RESUMO
The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.
Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TranscriptomaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17â¯kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17â¯kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (Pâ¯=â¯.0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.
Assuntos
Encéfalo/metabolismo , Doença de Parkinson/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Loci Gênicos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genéticaRESUMO
Pendrin (SLC26A4), a Cl-/anion exchanger, is expressed at high levels in kidney, thyroid, and inner ear epithelia, where it has an essential role in bicarbonate secretion/chloride reabsorption, iodide accumulation, and endolymph ion balance, respectively. Pendrin is expressed at lower levels in other tissues, such as airways and esophageal epithelia, where it is transcriptionally regulated by the inflammatory cytokines interleukin (IL)-4 and IL-13 through a signal transducer and activator of transcription 6 (STAT6)-mediated pathway. In the airway epithelium, increased pendrin expression during inflammatory diseases leads to imbalances in airway surface liquid thickness and mucin release, while, in the esophageal epithelium, dysregulated pendrin expression is supposed to impact the intracellular pH regulation system. In this review, we discuss some of the recent findings on interleukin-mediated transcriptional regulation of pendrin and how this dysregulation impacts airway and esophagus epithelial homeostasis during inflammatory diseases.
Assuntos
Mucosa Esofágica/metabolismo , Regulação da Expressão Gênica , Interleucinas/metabolismo , Mucosa Respiratória/metabolismo , Transportadores de Sulfato/genética , Transcrição Gênica , Animais , Biomarcadores , Mucosa Esofágica/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/metabolismo , Muco/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Transportadores de Sulfato/metabolismo , Transportadores de Sulfato/uso terapêuticoRESUMO
BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Animais , Cloretos/metabolismo , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos , Células NIH 3T3 , Ácido Niflúmico/química , Ácido Niflúmico/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype.
Assuntos
Perda Auditiva Neurossensorial/patologia , Proteínas de Membrana Transportadoras/genética , Aqueduto Vestibular/anormalidades , População Branca/genética , Adolescente , Adulto , Idoso , Alelos , Áustria , Criança , Conexina 26/genética , Retículo Endoplasmático/metabolismo , Feminino , Genótipo , Células HEK293 , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Transportadores de Sulfato , Aqueduto Vestibular/patologia , Adulto JovemRESUMO
Interleukin-13 (IL13) is a major player in the development of airway hyperresponsiveness in several respiratory disorders. Emerging data suggest that an increased expression of pendrin in airway epithelia is associated with elevated airway hyperreactivity in asthma. Here, we investigate the effect of IL13 on pendrin localization and function using bronchiolar NCI-H292 cells. The data obtained revealed that IL13 increases the cell surface expression of pendrin. This effect was paralleled by a significant increase in the intracellular pH, possibly via indirect stimulation of NHE. IL13 effect on pendrin localization and intracellular pH was reversed by theophylline, a bronchodilator compound used to treat asthma. IL13 upregulated RhoA activity, a crucial protein controlling actin dynamics, via G-alpha-13. Specifically, IL13 stabilized actin cytoskeleton and promoted co-localization and a direct molecular interaction between pendrin and F-actin in the plasma membrane region. These effects were reversed following exposure of cells to theophylline. Selective inhibition of Rho kinase, a downstream effector of Rho, reduced the IL13-dependent cell surface expression of pendrin. Together, these data indicate that IL13 increases pendrin abundance to the cell surface via Rho/actin signaling, an effect reversed by theophylline.
Assuntos
Actinas/metabolismo , Brônquios/metabolismo , Interleucina-13/metabolismo , Transdução de Sinais/fisiologia , Transportadores de Sulfato/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Asma/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
BACKGROUND/AIM: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (â¼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. METHODS: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. RESULTS: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. CONCLUSION: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.
Assuntos
Citocromo P-450 CYP2D6/genética , Variações do Número de Cópias de DNA , Técnicas de Genotipagem/métodos , Alelos , Estudos de Coortes , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Discriminante , Frequência do Gene , Genótipo , Haplótipos , Humanos , Fenótipo , Análise de Sequência de DNARESUMO
Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. METHODS: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. RESULTS: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis), created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. CONCLUSIONS: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription.
Assuntos
Brônquios/metabolismo , Ilhas de CpG , Metilação de DNA , Células Epiteliais/metabolismo , Interleucina-4/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Elementos de Resposta , Brônquios/citologia , Linhagem Celular , Epigênese Genética , Células Epiteliais/citologia , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Transportadores de SulfatoRESUMO
BACKGROUND: TPA Induced Sequence 7 acts as a transcriptional co-regulator controlling the expression of genes involved in differentiation of various cell types, including skeletal myoblasts. We and others have shown that TIS7 regulates adult myogenesis through MyoD, one of the essential myogenic regulatory factors. RESULTS: Here, we present data identifying ICln as the specific, novel protein downstream of TIS7 controlling myogenesis. We show that TIS7/ICln epigenetically regulate myoD expression controlling protein methyl transferase activity. In particular, ICln regulates MyoD expression via its interaction with PRMT5 by an epigenetic modification that utilizes symmetrical di-methylation of histone H3 on arginine 8. We provide multiple evidences that TIS7 directly binds DNA, which is a functional feature necessary for its role in transcriptional regulation. CONCLUSION: We present here a molecular insight into TIS7-specific control of MyoD gene expression and thereby skeletal muscle differentiation.
Assuntos
Diferenciação Celular/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas de Membrana/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Immunoblotting , Proteínas de Membrana/genética , Camundongos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Proteína MyoD/genética , Proteína MyoD/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Ressonância de Plasmônio de Superfície , Transcrição Gênica/genéticaRESUMO
Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants.
RESUMO
BACKGROUND/AIMS: Pendrin is a Cl-/I-/HCO3- exchanger playing a fundamental role in controlling blood pressure and airway function, therefore representing an attractive target for the treatment of hypertensive states and respiratory distresses. A review of the literature regarding the ability of some compounds (namely several known inhibitors of ion transport) to block pendrin activity revealed discordant findings. These incongruous findings may be due, in part, to the concentration of compound and/or the nature of the model system used in the study. METHODS: Pendrin activity was evaluated by measuring pendrin-dependent iodide influx following overexpression of the transporter in a human kidney cell line, in the presence of selected test compounds or the respective vehicles. RESULTS: Pendrin activity was significantly hampered by 0.1 mM 5-nitro-2-[(3-phenylpropyl)amino]benzoic acid (NPPB), niflumic acid and tenidap, but was resistant to 0.1 mM 4, 4'-diisothiocyano-2, 2'-stilbene-disulfonic acid (DIDS), furosemide and probenecid. CONCLUSIONS: The results of the present study indicate that clinically effective non-steroidal anti-inflammatory drugs (niflumic acid and tenidap) directly inhibit pendrin activity.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/química , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/metabolismo , Western Blotting , Linhagem Celular , Furosemida/química , Furosemida/metabolismo , Células HEK293 , Humanos , Indóis/química , Indóis/metabolismo , Rim/citologia , Rim/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Ácido Niflúmico/química , Ácido Niflúmico/metabolismo , Nitrobenzoatos/química , Nitrobenzoatos/metabolismo , Oxindóis , Plasmídeos/genética , Plasmídeos/metabolismo , Probenecid/química , Probenecid/metabolismo , Ligação Proteica , Espectrometria de Fluorescência , Transportadores de SulfatoRESUMO
The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.
Assuntos
Bicamadas Lipídicas , Vírus Sinciciais Respiratórios/metabolismo , Proteínas Virais/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação , Chlorocebus aethiops , Clonagem Molecular , Genes Virais , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Pironina/análogos & derivados , Pironina/farmacologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Células Vero , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
PGC-1α has been implicated in the pathogenesis of neurodegenerative disorders. Several single-nucleotide polymorphisms (SNPs) located in two separate haplotype blocks of PPARGC1A have shown associations with Huntington's disease (HD) and Parkinson's disease, but causative SNPs have not been identified. One SNP (rs7665116) was located in a highly conserved 233 bp region of intron 2. To determine whether rs7665116 is located in an alternative exon, we performed 5'-RLM-RACE from exon 3 and discovered multiple new transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2, but did not contain the conserved region harboring rs7665116. Using real-time polymerase chain reaction, RNase protection assays and northern blotting, we show that the majority of these transcripts are brain specific and are at least equally or perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons encode full-length brain-specific PGC-1α proteins that differ only at their N-termini (NTs) from PGC-1α, encoded by the reference sequence. Additional truncated isoforms containing these NTs that are similar to NT-PGC-1α exist. Other transcripts may encode potential dominant negative forms, as they are predicted to lack the second LXXLL motif that serves as an interaction site for several nuclear receptors. Furthermore, we show that the new promoter is active in neuronal cell lines and describe haplotypes encompassing this region that are associated with HD age of onset. The discovery of such a large PPARGC1A genomic locus and multiple isoforms in brain warrants further functional studies and may provide new tissue-specific targets for treating neurodegenerative diseases.
Assuntos
Encéfalo/metabolismo , Genoma Humano , Proteínas de Choque Térmico/genética , Doença de Huntington/epidemiologia , Doença de Huntington/genética , Fatores de Transcrição/genética , Idade de Início , Éxons , Genômica , Proteínas de Choque Térmico/metabolismo , Humanos , Íntrons , Dados de Sequência Molecular , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Hemocromatose/prevenção & controle , Sobrecarga de Ferro/tratamento farmacológico , Nifedipino/farmacologia , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Células COS , Bloqueadores dos Canais de Cálcio/uso terapêutico , Chlorocebus aethiops , Eletrofisiologia , Humanos , Immunoblotting , Ferro/metabolismo , Ferro/urina , Fígado/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Nifedipino/uso terapêutico , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Ion transporters are the molecular basis for ion homeostasis of the cell and the whole organism. The anion exchanger pendrin is only one of a number of examples where a complete or partial loss of function and/or deregulation of expression of ion transporters may lead or contribute to pathological conditions in humans. A complete understanding of the function of ion transporters in health and disease may pave the way for the identification of new and focused therapeutic approaches. Exchange of knowledge and connectivity between the experts in the feld of transport physiology is essential in facing these challenging tasks. The Lake Cumberland Biological Transport Group and the Pendrin Consortium are examples of scientific forums where investigators combine their efforts towards a better understanding of molecular pathophysiology of ion transport. This issue discusses the versatility of ion transporters involved in the regulation of cellular volume and other functions, such as the solute carrier (SLC) 12A gene family members SLC12A4-7, encoding the Na(+)-independent cation-chloride cotransporters commonly known as the K(+)-Cl(-) cotransporters KCC1-4, and the betaine/γ-aminobutyric acid transport system (BGT1, SLC6A12), just to name a few. The issue further addresses the pathophysiology of intestinal and respiratory epithelia and related therapeutic tools and techniques to investigate interactions between proteins and proteins and small compounds. Finally, the current knowledge and new findings on the expression, regulation and function of pendrin (SLC26A4) in the inner ear, kidney, airways and blood platelets are presented.
Assuntos
Transporte Biológico/genética , Transporte de Íons/genética , Proteínas de Membrana Transportadoras/metabolismo , Antiporters/genética , Homeostase , Humanos , Proteínas de Membrana Transportadoras/genética , Transportadores de SulfatoRESUMO
One of the most pressing challenges in the post genomic era is the identification and characterization of protein-protein interactions (PPIs), as these are essential in understanding the cellular physiology of health and disease. Experimental techniques suitable for characterizing PPIs (X-ray crystallography or nuclear magnetic resonance spectroscopy, among others) are usually laborious, time-consuming and often difficult to apply to membrane proteins, and therefore require accurate prediction of the candidate interacting partners. High-throughput experimental methods (yeast two-hybrid and affinity purification) succumb to the same shortcomings, and can also lead to high rates of false positive and negative results. Therefore, reliable tools for predicting PPIs are needed. The use of the operon structure in the eukaryote Caenorhabditis elegans genome is a valuable, though underserved, tool for identifying physically or functionally interacting proteins. Based on the concept that genes organized in the same operon may encode physically or functionally related proteins, this algorithm is easy to be applied and, importantly, gives a limited number of candidate partners of a given protein, allowing for focused experimental verification. Moreover, this approach can be successfully used to predict PPIs in the human system, including those of membrane proteins.
Assuntos
Proteínas de Membrana/metabolismo , Óperon/genética , Mapas de Interação de Proteínas/genética , Animais , Caenorhabditis elegans/genética , Biologia Computacional , Genoma , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: With the rise of antibiotic resistance, new alternatives are being sought to effectively modulate the characteristics of gut microbiota to obtain pathogen resistance without the use of antibiotics. In the past, an oligosaccharide derivative of carrots, galursan HF 7K (GHF7K), has been used clinically in Austria and recently in the fowl-industry to promote health. This study examined the potential role of GHF7K as a prebiotic to alter the gut microbiota in mice. METHODS: Mice were fed either a control diet (CT) or a diet containing 2% GHF7K in the water and chow for 2 weeks, and weight, food and water consumption, gut microbiota and ion composition of the intestinal fluid were examined. RESULTS: Dietary supplement of GHF7K did not alter mouse weight or daily food consumption. Additionally, no changes were observed in the total number of luminal or mucosa-associated bacteria populations in GHF7K-fed mice. GHF7K supplementation significantly altered the composition of luminal, and to a less extent, mucosa-associated bacterial populations at the level of the phyla, with region-specific differences. Similar to antibiotic use, Proteobacteria number was increased in the ileum and colon of GHF7K-fed mice, with no changes in the number of beneficial Lactobacillus and Bifidobacterium genera of phylum Firmicutes. Corresponding with the altered gut microbiota, changes in the ion composition of the intestinal fluid were observed. An increased Cl(-) concentration was observed in the duodenum and jejunum, while the Na(+) concentration was increased in the cecum of GHF7K-fed mice. Decreases were observed in the K(+) concentration in the cecum and distal colon. CONCLUSIONS: Dietary supplement of GHF7K is capable of altering the gut microbiota, which correlates to changes in the intestinal environment. These data suggest that GHF7K dietary supplement can purposefully be used to alter the gut microbiota, and thus could potentially represent an alternative approach to prophylactic antibiotic use.
Assuntos
Intestinos/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Ácido Pipemídico/administração & dosagem , Prebióticos , Animais , Antibacterianos/administração & dosagem , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Peso Corporal , Suplementos Nutricionais , Intestinos/microbiologia , CamundongosRESUMO
BACKGROUND/AIMS: We investigated if mixtures of the phosphatidylcholine (PC) lipids 1,2-dilauroyl-sn-glycero-3-phosphocholine (C12:0 PC; DLPC) and 1,2-diarachidoyl-sn-glycero-3-phosphocholine (C20:0 PC; DAPC), which differ by eight methylene groups in acyl chain length, lead to the spontaneous formation of distinct lipid rafts and asymmetric bilayers. METHODS: The experiments were performed using Atomic Force Microscopy (AFM). RESULTS: We show that DLPC and DAPC mixed at a molar ratio of 1:1 lead to the formation of single, double and triple bilayers with peaks at 6.14 ± 0.11, 13.27 ± 0.17 and 20.54 ± 0.46 nm, respectively (n=750). Within these formations discrete height steps of 0.92 nm can be resolved (n=422). CONCLUSION: The most frequently observed height steps value of 0.92 nm matches best with the calculated mean lipid hydrophobic thickness difference for asymmetric C12:0 PC and C20:0 PC lipid bilayers of 0.88 nm. This indicates the ability of DLPC and DAPC to form asymmetric lipid bilayers.
Assuntos
Bicamadas Lipídicas/química , Fosfatidilcolinas/química , Interações Hidrofóbicas e Hidrofílicas , Microscopia de Força AtômicaRESUMO
BACKGROUND: Human pendrin (SLC26A4, PDS) is an integral membrane protein acting as an electroneutral anion exchanger. Loss of function mutations in pendrin protein cause Pendred syndrome, a disorder characterized by sensorineural deafness and a partial iodide organification defect that may lead to thyroid goiter. Additionally, pendrin up-regulation could play a role in the pathogenesis of several diseases including bronchial asthma and chronic obstructive pulmonary disease (COPD). Therefore, monitoring the plasma membrane abundance and trafficking of pendrin in the context of a living cell is crucially important. METHODS: Trafficking of pendrin to the plasma membrane was monitored by fluorescence resonance energy transfer (FRET), a physical phenomenon occurring between two fluorophores (the FRET donor and acceptor) located in close spatial proximity. Because the efficiency of the energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, FRET is extremely sensitive to small changes in distance between the donor and acceptor and is therefore a powerful tool to determine protein-protein interactions. RESULTS: FRET studies revealed that forskolin-induced cAMP production is associated with a significant increase of pendrin expression at plasma membrane, which is paralleled by a decrease in intracellular pH. Pendrin transposition to the membrane is accompanied with a partial depolymerization of actin cytoskeleton via Rho-GTPase inhibition. CONCLUSION: Trafficking to the plasma membrane is critical in the regulation of pendrin activity. Therefore, reliable tools for monitoring and quantifying this phenomenon are highly desirable.
Assuntos
Brônquios/efeitos dos fármacos , Colforsina/farmacologia , Bócio Nodular/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Membrana Transportadoras/metabolismo , Actinas/metabolismo , Brônquios/citologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Transferência Ressonante de Energia de Fluorescência , Bócio Nodular/patologia , Perda Auditiva Neurossensorial/patologia , Humanos , Proteínas de Membrana Transportadoras/genética , Mutação , Transporte Proteico/efeitos dos fármacos , Transportadores de SulfatoRESUMO
BACKGROUND: The anion exchanger pendrin (SLC26A4) is required for proper development of the inner ear, and contributes to iodide organification in thyroid glands as well as anion transport in various epithelia, such as airways and renal tubules. SLC26A4 deficiency leads to Pendred syndrome, which is characterized by hearing loss with enlarged vestibular aqueducts and variable hypothyroidism and goiter. Pendrin expression in kidney, heart, lung and thyroid is up-regulated by the mineralocorticoid deoxycorticosterone (DOCA). Platelets express anion exchangers but virtually nothing is known about the molecular identity and regulation of those carriers. Other carriers such as the Na(+)/H(+) exchanger are regulated by the mineralocorticoid-sensitive serum and glucocorticoid inducible kinase SGK1. METHODS: The present study utilized i) quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify the transcript levels of Slc26a4 as compared to Gapdh and ii) western blotting to assess Slc26a4 protein abundance in murine platelets from gene-targeted mice lacking Sgk1 (sgk1(-/-)) and respective wild type animals (sgk1(+/+)) treated without or with a subcutaneous injection of 2.5 mg DOCA for 3 h, or in sgk1(+/+) platelets with or without in vitro treatment for 1 h with 10 µg/ml DOCA. RESULTS: Slc26a4 was expressed in platelets, and in vitro DOCA treatment increased Slc26a4 mRNA levels in platelets isolated from sgk1(+/+) mice. Moreover, in vivo DOCA treatment significantly up-regulated Slc26a4 mRNA levels in platelets isolated from sgk1(+/+) but not sgk1(-/-) mice. An increase in Sgk1 mRNA levels paralleled that of Slc26a4 mRNA levels in platelets of sgk1(+/+) mice. In addition, DOCA treatment further increased Slc26a4 protein abundance in platelets isolated from sgk1(+/+) mice. CONCLUSIONS: Pendrin is expressed in platelets and is presumably regulated by SGK1 and mineralocorticoids.