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1.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672907

RESUMO

HKT channels are a plant protein family involved in sodium (Na+) and potassium (K+) uptake and Na+-K+ homeostasis. Some HKTs underlie salt tolerance responses in plants, while others provide a mechanism to cope with short-term K+ shortage by allowing increased Na+ uptake under K+ starvation conditions. HKT channels present a functionally versatile family divided into two classes, mainly based on a sequence polymorphism found in the sequences underlying the selectivity filter of the first pore loop. Physiologically, most class I members function as sodium uniporters, and class II members as Na+/K+ symporters. Nevertheless, even within these two classes, there is a high functional diversity that, to date, cannot be explained at the molecular level. The high complexity is also reflected at the regulatory level. HKT expression is modulated at the level of transcription, translation, and functionality of the protein. Here, we summarize and discuss the structure and conservation of the HKT channel family from algae to angiosperms. We also outline the latest findings on gene expression and the regulation of HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Simportadores/metabolismo , Proteínas de Transporte de Cátions/classificação , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas , Transporte de Íons , Magnoliopsida/genética , Magnoliopsida/metabolismo , Microalgas/genética , Microalgas/metabolismo , Filogenia , Proteínas de Plantas/genética , Simportadores/classificação , Simportadores/genética
2.
Neurosci Lett ; 750: 135810, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33705929

RESUMO

Although clinical efficacy of waggle needling has been confirmed, therapeutic mechanisms still remain poorly understood. Reduction of GABA was involved in the etiology of spasticity. Recently, accumulated evidences suggest that the inhibitory effect of GABA is determined by low intracellular chloride concentration, which is predominantly mediated by KCC2. This study was designed to investigate whether KCC2-GABAA pathway was involved in the mechanism underlying acupuncture intervention in rats with middle cerebral artery occlusion (MCAO). Three days after modeling, the rats received waggle needling, routine needling and placebo needling for 7 consecutive days. After treatment, the muscle spasticity, motor function and infarct volumes were tested. KCC2 and GABAAγ2 levels were detected via western blotting, RT-PCR and immunofluorescence. KCC2 antagonist and agonist were administered after the last intervention. We found that acupuncture, particularly waggle needling, could remarkably alleviate muscle spasticity, reverse motor deficits and reduce cerebral infraction in MCAO rats, possibly due to its effects on up-regulating expressions of KCC2 and GABAAγ2 in the cortical infarct regions. However, the effects were blocked by KCC2 antagonist. In summary, this study suggests that improvements in muscle spasticity and motor function induced by waggle needling correlates with the activation of KCC2-GABAA pathway.


Assuntos
Terapia por Acupuntura/métodos , Infarto da Artéria Cerebral Média/terapia , Músculo Esquelético/metabolismo , Reabilitação do Acidente Vascular Cerebral/métodos , Pontos de Acupuntura , Animais , Infarto da Artéria Cerebral Média/reabilitação , Masculino , Espasticidade Muscular/terapia , Músculo Esquelético/fisiopatologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Simportadores/genética , Simportadores/metabolismo , Regulação para Cima
3.
Am J Physiol Cell Physiol ; 320(5): C722-C730, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33596149

RESUMO

Hereditary motor sensory neuropathy (HMSN/ACC) with agenesis of the corpus callosum (ACC) has been documented in the French-derived populations of Charlevoix and Saguenay/Lac St. Jean in Quebec, Canada, as well as a few sporadic families throughout the world. HMSN/ACC occurs because of loss-of-function mutations in the potassium-chloride cotransporter 3 (KCC3). In HMSN/ACC, motor deficits occur early in infancy with rapid and continual deterioration of motor and sensory fibers into juvenile and adulthood. Genetic work in mice has demonstrated that the disease is caused by loss of KCC3 function in neurons and particularly parvalbumin (PV)-expressing neurons. Currently, there are no treatments or cures for HMSN/ACC other than pain management. As genetic counseling in Quebec has increased as a preventative strategy, most individuals with HSMN/ACC are now adults. The onset of the disease is unknown. In particular, it is unknown if the disease starts early during development and whether it can be reversed by restoring KCC3 function. In this study, we used two separate mouse models that when combined to the PV-CreERT2 tamoxifen-inducible system allowed us to 1) disrupt KCC3 expression in adulthood or juvenile periods; and 2) reintroduce KCC3 expression in mice that first develop with a nonfunctional cotransporter. We show that disrupting or reintroducing KCC3 in the adult mouse has no effect on locomotor behavior, indicating that expression of KCC3 is critical during embryonic development and/or the perinatal period and that once the disease has started, reexpressing a functional cotransporter fails to change the course of HMSN/ACC.


Assuntos
Agenesia do Corpo Caloso/terapia , Comportamento Animal , Gânglios Espinais/metabolismo , Terapia Genética , Atividade Motora , Doenças do Sistema Nervoso Periférico/terapia , Simportadores/metabolismo , Fatores Etários , Agenesia do Corpo Caloso/genética , Agenesia do Corpo Caloso/metabolismo , Agenesia do Corpo Caloso/fisiopatologia , Animais , Modelos Animais de Doenças , Feminino , Gânglios Espinais/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento , Predisposição Genética para Doença , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Parvalbuminas/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologia , Fenótipo , Equilíbrio Postural , Teste de Desempenho do Rota-Rod , Simportadores/genética
4.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557247

RESUMO

The effect of a cellular prion protein (PrPc) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrPc in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp-/- mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na+/K+ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp-/- vs. WT control group, without a substantial change in the Na+/K+ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp-/- vs. WT mice. The present study demonstrates that a lack of PrPc leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp-/- vs. WT mice. Our findings provide evidence that PrPc might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.


Assuntos
Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Ácido Láctico/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Priônicas/fisiologia , RNA Mensageiro/metabolismo , Simportadores/metabolismo , Animais , Transporte Biológico , Glicólise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos/genética , RNA Mensageiro/genética , Simportadores/genética
5.
Nature ; 591(7848): 157-161, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33597751

RESUMO

Citrate is best known as an intermediate in the tricarboxylic acid cycle of the cell. In addition to this essential role in energy metabolism, the tricarboxylate anion also acts as both a precursor and a regulator of fatty acid synthesis1-3. Thus, the rate of fatty acid synthesis correlates directly with the cytosolic concentration of citrate4,5. Liver cells import citrate through the sodium-dependent citrate transporter NaCT (encoded by SLC13A5) and, as a consequence, this protein is a potential target for anti-obesity drugs. Here, to understand the structural basis of its inhibition mechanism, we determined cryo-electron microscopy structures of human NaCT in complexes with citrate or a small-molecule inhibitor. These structures reveal how the inhibitor-which binds to the same site as citrate-arrests the transport cycle of NaCT. The NaCT-inhibitor structure also explains why the compound selectively inhibits NaCT over two homologous human dicarboxylate transporters, and suggests ways to further improve the affinity and selectivity. Finally, the NaCT structures provide a framework for understanding how various mutations abolish the transport activity of NaCT in the brain and thereby cause epilepsy associated with mutations in SLC13A5 in newborns (which is known as SLC13A5-epilepsy)6-8.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Ácido Cítrico/metabolismo , Microscopia Crioeletrônica , Malatos/farmacologia , Fenilbutiratos/farmacologia , Simportadores/antagonistas & inibidores , Simportadores/química , Sítios de Ligação , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Ácido Cítrico/química , Transportadores de Ácidos Dicarboxílicos/química , Transportadores de Ácidos Dicarboxílicos/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Humanos , Malatos/química , Modelos Moleculares , Mutação , Fenilbutiratos/química , Multimerização Proteica , Sódio/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/genética , Simportadores/genética , Simportadores/ultraestrutura
6.
Int J Mol Sci ; 22(3)2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33513812

RESUMO

Stroke is one of the major culprits responsible for morbidity and mortality worldwide, and the currently available pharmacological strategies to combat this global disease are scanty. Cation-chloride cotransporters (CCCs) are expressed in several tissues (including neurons) and extensively contribute to the maintenance of numerous physiological functions including chloride homeostasis. Previous studies have implicated two CCCs, the Na+-K+-Cl- and K+-Cl- cotransporters (NKCCs and KCCs) in stroke episodes along with their upstream regulators, the with-no-lysine kinase (WNKs) family and STE20/SPS1-related proline/alanine rich kinase (SPAK) or oxidative stress response kinase (OSR1) via a signaling pathway. As the WNK-SPAK/OSR1 pathway reciprocally regulates NKCC and KCC, a growing body of evidence implicates over-activation and altered expression of NKCC1 in stroke pathology whilst stimulation of KCC3 during and even after a stroke event is neuroprotective. Both inhibition of NKCC1 and activation of KCC3 exert neuroprotection through reduction in intracellular chloride levels and thus could be a novel therapeutic strategy. Hence, this review summarizes the current understanding of functional regulations of the CCCs implicated in stroke with particular focus on NKCC1, KCC3, and WNK-SPAK/OSR1 signaling and discusses the current and potential pharmacological treatments for stroke.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Acidente Vascular Cerebral/metabolismo , Simportadores/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Homeostase , Humanos , Neurônios/metabolismo , Neurônios/patologia , Fosforilação , Transdução de Sinais , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Simportadores de Cloreto de Sódio-Potássio/genética , Acidente Vascular Cerebral/fisiopatologia , Simportadores/genética
7.
Viruses ; 13(1)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445753

RESUMO

An estimated two billion people worldwide have been infected with hepatitis B virus (HBV). Despite the high infectivity of HBV in vivo, a lack of easily infectable in vitro culture systems hinders studies of HBV. Overexpression of the sodium taurocholate co-transporting polypeptide (NTCP) bile acid transporter in hepatoma cells improved infection efficiency. We report here a hepatoma cell culture system that does not require dimethyl sulfoxide (DMSO) for HBV infection. We overexpressed NTCP in Huh7.5 cells and allowed these cells to differentiate in a medium supplemented with human serum (HS) instead of fetal bovine serum (FBS). We show that human serum culture enhanced HBV infection in Huh7.5-NTCP cells, e.g., in HS cultures, HBV pgRNA levels were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. Human serum culture increased levels of hepatocyte differentiation markers, such as albumin secretion, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by culture in human serum may contribute to viral entry. Our study demonstrates an in vitro HBV infection of Huh7.5-NTCP cells without the use of potentially toxic DMSO.


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Replicação Viral , Biomarcadores , Linhagem Celular , Células Cultivadas , Dimetil Sulfóxido/farmacologia , Expressão Gênica , Vetores Genéticos/genética , Vírus da Hepatite B/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
8.
J Mol Biol ; 433(4): 166764, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33359100

RESUMO

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.


Assuntos
Simulação de Dinâmica Molecular , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Conformação Proteica , Simportadores/química , Transporte Biológico , Ativação do Canal Iônico , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Micelas , Mutação , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Sódio/química , Sódio/metabolismo , Análise Espectral , Relação Estrutura-Atividade , Simportadores/genética , Simportadores/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(36): 22544-22551, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32826330

RESUMO

Obesity is a major health problem worldwide, given its growing incidence and its association with a variety of comorbidities. Weight gain results from an increase in energy intake without a concomitant increase in energy expenditure. To combat the obesity epidemic, many studies have focused on the pathways underlying satiety and hunger signaling, while other studies have concentrated on the mechanisms involved in energy expenditure, most notably adaptive thermogenesis. Hypothyroidism in humans is typically associated with a decreased basal metabolic rate, lower energy expenditure, and weight gain. However, hypothyroid mouse models have been reported to have a leaner phenotype than euthyroid controls. To elucidate the mechanism underlying this phenomenon, we used a drug-free mouse model of hypothyroidism: mice lacking the sodium/iodide symporter (NIS), the plasma membrane protein that mediates active iodide uptake in the thyroid. In addition to being leaner than euthyroid mice, owing in part to reduced food intake, these hypothyroid mice show signs of compensatory up-regulation of the skeletal-muscle adaptive thermogenic marker sarcolipin, with an associated increase in fatty acid oxidation (FAO). Neither catecholamines nor thyroid-stimulating hormone (TSH) are responsible for sarcolipin expression or FAO stimulation; rather, thyroid hormones are likely to negatively regulate both processes in skeletal muscle. Our findings indicate that hypothyroidism in mice results in a variety of metabolic changes, which collectively lead to a leaner phenotype. A deeper understanding of these changes may make it possible to develop new strategies against obesity.


Assuntos
Hipotireoidismo/metabolismo , Músculo Esquelético/metabolismo , Termogênese/fisiologia , Animais , Modelos Animais de Doenças , Ingestão de Alimentos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Musculares/metabolismo , Fenótipo , Proteolipídeos/metabolismo , Simportadores/genética , Simportadores/metabolismo
10.
Arterioscler Thromb Vasc Biol ; 40(10): 2468-2480, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32787517

RESUMO

OBJECTIVE: The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells. CONCLUSIONS: SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.


Assuntos
Canais de Potássio KCNQ/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Simportadores/metabolismo , Vasoconstrição , Animais , Células CHO , Cricetulus , Canais de Potássio KCNQ/genética , Potenciais da Membrana , Artérias Mesentéricas/metabolismo , Ligação Proteica , Ratos , Artéria Renal/metabolismo , Transdução de Sinais , Simportadores/genética , Técnicas de Cultura de Tecidos
11.
Eur J Endocrinol ; 183(5): K1-K5, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32805706

RESUMO

Background: Among patients with congenital hypothyroidism, 35% have dyshormonogenesis (DH) with thyroid gland in situ with or without goiter. The majority of DH cases are due to mutations in genes involved in thyroid hormone production as TG, TPO, SLC5A5/NIS, SLC26A4/PDS, IYD/DEHAL1, DUOX2, and DUOXA2, and are usually inherited on an autosomal recessive basis. Most previously reported cases of fetal hypothyroidism and goiter were related to TG or TPO mutations and recently DUOXA2. Patient: In a male patient with antenatal goiter treated with intraamniotic levothyroxine injections, whose long-term follow-up is described in detail, two novel NIS mutations were detected. Mutations of NIS were located in exon 1 (c.52G>A, p.G18R) and exon 13 (c.1546C>T, p.R516X), each mutation was inherited from parents, who are healthy carriers. The p.G18R mutation affecting the first transmembrane domain of the protein can be responsible for deficient iodide uptake. However, the second is a nonsense mutation leading probably to mRNA degradation. In addition, the patient has undergone a thyroidectomy and we have studied the thyroid tissue. The thyroid histology showed heterogeneity with large follicles, epithelial hyperplasia and many areas of fibrosis. Immunohistochemistry with NIS specific antibody showed NIS staining at the basolateral plasma membrane of the thyrocytes. Conclusions: We report the first case of fetal goitrous hypothyroidism due to two novel NIS mutations with access to thyroid tissue of the patient, specific histology studies and long-term follow-up. This case expands our knowledge and provides further insights on molecular causes of fetal goiter in humans.


Assuntos
Hipotireoidismo Congênito/genética , Bócio/genética , Mutação , Simportadores/genética , Adolescente , Criança , Pré-Escolar , Hipotireoidismo Congênito/tratamento farmacológico , Bócio/tratamento farmacológico , Humanos , Lactente , Recém-Nascido , Masculino , Diagnóstico Pré-Natal , Tiroxina/uso terapêutico , Resultado do Tratamento , Adulto Jovem
12.
Proc Natl Acad Sci U S A ; 117(33): 20159-20170, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747553

RESUMO

Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6 -methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.


Assuntos
Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Vacinas Anticâncer/imunologia , Lactatos/metabolismo , Melanoma/metabolismo , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/fisiologia , Homólogo AlkB 5 da RNA Desmetilase/genética , Anticorpos , Citocinas/genética , Citocinas/metabolismo , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/terapia , Metiltransferases/genética , Metiltransferases/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Células Supressoras Mieloides/fisiologia , Sítios de Splice de RNA , Processamento de RNA , Simportadores/genética , Simportadores/metabolismo , Transcriptoma , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
13.
PLoS One ; 15(7): e0236113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32687511

RESUMO

Loss of function mutations in the gene encoding the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) lead to severe neurodevelopmental defects in humans associated with a specific thyroid hormone phenotype manifesting high serum 3,5,3'-triiodothyronine (T3) and low thyroxine (T4) levels. Patients present a paradoxical state of peripheral hyperthyroidism and brain hypothyroidism, this last one most likely arising from impaired thyroid hormone transport across the brain barriers. The administration of thyroid hormones by delivery pathways that bypass the brain barriers, such as the intranasal delivery route, offers the possibility to improve the neurological defects of MCT8-deficient patients. In this study, the thyroid hormones T4 and T3 were administrated intranasally in different mouse models of MCT8 deficiency. We have found that, under the present formulation, intranasal administration of thyroid hormones does not increase the content of thyroid hormones in the brain and further raises the peripheral thyroid hormone levels. Our data suggests intranasal delivery of thyroid hormones is not a suitable therapeutic strategy for MCT8 deficiency, although alternative formulations could be considered in the future to improve the nose-to-brain transport.


Assuntos
Transportadores de Ácidos Monocarboxílicos/deficiência , Simportadores/deficiência , Hormônios Tireóideos/administração & dosagem , Hormônios Tireóideos/farmacologia , Administração Intranasal , Animais , Encéfalo/citologia , Camundongos , Transportadores de Ácidos Monocarboxílicos/genética , Mutação , Transdução de Sinais/efeitos dos fármacos , Simportadores/genética , Hormônios Tireóideos/sangue
14.
Biomed Khim ; 66(3): 185-195, 2020 May.
Artigo em Russo | MEDLINE | ID: mdl-32588824

RESUMO

Inhibition of the apical sodium-dependent bile acid transporter (ASBT, also known as IBAT - ileal bile acid transporter, SLC10A2) leads to disruption of the enterohepatic circulation of bile acids and their excretion with fecal masses. This is accompanied by cholesterol utilization for synthesis of new bile acids. ASBT inhibitors are promising drugs for the treatment of such diseases as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes mellitus, necrotic enterocolitis, chronic constipation, atherosclerosis. To date the most known chemically synthesized inhibitors are: A3309, SHP626, A4250, 264W94, GSK2330672, SC-435. All of them are at different stages of clinical trials, which confirm the high efficacy and good tolerance of these inhibitors. Current trends in this field also include directed chemical synthesis of ASBT inhibitors, as well as their search among substances of plant origin.


Assuntos
Diabetes Mellitus Tipo 2 , Transportadores de Ânions Orgânicos Dependentes de Sódio , Simportadores , Ácidos e Sais Biliares , Ensaios Clínicos como Assunto , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/farmacologia , Simportadores/genética
15.
Am J Physiol Cell Physiol ; 319(2): C359-C370, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32520610

RESUMO

SLC4A11 is the only member of the SLC4 family that transports protons rather than bicarbonate. SLC4A11 is expressed in corneal endothelial cells, and its mutation causes corneal endothelial dystrophy, although the mechanism of pathogenesis is unknown. We previously demonstrated that the magnitude of the H+ conductance (Gm) mediated by SLC4A11 is increased by rises in intracellular as well as extracellular pH (pHi and pHe). To better understand this feature and whether it is altered in disease, we studied the pH dependence of wild-type and mutant mouse Slc4a11 expressed in Xenopus oocytes. Using voltage-clamp circuitry in conjunction with a H+-selective microelectrode and a microinjector loaded with NaHCO3, we caused incremental rises in oocyte pHi and measured the effect on Gm. We find that the rise of Gm has a steeper pHi dependence at pHe =8.50 than at pHe =7.50. Data gathered at pHe =8.50 can be fit to the Hill equation enabling the calculation of a pK value that reports pHi dependence. We find that mutation of lysine residues that are close to the first transmembrane span (TM1) causes an alkaline shift in pK. Furthermore, two corneal-dystrophy-causing mutations close to the extracellular end of TM1, E399K and T401K (E368K and T370K in mouse), cause an acidic shift in pK, while a third mutation in the fourth intracellular loop, R804H (R774H in mouse), causes an alkaline shift in pK. This is the first description of determinants of SLC4A11 pH dependence and the first indication that a shift in pH dependence could modify disease expressivity in some cases of corneal dystrophy.


Assuntos
Proteínas de Transporte de Ânions/genética , Transporte Biológico/genética , Distrofias Hereditárias da Córnea/genética , Lisina/genética , Simportadores/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Bicarbonatos/metabolismo , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Modelos Animais de Doenças , Epitélio Posterior/metabolismo , Epitélio Posterior/patologia , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons/genética , Lisina/metabolismo , Camundongos , Mutação/genética , Oócitos/metabolismo , Oócitos/patologia , Sódio , Xenopus/genética
16.
Lancet Diabetes Endocrinol ; 8(7): 594-605, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32559475

RESUMO

BACKGROUND: Disordered thyroid hormone transport, due to mutations in the SLC16A2 gene encoding monocarboxylate transporter 8 (MCT8), is characterised by intellectual and motor disability resulting from cerebral hypothyroidism and chronic peripheral thyrotoxicosis. We sought to systematically assess the phenotypic characteristics and natural history of patients with MCT8 deficiency. METHODS: We did an international, multicentre, cohort study, analysing retrospective data from Jan 1, 2003, to Dec 31, 2019, from patients with MCT8 deficiency followed up in 47 hospitals in 22 countries globally. The key inclusion criterion was genetically confirmed MCT8 deficiency. There were no exclusion criteria. Our primary objective was to analyse the overall survival of patients with MCT8 deficiency and document causes of death. We also compared survival between patients who did or did not attain full head control by age 1·5 years and between patients who were or were not underweight by age 1-3 years (defined as a bodyweight-for-age Z score <-2 SDs or <5th percentile according to WHO definition). Other objectives were to assess neurocognitive function and outcomes, and clinical parameters including anthropometric characteristics, biochemical markers, and neuroimaging findings. FINDINGS: Between Oct 14, 2014, and Jan 17, 2020, we enrolled 151 patients with 73 different MCT8 (SLC16A2) mutations. Median age at diagnosis was 24·0 months (IQR 12·0-60·0, range 0·0-744·0). 32 (21%) of 151 patients died; the main causes of mortality in these patients were pulmonary infection (six [19%]) and sudden death (six [19%]). Median overall survival was 35·0 years (95% CI 8·3-61·7). Individuals who did not attain head control by age 1·5 years had an increased risk of death compared with patients who did attain head control (hazard ratio [HR] 3·46, 95% CI 1·76-8·34; log-rank test p=0·0041). Patients who were underweight during age 1-3 years had an increased risk for death compared with patients who were of normal bodyweight at this age (HR 4·71, 95% CI 1·26-17·58, p=0·021). The few motor and cognitive abilities of patients did not improve with age, as evidenced by the absence of significant correlations between biological age and scores on the Gross Motor Function Measure-88 and Bayley Scales of Infant Development III. Tri-iodothyronine concentrations were above the age-specific upper limit in 96 (95%) of 101 patients and free thyroxine concentrations were below the age-specific lower limit in 94 (89%) of 106 patients. 59 (71%) of 83 patients were underweight. 25 (53%) of 47 patients had elevated systolic blood pressure above the 90th percentile, 34 (76%) of 45 patients had premature atrial contractions, and 20 (31%) of 64 had resting tachycardia. The most consistent MRI finding was a global delay in myelination, which occurred in 13 (100%) of 13 patients. INTERPRETATION: Our description of characteristics of MCT8 deficiency in a large patient cohort reveals poor survival with a high prevalence of treatable underlying risk factors, and provides knowledge that might inform clinical management and future evaluation of therapies. FUNDING: Netherlands Organisation for Health Research and Development, and the Sherman Foundation.


Assuntos
Biomarcadores/análise , Transtornos Mentais/patologia , Transportadores de Ácidos Monocarboxílicos/deficiência , Doenças Musculares/patologia , Transtornos do Neurodesenvolvimento/patologia , Simportadores/deficiência , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Agências Internacionais , Masculino , Transtornos Mentais/etiologia , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/genética , Doenças Musculares/etiologia , Mutação , Transtornos do Neurodesenvolvimento/etiologia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Simportadores/genética , Adulto Jovem
17.
Plant Mol Biol ; 103(4-5): 561-580, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32405802

RESUMO

KEY MESSAGE: CmHKT1;1 selectively exports Na+ from plant cells. Upon NaCl stress, its expression increased in a salt-tolerant melon cultivar. Overexpression of CmHKT1;1 increased transgenic Arabidopsis salt tolerance through improved K+/Na+ balance. High-affinity K+ transporters (HKTs) are thought to be involved in reducing Na+ in plant shoots under salt stress and modulating salt tolerance, but their function in a moderately salt-tolerant species of melon (Cucumis melo L.) remains unclear. In this study, a Na+ transporter gene, CmHKT1;1 (GenBank accession number: MK986658), was isolated from melons based on genome data. The transcript of CmHKT1;1 was relatively more abundant in roots than in stems or leaves from melon seedlings. The tobacco transient expression system showed that CmHKT1;1 was plasma-membrane localized. Upon salt stress, CmHKT1;1 expression was more strongly upregulated in a salt-tolerant melon cultivar, 'Bingxuecui' (BXC) compared with a salt-sensitive cultivar, 'Yulu' (YL). Electrophysiological evidence demonstrated that CmHKT1;1 only transported Na+, rather than K+, when expressed in Xenopus laevis oocytes. Overexpression of CmHKT1;1 increased salt sensitivity in Saccharomyces cerevisiae and salt tolerance in Arabidopsis thaliana. Under NaCl treatments, transgenic Arabidopsis plants accumulated significantly lower concentrations of Na+ in shoots than wild type plants and showed a better K+/Na+ balance, leading to better Fv/Fm, root length, biomass, and enhanced plant growth. The CmHKT1;1 gene may serve as a useful candidate for improving crop salt tolerance.


Assuntos
Arabidopsis/metabolismo , Cucumis melo/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Clorofila/análise , Clonagem Molecular , Cucumis melo/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Saccharomyces cerevisiae/genética , Tolerância ao Sal , Plântula/genética , Plântula/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Cloreto de Sódio/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/fisiologia , Simportadores/genética , Simportadores/metabolismo , Tabaco/genética , Tabaco/metabolismo
18.
PLoS One ; 15(5): e0226472, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379828

RESUMO

The ParB-parS partition complexes that bacterial replicons use to ensure their faithful inheritance also find employment in visualization of DNA loci, as less intrusive alternatives to fluorescent repressor-operator systems. The ability of ParB molecules to interact via their N-terminal domains and to bind to non-specific DNA enables expansion of the initial complex to a size both functional in partition and, via fusion to fluorescent peptides, visible by light microscopy. We have investigated whether it is possible to dispense with the need to insert parS in the genomic locus of interest, by determining whether ParB fused to proteins that bind specifically to natural DNA sequences can still assemble visible complexes. In yeast cells, coproduction of fusions of ParB to a fluorescent peptide and to a TALE protein targeting an endogenous sequence did not yield visible foci; nor did any of several variants of these components. In E.coli, coproduction of fusions of SopB (F plasmid ParB) to fluorescent peptide, and to dCas9 together with specific guide RNAs, likewise yielded no foci. The result of coproducing analogous fusions of SopB proteins with distinct binding specificities was also negative. Our observations imply that in order to assemble higher order partition complexes, ParB proteins need specific activation through binding to their cognate parS sites.


Assuntos
Proteínas de Bactérias/metabolismo , Centrômero/química , Centrômero/metabolismo , DNA Bacteriano/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Bases , Sítios de Ligação , Proteína 9 Associada à CRISPR , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismo
19.
PLoS One ; 15(5): e0232846, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32380514

RESUMO

The structure of lactose permease, stabilized in a periplasmic open conformation by two Gly to Trp replacements (LacYww) and complexed with a nanobody directed against this conformation, provides the highest resolution structure of the symporter. The nanobody binds in a different manner than two other nanobodies made against the same mutant, which also bind to the same general region on the periplasmic side. This region of the protein may represent an immune hotspot. The CDR3 loop of the nanobody is held by hydrogen bonds in a conformation that partially blocks access to the substrate-binding site. As a result, kon and koff for galactoside binding to either LacY or the double mutant complexed with the nanobody are lower than for the other two LacY/nanobody complexes though the Kd values are similar, reflecting the fact that the nanobodies rigidify structures along the pathway. While the wild-type LacY/nanobody complex clearly stabilizes a similar 'extracellular open' conformation in solution, judged by binding kinetics, the complex with wild-type LacY did not yet crystallize, suggesting the nanobody does not bind strongly enough to shift the equilibrium to stabilize a periplasmic side-open conformation suitable for crystallization. However, the similarity of the galactoside binding kinetics for the nanobody-bound complexes with wild type LacY and with LacYWW indicates that they have similar structures, showing that the reported co-structures reliably show nanobody interactions with LacY.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transporte de Monossacarídeos/química , Anticorpos de Domínio Único/química , Simportadores/química , Substituição de Aminoácidos , Reações Antígeno-Anticorpo , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Galactose/metabolismo , Glicina/química , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/imunologia , Mutação de Sentido Incorreto , Mutação Puntual , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Anticorpos de Domínio Único/imunologia , Relação Estrutura-Atividade , Simportadores/genética , Simportadores/imunologia , Tiogalactosídeos/química , Triptofano/química
20.
Nat Struct Mol Biol ; 27(6): 533-539, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32451489

RESUMO

The Na+/I- symporter (NIS), the plasma membrane protein that actively transports I- (stoichiometry 2Na+:1I-) in thyroid physiology and radioiodide-based thyroid cancer treatment, also transports the environmental pollutant perchlorate (stoichiometry 1Na+:1ClO4-), which competes with I- for transport. Until now, the mechanism by which NIS transports different anion substrates with different stoichiometries has remained unelucidated. We carried out transport measurements and analyzed these using a statistical thermodynamics-based equation and electrophysiological experiments to show that the different stoichiometry of ClO4- transport is due to ClO4- binding to a high-affinity non-transport allosteric site that prevents Na+ from binding to one of its two sites. Furthermore, low concentrations of ClO4- inhibit I- transport not only by competition but also, critically, by changing the stoichiometry of I- transport to 1:1, which greatly reduces the driving force. The data reveal that ClO4- pollution in drinking water is more dangerous than previously thought.


Assuntos
Percloratos/metabolismo , Simportadores/química , Simportadores/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Ânions/química , Ânions/metabolismo , Sítios de Ligação , Transporte Biológico , Cães , Eletrofisiologia/métodos , Feminino , Humanos , Iodo/metabolismo , Células Madin Darby de Rim Canino , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Percloratos/química , Ratos , Sódio/metabolismo , Simportadores/genética , Termodinâmica , Xenopus laevis
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