , Transtornos da Motilidade Ocular/patologia , Trocadores de Sódio-Hidrogênio/genética , Adulto , Animais , Atrofia , Cricetulus , Dendritos/patologia , Vesículas Extracelulares/metabolismo , Células HeLa , Hipocampo/patologia , Humanos , Masculino , Mutação de Sentido Incorreto , Trocadores de Sódio-Hidrogênio/química , Adulto Jovem
18.
Biochim Biophys Acta Biomembr ; 1861(1): 191-200, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071192

RESUMO

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pHi. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431-443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.


Assuntos
Trocador 1 de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/química , Alanina/química , Animais , Membrana Celular/química , Citoplasma/química , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Espectroscopia de Ressonância Magnética , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Domínios Proteicos , Isoformas de Proteínas/química , Estrutura Secundária de Proteína , Prótons
19.
Biochem Cell Biol ; 97(3): 307-314, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30011386

RESUMO

The Vc-NhaP2 antiporter from Vibrio cholerae exchanges H+ for K+ or Na+ but not for the smaller Li+. The molecular basis of this unusual selectivity remains unknown. Phyre2 and Rosetta software were used to generate a structural model of the Vc-NhaP2. The obtained model suggested that a cluster of residues from different transmembrane segments (TMSs) forms a putative cation-binding pocket in the middle of the membrane: D133 and T132 from TMS V together with D162 and E157 of TMS VI. The model also suggested that L257, G258, and N259 from TMS IX together with T276, D273, Q280, and Y251 from TMS X as well as L289 and L342 from TMS XII form a transmembrane pathway for translocated ions with a built-in filter determining cation selectivity. Alanine-scanning mutagenesis of the identified residues verified the model by showing that structural modifications of the pathway resulted in altered cation selectivity and transport activity. In particular, L257A, G258A, Q280A, and Y251A variants gained Li+/H+ antiport capacity that was absent in the nonmutated antiporter. T276A, D273A, and L289A variants exclusively exchanged K+ for H+, while a L342A variant mediated Na+/H+ exchange only, thus maintaining strict alkali cation selectivity.


Assuntos
Proteínas de Bactérias/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cátions/química , Cátions/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Vibrio cholerae/química
20.
Cell ; 173(7): 1636-1649.e16, 2018 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-29754813

RESUMO

Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing complex I are homologous respiratory complexes with a common ancestor, but a structural basis for their evolutionary relationship is lacking. Here, we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophile Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of complex I while its membrane-embedded ion-translocation module can be divided into a H+- and a Na+-translocating unit. The H+-translocating unit is rotated 180° in-membrane with respect to its counterpart in complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na+-translocating unit, absent in complex I, resembles that found in the Mrp H+/Na+ antiporter and enables hydrogen gas evolution by MBH to establish a Na+ gradient for ATP synthesis near 100°C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.


Assuntos
Proteínas Arqueais/metabolismo , Hidrogenase/metabolismo , Pyrococcus furiosus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Evolução Molecular , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Sódio/química , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
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