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1.
Invest Ophthalmol Vis Sci ; 62(12): 4, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34499705

RESUMO

Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown. Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking. Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts. Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.


Assuntos
Amônia/farmacologia , Proteínas de Transporte de Ânions/genética , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/metabolismo , Mitocôndrias/patologia , Proteômica/métodos , Simportadores/genética , Animais , Proteínas de Transporte de Ânions/metabolismo , Células Cultivadas , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/patologia , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Chaperonas Moleculares/efeitos dos fármacos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Transporte Proteico , Simportadores/metabolismo
2.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502056

RESUMO

Skeletal tissue involves systemic adipose tissue metabolism and energy expenditure. MicroRNA signaling controls high-fat diet (HFD)-induced bone and fat homeostasis dysregulation remains uncertain. This study revealed that transgenic overexpression of miR-29a under control of osteocalcin promoter in osteoblasts (miR-29aTg) attenuated HFD-mediated body overweight, hyperglycemia, and hypercholesterolemia. HFD-fed miR-29aTg mice showed less bone mass loss, fatty marrow, and visceral fat mass together with increased subscapular brown fat mass than HFD-fed wild-type mice. HFD-induced O2 underconsumption, respiratory quotient repression, and heat underproduction were attenuated in miR-29aTg mice. In vitro, miR-29a overexpression repressed transcriptomic landscapes of the adipocytokine signaling pathway, fatty acid metabolism, and lipid transport, etc., of bone marrow mesenchymal progenitor cells. Forced miR-29a expression promoted osteogenic differentiation but inhibited adipocyte formation. miR-29a signaling promoted brown/beige adipocyte markers Ucp-1, Pgc-1α, P2rx5, and Pat2 expression and inhibited white adipocyte markers Tcf21 and Hoxc9 expression. The microRNA also reduced peroxisome formation and leptin expression during adipocyte formation and downregulated HFD-induced leptin expression in bone tissue. Taken together, miR-29a controlled leptin signaling and brown/beige adipocyte formation of osteogenic progenitor cells to preserve bone anabolism, which reversed HFD-induced energy underutilization and visceral fat overproduction. This study sheds light on a new molecular mechanism by which bone integrity counteracts HFD-induced whole-body fat overproduction.


Assuntos
Gordura Intra-Abdominal/metabolismo , Leptina/genética , MicroRNAs/metabolismo , Osteoblastos/metabolismo , Osteoporose/metabolismo , Adipócitos/citologia , Adipócitos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Dieta Hiperlipídica/efeitos adversos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Leptina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Osteoblastos/citologia , Osteoporose/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Peroxissomos/metabolismo , Receptores Purinérgicos P2X5/genética , Receptores Purinérgicos P2X5/metabolismo , Simportadores/genética , Simportadores/metabolismo , Termogênese , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
3.
J Agric Food Chem ; 69(31): 8714-8725, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34323067

RESUMO

Upregulated de novo lipogenesis (DNL) plays a pivotal role in the progress of the nonalcoholic fatty liver disease (NAFLD). Cytoplasmic citrate flux, mediated by plasma membrane citrate transporter (SLC13A5), mitochondrial citrate carrier (SLC25A1), and ATP-dependent citrate lyase (ACLY), determines the central carbon source for acetyl-CoA required in DNL. Curcumin, a widely accepted dietary polyphenol, can attenuate lipid accumulation in NAFLD. Here, we first investigated the lipid-lowering effect of curcumin against NAFLD in oleic and palmitic acid (OPA)-induced primary mouse hepatocytes and high-fat plus high-fructose diet (HFHFD)-induced mice. Curcumin profoundly attenuated OPA- or HFHFD-induced hyperlipidemia and aberrant hepatic lipid deposition via modulating the expression and function of SLC13A5 and ACLY. The possible mechanism of curcumin on the citrate pathway was investigated using HepG2 cells, HEK293T cells transfected with human SLC13A5, and recombinant human ACLY. In OPA-stimulated HepG2 cells, curcumin rectified the dysregulated expression of SLC13A5/ACLY possibly via the AMPK-mTOR signaling pathway. Besides, curcumin also functionally inhibited both citrate transport and metabolism mediated by SLC13A5 and ACLY, respectively. These findings confirm that curcumin improves the lipid accumulation in the liver by blocking citrate disposition and hence may be used to prevent NAFLD.


Assuntos
Curcumina , Hepatopatia Gordurosa não Alcoólica , Transportadores de Ânions Orgânicos , Simportadores , ATP Citrato (pro-S)-Liase/metabolismo , Animais , Ácido Cítrico , Curcumina/farmacologia , Transportadores de Ácidos Dicarboxílicos , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Camundongos , Proteínas Mitocondriais , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Simportadores/metabolismo
4.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199867

RESUMO

NIS is a potent iodide transporter encoded by the SLC5A5 gene. Its expression is reduced in papillary thyroid carcinoma (PTC). In this study we analyzed the impact of miR-181a-5p on NIS expression in the context of PTC. We used real-time PCR to analyze the expression of SLC5A5 and miR-181a-5p in 49 PTC/normal tissue pairs. Luciferase assays and mutagenesis were performed to confirm direct binding of miR-181a-5p to the 3'UTR of SLC5A5 and identify the binding site. The impact of modulation of miR-181a-5p using appropriate plasmids on endogenous NIS and radioactive iodine accumulation was verified. We confirmed downregulation of SLC5A5 and concomitant upregulation of miR-181a-5p in PTC. Broadly used algorithms did not predict the binding site of miR-181a-5p in 3'UTR of SLC5A5, but we identified and confirmed the binding site through mutagenesis using luciferase assays. In MCF7 and HEK293-flhNIS cell lines, transfection with mir-181a-expressing plasmid decreased endogenous SLC5A5, whereas silencing of miR-181a-5p increased it. We observed similar tendencies in protein expression and radioactive iodine accumulation. This study shows for the first time that miR-181a-5p directly regulates SLC5A5 expression in the context of PTC and may decrease efficacy of radioiodine treatment. Accordingly, miR-181a-5p may serve as an emerging target to enhance the efficacy of radioactive iodine therapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Simportadores/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Simportadores/genética , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Células Tumorais Cultivadas
5.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
6.
Nat Commun ; 12(1): 4649, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330925

RESUMO

The bacterium Vibrio cholerae can colonize the human intestine and cause cholera, but spends much of its life cycle in seawater. The pathogen must adapt to substantial environmental changes when moving between seawater and the human intestine, including different availability of carbon sources such as fructose. Here, we use in vitro experiments as well as mouse intestinal colonization assays to study the mechanisms used by pandemic V. cholerae to adapt to these environmental changes. We show that a LacI-type regulator (FruI) and a fructose/H+ symporter (FruT) are important for fructose uptake at low fructose concentrations, as those found in seawater. FruT is downregulated by FruI, which is upregulated when O2 concentrations are low (as in the intestine) by ArcAB, a two-component system known to respond to changes in oxygen levels. As a result, the bacteria predominantly use FruT for fructose uptake under seawater conditions (low fructose, high O2), and use a known fructose phosphotransferase system (PTS, Fpr) for fructose uptake under conditions found in the intestine. PTS activity leads to reduced levels of intracellular cAMP, which in turn upregulate virulence genes. Our results indicate that the FruT/FruI system may be important for survival of pandemic V. cholerae in seawater.


Assuntos
Proteínas de Bactérias/metabolismo , Frutose/metabolismo , Simportadores/metabolismo , Vibrio cholerae/metabolismo , Animais , Proteínas de Bactérias/genética , Cólera/epidemiologia , Cólera/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Genômica/métodos , Humanos , Masculino , Camundongos , Viabilidade Microbiana/genética , Pandemias , Regiões Promotoras Genéticas/genética , Ligação Proteica , Água do Mar/microbiologia , Simportadores/genética , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Virulência/genética
7.
Toxicology ; 459: 152853, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252480

RESUMO

Oxaliplatin (OXA) is a third-generation platinum drug; however, its application is greatly limited due to the severe peripheral neurotoxicity. This study aims to confirm the transport mechanism of OXA and to explore whether L-tetrahydropalmatine (L-THP) would alleviate OXA-induced peripheral neurotoxicity by selectively inhibiting these uptake transporters in vitro and in vivo. Our results revealed that organic cation transporter 2 (OCT2), organic cation/carnitine transporter 1 (OCTN1) and organic cation/carnitine transporter 2 (OCTN2) were involved in the uptake of OXA in dorsal root ganglion (DRG) neurons and mitochondria, respectively. L-THP (1-100 µM) reduced OXA (40 µM) induced cytotoxicity in MDCK-hOCT2 (Madin-Darby canine kidney, MDCK), MDCK-hOCTN1, MDCK-hOCTN2, and rat primary DRG cells, and decreased the accumulation of OXA in above cells and rat DRG mitochondria, but did not affect its efflux from MDCK-hMRP2 cells. Furthermore, Co-administration of L-THP (5-20 mg/kg for mice, 10-40 mg/kg for rats; twice a week, iv or ig) attenuated OXA (8 mg/kg for mice, 4 mg/kg for rats; twice a week, iv) induced peripheral neurotoxicity and reduced the platinum concentration in the DRG. Whereas, L-THP (1-100 µM for cells; 10-20 mg/kg for mice) did not impair the antitumour efficacy of OXA (40 µM for cells; 8 mg/kg for mice) in HT29 tumour-bearing nude mice nor in tumour cells (HT29 and SW620 cells). In conclusion, OCT2, OCTN1 and OCTN2 contribute to OXA uptake in the DRG and mitochondria. L-THP attenuates OXA-induced peripheral neurotoxicity via inhibiting OXA uptake but without impairing the antitumour efficacy of OXA. L-THP is a potential candidate drug to attenuate OXA-induced peripheral neurotoxicity.


Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Alcaloides de Berberina/farmacologia , Proteínas de Transporte/antagonistas & inibidores , Gânglios Espinais/metabolismo , Mitocôndrias/metabolismo , Oxaliplatina/farmacocinética , Oxaliplatina/toxicidade , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/prevenção & controle , Animais , Comportamento Animal/efeitos dos fármacos , Cães , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Células HT29 , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Transportador 2 de Cátion Orgânico/antagonistas & inibidores , Ratos , Membro 5 da Família 22 de Carreadores de Soluto/antagonistas & inibidores , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Simportadores/antagonistas & inibidores , Simportadores/metabolismo
8.
Am J Physiol Regul Integr Comp Physiol ; 321(3): R469-R481, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34189959

RESUMO

Chronic intermittent hypoxia (CIH) is associated with diurnal hypertension, increased sympathetic nerve activity (SNA), and increases in circulating angiotensin II (ANG II). In rats, CIH increases angiotensin type 1 (AT1a) receptor expression in the median preoptic nucleus (MnPO), and pharmacological blockade or viral knockdown of this receptor prevents CIH-dependent increases in diurnal blood pressure. The current study investigates the role of AT1a receptor in modulating the activity of MnPO neurons following 7 days of CIH. Male Sprague-Dawley rats received MnPO injections of an adeno-associated virus with an shRNA against the AT1a receptor or a scrambled control. Rats were then exposed to CIH for 8 h a day for 7 days. In vitro, loose patch recordings of spontaneous action potential activity were made from labeled MnPO neurons in response to brief focal application of ANG II or the GABAA receptor agonist muscimol. In addition, MnPO K-Cl cotransporter isoform 2 (KCC2) protein expression was assessed using Western blot. CIH impaired the duration but not the magnitude of ANG II-mediated excitation in the MnPO. Both CIH and AT1a knockdown also impaired GABAA-mediated inhibition, and CIH with AT1a knockdown produced GABAA-mediated excitation. Recordings using the ratiometric Cl- indicator ClopHensorN showed CIH was associated with Cl- efflux in MnPO neurons that was associated with decreased KCC2 phosphorylation. The combination of CIH and AT1a knockdown attenuated reduced KCC2 phosphorylation seen with CIH alone. The current study shows that CIH, through the activity of AT1a receptors, can impair GABAA-mediated inhibition in the MnPO and contribute to sustained hypertension.


Assuntos
Pressão Sanguínea , Neurônios GABAérgicos/metabolismo , Hipertensão/metabolismo , Hipóxia/metabolismo , Inibição Neural , Área Pré-Óptica/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de GABA-A/metabolismo , Síndromes da Apneia do Sono/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Hipertensão/genética , Hipertensão/fisiopatologia , Hipóxia/genética , Hipóxia/fisiopatologia , Masculino , Fosforilação , Área Pré-Óptica/fisiopatologia , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , Síndromes da Apneia do Sono/genética , Síndromes da Apneia do Sono/fisiopatologia , Simportadores/metabolismo , Fatores de Tempo
9.
J Gen Physiol ; 153(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34110360

RESUMO

MelB catalyzes the obligatory cotransport of melibiose with Na+, Li+, or H+. Crystal structure determination of the Salmonella typhimurium MelB (MelBSt) has revealed a typical major facilitator superfamily (MFS) fold at a periplasmic open conformation. Cooperative binding of Na+ and melibiose has been previously established. To determine why cotranslocation of sugar solute and cation is obligatory, we analyzed each binding in the thermodynamic cycle using three independent methods, including the determination of melting temperature by circular dichroism spectroscopy, heat capacity change (ΔCp), and regulatory phosphotransferase EIIAGlc binding with isothermal titration calorimetry (ITC). We found that MelBSt thermostability is increased by either substrate (Na+ or melibiose) and observed a cooperative effect of both substrates. ITC measurements showed that either binary formation yields a positive sign in the ΔCp, suggesting MelBSt hydration and a likely widening of the periplasmic cavity. Conversely, formation of a ternary complex yields negative values in ΔCp, suggesting MelBSt dehydration and cavity closure. Lastly, we observed that EIIAGlc, which has been suggested to trap MelBSt at an outward-open state, readily binds to the MelBSt apo state at an affinity similar to MelBSt/Na+. However, it has a suboptimal binding to the ternary state, implying that MelBSt in the ternary complex may be conformationally distant from the EIIAGlc-preferred outward-facing conformation. Our results consistently support the notion that binding of one substrate (Na+ or melibiose) favors MelBSt at open states, whereas the cooperative binding of both substrates triggers the alternating-access process, thus suggesting this conformational regulation could ensure the obligatory cotransport.


Assuntos
Melibiose , Simportadores , Ligação Proteica , Salmonella typhimurium/metabolismo , Sódio/metabolismo , Simportadores/metabolismo
10.
Am J Physiol Gastrointest Liver Physiol ; 321(2): G123-G133, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34077272

RESUMO

Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.


Assuntos
Biotina/metabolismo , Metilação de DNA , Epigênese Genética , Etanol/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Acetaldeído/farmacologia , Animais , Células CACO-2 , Células Cultivadas , Etanol/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos/farmacologia , Ácidos Palmíticos/farmacologia , Simportadores/genética , Simportadores/metabolismo
11.
Int J Mol Sci ; 22(10)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34069814

RESUMO

Osteogenesis imperfecta (OI) is a bone fragility disorder that is usually caused by mutations affecting collagen type I. We compared the calvaria bone tissue transcriptome of male 10-week-old heterozygous Jrt (Col1a1 mutation) and homozygous oim mice (Col1a2 mutation) to their respective littermate results. We found that Jrt and oim mice shared 185 differentially expressed genes (upregulated: 106 genes; downregulated: 79 genes). A total of seven genes were upregulated by a factor of two or more in both mouse models (Cyp2e1, Slc13a5, Cgref1, Smpd3, Ifitm5, Cthrc1 and Rerg). One gene (Gypa, coding for a blood group antigen) was downregulated by a factor of two or more in both OI mouse models. Overrepresentation analyses revealed that genes involved in 'ossification' were significantly overrepresented among upregulated genes in both Jrt and oim mice, whereas hematopoietic genes were downregulated. Several genes involved in Wnt signaling and transforming growth factor beta signaling were upregulated in oim mice, but less so in Jrt mice. Thus, this study identified a set of genes that are dysregulated across various OI mouse models and are likely to play an important role in the pathophysiology of this disorder.


Assuntos
Osteogênese Imperfeita/genética , Crânio/metabolismo , Animais , Colágeno Tipo I/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Modelos Animais de Doenças , Fêmur/metabolismo , Perfilação da Expressão Gênica/métodos , Heterozigoto , Homozigoto , Masculino , Camundongos , Mutação , Osteogênese , Osteogênese Imperfeita/metabolismo , Osteogênese Imperfeita/fisiopatologia , Crânio/fisiologia , Esfingomielina Fosfodiesterase/metabolismo , Simportadores/metabolismo , Transcriptoma/genética
12.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064332

RESUMO

Spinal cord injury (SCI) leads to numerous chronic and debilitating functional deficits that greatly affect quality of life. While many pharmacological interventions have been explored, the current unsurpassed therapy for most SCI sequalae is exercise. Exercise has an expansive influence on peripheral health and function, and by activating the relevant neural pathways, exercise also ameliorates numerous disorders of the central nervous system (CNS). While the exact mechanisms by which this occurs are still being delineated, major strides have been made in the past decade to understand the molecular underpinnings of this essential treatment. Exercise rapidly and prominently affects dendritic sprouting, synaptic connections, neurotransmitter production and regulation, and ionic homeostasis, with recent literature implicating an exercise-induced increase in neurotrophins as the cornerstone that binds many of these effects together. The field encompasses vast complexity, and as the data accumulate, disentangling these molecular pathways and how they interact will facilitate the optimization of intervention strategies and improve quality of life for individuals affected by SCI. This review describes the known molecular effects of exercise and how they alter the CNS to pacify the injury environment, increase neuronal survival and regeneration, restore normal neural excitability, create new functional circuits, and ultimately improve motor function following SCI.


Assuntos
Exercício Físico , Regulação da Expressão Gênica , Regeneração Nervosa/genética , Plasticidade Neuronal/genética , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Humanos , Neurotrofina 3/genética , Neurotrofina 3/metabolismo , Qualidade de Vida , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/reabilitação , Simportadores/genética , Simportadores/metabolismo
13.
Nature ; 595(7866): 315-319, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34135507

RESUMO

Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources1-6. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner7,8. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.


Assuntos
Transporte Biológico , Barreira Hematoencefálica/metabolismo , Microscopia Crioeletrônica , Ácidos Graxos Ômega-3/metabolismo , Simportadores/química , Simportadores/metabolismo , Animais , Sítios de Ligação , Galinhas , Ácidos Graxos Ômega-3/química , Espectrometria de Massas , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Sódio/metabolismo , Simportadores/ultraestrutura
14.
J Mol Biol ; 433(16): 167056, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34022207

RESUMO

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl- with K+ and/or Na+ in an electroneutral manner. They play important roles in myriad fundamental physiological processes--from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis--and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na+/K+/Cl- cotransporter NKCC1 and the K+/Cl- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.


Assuntos
Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/química , Simportadores/metabolismo , Sítios de Ligação , Transporte de Íons , Ligação Proteica , Multimerização Proteica , Relação Estrutura-Atividade
15.
PLoS One ; 16(5): e0251765, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33999935

RESUMO

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.


Assuntos
Inibidores da Angiogênese/farmacologia , Membrana Corioalantoide , Neoplasias Experimentais , Neovascularização Patológica , Consumo de Oxigênio/efeitos dos fármacos , Pirimidinonas/farmacologia , Tiofenos/farmacologia , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/metabolismo , Membrana Corioalantoide/patologia , Cães , Humanos , Camundongos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Simportadores/antagonistas & inibidores , Simportadores/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Exp Neurol ; 342: 113734, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33945789

RESUMO

Disruptions to either sulfate supply or sulfation enzymes can affect brain development and have long-lasting effects on brain function, yet our understanding of the molecular mechanisms governing this are incomplete. Perineuronal nets (PNNs) are highly sulfated, specialized extracellular matrix structures that regulate the maturation of synaptic connections and neuronal plasticity. We have previously shown that mice heterozygous for the brain sulfate transporter Slc13a4 have abnormal social interactions, memory, exploratory behaviors, stress and anxiety of postnatal origin, pointing to potential deficits in PNN biology, and implicate SLC13A4 as a critical factor required for regulating normal synaptic connectivity and function. Here, we sought to investigate aberrant PNN formation as a potential mechanism contributing to the functional deficits displayed by Slc13a4+/- mice. Following social interactions, we reveal reduced neuronal activation in the somatosensory cortex of Slc13a4+/- mice, and altered inhibitory and excitatory postsynaptic currents. In line with this, we found a reduction in parvalbumin-expressing neurons decorated with PNNs, as well as reduced expression of markers for PNN maturation. Finally, we reveal that postnatal administration of N-acetylcysteine prevented PNN abnormalities from manifesting in Slc13a4+/- adult animals. Collectively, these data highlight a central role for postnatal SLC13A4 in normal PNN formation, circuit function and subsequent animal behavior.


Assuntos
Acetilcisteína/administração & dosagem , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/metabolismo , Transportadores de Sulfato/metabolismo , Simportadores/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Feminino , Sequestradores de Radicais Livres/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/metabolismo , Transportadores de Sulfato/genética , Simportadores/genética
17.
Artigo em Inglês | MEDLINE | ID: mdl-33932583

RESUMO

Transporters expressed by hepatocytes and enterocytes play a critical role in maintaining the enterohepatic circulation of bile acids. The sodium taurocholate cotransporting polypeptide (NTCP), exclusively expressed at the basolateral side of hepatocytes, mediates the uptake of conjugated bile acids. In conditions where bile flow is impaired (cholestasis), pharmacological inhibition of NTCP-mediated bile acid influx is suggested to reduce hepatocellular damage due to bile acid overload. Furthermore, NTCP has been shown to play an important role in hepatitis B virus (HBV) and hepatitis Delta virus (HDV) infection by functioning as receptor for viral entry into hepatocytes. This review provides a summary of current molecular insight into the regulation of NTCP expression at the plasma membrane, hepatic bile acid transport, and NTCP-mediated viral infection.


Assuntos
Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/fisiologia , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Internalização do Vírus , Animais , Transporte Biológico , Humanos
18.
Theranostics ; 11(13): 6251-6277, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995657

RESUMO

The advanced, metastatic differentiated thyroid cancers (DTCs) have a poor prognosis mainly owing to radioactive iodine (RAI) refractoriness caused by decreased expression of sodium iodide symporter (NIS), diminished targeting of NIS to the cell membrane, or both, thereby decreasing the efficacy of RAI therapy. Genetic aberrations (such as BRAF, RAS, and RET/PTC rearrangements) have been reported to be prominently responsible for the onset, progression, and dedifferentiation of DTCs, mainly through the activation of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling pathways. Eventually, these alterations result in a lack of NIS and disabling of RAI uptake, leading to the development of resistance to RAI therapy. Over the past decade, promising approaches with various targets have been reported to restore NIS expression and RAI uptake in preclinical studies. In this review, we summarized comprehensive molecular mechanisms underlying the dedifferentiation in RAI-refractory DTCs and reviews strategies for restoring RAI avidity by tackling the mechanisms.


Assuntos
Radioisótopos do Iodo/uso terapêutico , Proteínas de Neoplasias/metabolismo , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Autofagia , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Humanos , Radioisótopos do Iodo/farmacocinética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores da Tireotropina/metabolismo , Proteínas Recombinantes/uso terapêutico , Terapia de Salvação , Transdução de Sinais , Simportadores/genética , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/radioterapia , Tireotropina/metabolismo , Tireotropina/uso terapêutico
19.
J Biol Chem ; 297(1): 100775, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34022218

RESUMO

Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-13C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1-dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.


Assuntos
L-Lactato Desidrogenase/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ácido Pirúvico/farmacologia , Simportadores/metabolismo , Ácidos/metabolismo , Tampões (Química) , Isótopos de Carbono , Extratos Celulares , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Espaço Extracelular/química , Glicólise/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/biossíntese , Especificidade por Substrato/efeitos dos fármacos
20.
J Biol Chem ; 296: 100793, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019872

RESUMO

K+-Cl- cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell-volume regulation. KCCs exhibit significant variations in K+ affinities, yet recent atomic structures demonstrated that K+- and Cl--binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity. Here, we used the extracellular loop 2 (EL2) that links transmembrane helices (TMs) 3 and 4, as a mechanism to modulate ECD folding. We compared consequences of point mutations in the K+-binding site on the function of WT KCC2 and in a KCC2 variant, in which EL2 was structurally altered by insertion of a IFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAHAAA (3xHA) tag (36 amino acids). In WT KCC2, individual mutations of five residues in the K+-binding site resulted in a 2- to 3-fold decreased transport rate. However, the same mutations in the KCC2 variant with EL2 structurally altered by insertion of a 3xHA tag had no effect on transport activity. Homology models of mouse KCC2 with the 3xHA tag inserted into EL2 using ab initio prediction were generated. The models suggest subtle conformational changes occur in the ECD upon EL2 modification. These data suggest that a conformational change in the ECD, for example, by interaction with EL2, might be an elegant way to modulate the K+ affinity of the different isoforms in the KCC subfamily.


Assuntos
Simportadores/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Transporte de Íons , Cinética , Camundongos , Modelos Moleculares , Potássio/metabolismo , Conformação Proteica , Simportadores/química
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