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1.
Int J Mol Sci ; 22(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34360794

RESUMO

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.


Assuntos
Neurônios Motores/enzimologia , Atrofia Muscular Espinal/enzimologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Ativação Enzimática , Camundongos , Camundongos Transgênicos , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Junção Neuromuscular/genética , Junção Neuromuscular/patologia , Receptores Proteína Tirosina Quinases/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo
2.
J Enzyme Inhib Med Chem ; 36(1): 1783-1797, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340630

RESUMO

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.


Assuntos
Anidrases Carbônicas/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Anidrases Carbônicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/química , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Indóis/química , Isoenzimas/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Modelos Moleculares , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
3.
Proc Natl Acad Sci U S A ; 118(36)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34426525

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [K i] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (K i = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (K i = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.


Assuntos
Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases 3C de Coronavírus/genética , Descoberta de Drogas/métodos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Animais , COVID-19/tratamento farmacológico , COVID-19/virologia , Células Cultivadas , Proteases 3C de Coronavírus/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Engenharia Genética , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , SARS-CoV-2/metabolismo , Relação Estrutura-Atividade , Replicação Viral
4.
Nat Commun ; 12(1): 4826, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376696

RESUMO

Loss-of-function mutations in NEK1 gene, which encodes a serine/threonine kinase, are involved in human developmental disorders and ALS. Here we show that NEK1 regulates retromer-mediated endosomal trafficking by phosphorylating VPS26B. NEK1 deficiency disrupts endosomal trafficking of plasma membrane proteins and cerebral proteome homeostasis to promote mitochondrial and lysosomal dysfunction and aggregation of α-synuclein. The metabolic and proteomic defects of NEK1 deficiency disrupts the integrity of blood-brain barrier (BBB) by promoting lysosomal degradation of A20, a key modulator of RIPK1, thus sensitizing cerebrovascular endothelial cells to RIPK1-dependent apoptosis and necroptosis. Genetic inactivation of RIPK1 or metabolic rescue with ketogenic diet can prevent postnatal lethality and BBB damage in NEK1 deficient mice. Inhibition of RIPK1 reduces neuroinflammation and aggregation of α-synuclein in the brains of NEK1 deficient mice. Our study identifies a molecular mechanism by which retromer trafficking and metabolism regulates cerebrovascular integrity, cerebral proteome homeostasis and RIPK1-mediated neuroinflammation.


Assuntos
Barreira Hematoencefálica/metabolismo , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Quinase 1 Relacionada a NIMA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/metabolismo , Quinase 1 Relacionada a NIMA/genética , Necroptose/genética , Fosforilação , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 41(9): 2469-2482, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34320834

RESUMO

Objective: Critical limb ischemia is a major complication of diabetes characterized by insufficient collateral vessel development and proper growth factor signaling unresponsiveness. Although mainly deactivated by hypoxia, phosphatases are important players in the deregulation of proangiogenetic pathways. Previously, SHP-1 (Scr homology 2-containing phosphatase-1) was found to be associated with the downregulation of growth factor actions in the diabetic muscle. Thus, we aimed to gain further understanding of the impact of SHP-1 on smooth muscle cell (SMC) function under hypoxic and diabetic conditions. Approach and Results: Despite being inactivated under hypoxic conditions, high glucose level exposure sustained SHP-1 phosphatase activity in SMC and increased its interaction with PDGFR (platelet-derived growth factor receptor)-ß, thus reducing PDGF proangiogenic actions. Overexpression of an inactive form of SHP-1 fully restored PDGF-induced proliferation, migration, and signaling pathways in SMC exposed to high glucose and hypoxia. Nondiabetic and diabetic mice with deletion of SHP-1 specifically in SMC were generated. Ligation of the femoral artery was performed, and blood flow was measured for 4 weeks. Blood flow reperfusion, vascular density and maturation, and limb survival were all improved while vascular apoptosis was attenuated in diabetic SMC-specific SHP-1 null mice as compared to diabetic mice. Conclusions: Diabetes and high glucose level exposure maintained SHP-1 activity preventing hypoxia-induced PDGF actions in SMC. Specific deletion of SHP-1 in SMC partially restored blood flow reperfusion in the diabetic ischemic limb. Therefore, local modulation of SHP-1 activity in SMC could represent a potential therapeutic avenue to improve the proangiogenic properties of SMC under ischemia and diabetes.


Assuntos
Indutores da Angiogênese/farmacologia , Diabetes Mellitus Experimental/enzimologia , Angiopatias Diabéticas/enzimologia , Membro Posterior/irrigação sanguínea , Isquemia/enzimologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Glicemia/metabolismo , Estudos de Casos e Controles , Bovinos , Hipóxia Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Ativação Enzimática , Humanos , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Transdução de Sinais
6.
Methods Mol Biol ; 2350: 43-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331278

RESUMO

Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.


Assuntos
Técnicas Biossensoriais/métodos , Imunofluorescência/métodos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte , Linhagem Celular , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência/métodos , Genes Reporter , Camundongos , Ligação Proteica , Proteínas rho de Ligação ao GTP/genética
7.
Nutrients ; 13(7)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209137

RESUMO

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor.


Assuntos
Hipotálamo/metabolismo , Inositol/análogos & derivados , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Administração Oral , Animais , Glicemia/metabolismo , Ativação Enzimática/efeitos dos fármacos , Glucagon/sangue , Homeostase , Hipotálamo/efeitos dos fármacos , Inositol/administração & dosagem , Inositol/sangue , Inositol/química , Inositol/farmacologia , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina , Fator de Crescimento Insulin-Like I/metabolismo , Masculino , Fosforilação/efeitos dos fármacos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
8.
Chem Biol Interact ; 345: 109572, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34217687

RESUMO

Oxidized low-density lipoprotein (ox-LDL) not only causes hyperlipidemia and contributes to atherosclerosis but also induces the endothelial dysfunction that leads to cardiovascular diseases. The nuclear factor-kappa B (NF-κB) pathway plays a key role in many chronic disorders and is a transcriptional factor in various inflammatory responses. The present study aimed to investigate the synergistic effects of pelargonic acid vanillylamide (PAVA) and rosuvastatin (RSV) on ox-LDL-induced inflammatory responses in human vascular endothelial cells (HUV-EC-C). HUV-EC-C were pretreated with PAVA or RSV and their combination for 2 h followed by ox-LDL for 24 h. The MTT assay was used to measure mitochondrial function. The DCFH-DA assay was used to evaluate oxidative phosphorylation, and western blotting was used to measured NF-κB/NLRP3 and related signaling pathways in HUV-EC-C. Ox-LDL induced lectin-type oxidized LDL receptor 1 (LOX-1) expression, NADPH oxidase 4 activation, and the overexpression of reactive oxygen species, which were inhibited by pretreatment with the combination of PAVA and RSV. Moreover, PAVA and RSV inhibited ox-LDL-induced NF-κBp65 activation. Ox-LDL induced NF-κB/NLRP3 pathway activation by inducing C-reactive protein expression, NLRP3 activation, caspase-1 activation, and IL-1ß secretion, which were inhibited by pretreatment with the combination of PAVA and RSV. The combination of PAVA and RSV reduced ox-LDL-induced recruitment of monocytes to the site of inflammation, inhibited activation of the NLRP3 inflammasome, and ameliorated the impairment of cell-cell junctions through the NF-κB pathway. Our results suggest that the synergistic effects of PAVA and RSV provide a novel mechanism for the treatment of cardiovascular diseases.


Assuntos
Benzilaminas/farmacologia , Citoproteção/efeitos dos fármacos , Ácidos Graxos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Junções Intercelulares/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamassomos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204583

RESUMO

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.


Assuntos
Doença de Fabry/genética , Predisposição Genética para Doença , Mutação , alfa-Galactosidase/genética , Alelos , Animais , Terapia Combinada/efeitos adversos , Terapia Combinada/métodos , Gerenciamento Clínico , Ativação Enzimática , Doença de Fabry/diagnóstico , Doença de Fabry/metabolismo , Doença de Fabry/terapia , Humanos , Relação Estrutura-Atividade , Resultado do Tratamento , alfa-Galactosidase/química , alfa-Galactosidase/metabolismo
10.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203724

RESUMO

Numerous studies have shown that hedgehog inhibitors (iHHs) only partially block the growth of tumor cells, especially in vivo. Leukemia often expands in a nutrient-depleted environment (bone marrow and thymus). In order to identify putative signaling pathways implicated in the adaptive response to metabolically adverse conditions, we executed quantitative phospho-proteomics in T-cell acute lymphoblastic leukemia (T-ALL) cells subjected to nutrient-depleted conditions (serum starvation). We found important modulations of peptides phosphorylated by critical signaling pathways including casein kinase, mammalian target of rapamycin, and 5'AMP-activated kinase (AMPK). Surprisingly, in T-ALL cells, AMPK signaling was the most consistently downregulated pathway under serum-depleted conditions, and this coincided with increased GLI1 expression and sensitivity to iHHs, especially the GLI1/2 inhibitor GANT-61. Increased sensitivity to GANT-61 was also found following genetic inactivation of the catalytic subunit of AMPK (AMPKα1) or pharmacological inhibition of AMPK by Compound C. Additionally, patient-derived xenografts showing high GLI1 expression lacked activated AMPK, suggesting an important role for this signaling pathway in regulating GLI1 protein levels. Further, joint targeting of HH and AMPK signaling pathways in T-ALL cells by GANT-61 and Compound C significantly increased the therapeutic response. Our results suggest that metabolic adaptation that occurs under nutrient starvation in T-ALL cells increases responsiveness to HH pathway inhibitors through an AMPK-dependent mechanism and that joint therapeutic targeting of AMPK signaling and HH signaling could represent a valid therapeutic strategy in rapidly expanding tumors where nutrient availability becomes limiting.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Hedgehog/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Morte Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Células Jurkat , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína GLI1 em Dedos de Zinco/metabolismo
11.
Science ; 373(6550): 77-81, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210880

RESUMO

Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix-degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA-guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.


Assuntos
Astrócitos/fisiologia , Período Crítico Psicológico , Plasticidade Neuronal , Córtex Visual/crescimento & desenvolvimento , Animais , Astrócitos/metabolismo , Conexina 30/metabolismo , Ativação Enzimática , GTP Fosfo-Hidrolases/metabolismo , Interneurônios/metabolismo , Interneurônios/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinapses/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
12.
Science ; 373(6550): 118-121, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34210888

RESUMO

Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases. The widened catalytic chamber, harboring a dimethylated nickel-containing F430 cofactor, would adapt the chemistry of methyl-coenzyme M reductases for a two-carbon substrate. A sulfur from methionine replaces the oxygen from a canonical glutamine as the nickel lower-axial ligand, a feature conserved in thermophilic ethanotrophs. Specific loop extensions, a four-helix bundle dilatation, and posttranslational methylations result in the formation of a 33-angstrom-long hydrophobic tunnel, which guides the ethane to the buried active site as confirmed with xenon pressurization experiments.


Assuntos
Proteínas Arqueais/química , Etano/química , Methanosarcinales/enzimologia , Oxirredutases/química , Cristalografia por Raios X , Ativação Enzimática , Sequências Hélice-Alça-Hélice , Metilação , Processamento de Proteína Pós-Traducional
13.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299188

RESUMO

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and ß1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and ß1 subunits, mediated via the Ca-activated PKC pathway.


Assuntos
Cálcio/metabolismo , Enterite/enzimologia , Células Epiteliais/enzimologia , Intestinos/enzimologia , Leucotrieno D4/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sobrevivência Celular/fisiologia , Células Cultivadas , Enterite/tratamento farmacológico , Enterite/patologia , Ativação Enzimática , Células Epiteliais/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos
14.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202757

RESUMO

The aim of this study was to investigate the effect of the application of homocysteine as well as its effect under the condition of aerobic physical activity on the activities of matrix metalloproteinases (MMP), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in cardiac tissue and on hepato-renal biochemical parameters in sera of rats. Male Wistar albino rats were divided into four groups (n = 10, per group): C: 0.9% NaCl 0.2 mL/day subcutaneous injection (s.c.); H: homocysteine 0.45 µmol/g b.w./day s.c.; CPA saline (0.9% NaCl 0.2 mL/day s.c.) and a program of physical activity on a treadmill; and HPA homocysteine (0.45 µmol/g b.w./day s.c.) and a program of physical activity on a treadmill. Subcutaneous injection of substances was applied 2 times a day at intervals of 8 h during the first two weeks of experimental protocol. Hcy level in serum was significantly higher in the HPA group compared to the CPA group (p < 0.05). Levels of glucose, proteins, albumin, and hepatorenal biomarkers were higher in active groups compared with the sedentary group. It was demonstrated that the increased activities of LDH (mainly caused by higher activity of isoform LDH2) and mMDH were found under the condition of homocysteine-treated rats plus aerobic physical activity. Independent application of homocysteine did not lead to these changes. Physical activity leads to activation of MMP-2 isoform and to increased activity of MMP-9 isoform in both homocysteine-treated and control rats.


Assuntos
Hiper-Homocisteinemia/metabolismo , Rim/metabolismo , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Malato Desidrogenase/metabolismo , Metaloproteinases da Matriz/metabolismo , Miocárdio/metabolismo , Condicionamento Físico Animal , Animais , Biomarcadores , Pesos e Medidas Corporais , Ativação Enzimática , Hiper-Homocisteinemia/etiologia , Miocárdio/enzimologia , Especificidade de Órgãos , Ratos , Fatores de Tempo
15.
Nat Commun ; 12(1): 4542, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315871

RESUMO

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.


Assuntos
Formaldeído/metabolismo , Nucleotídeos/metabolismo , Thermotoga maritima/enzimologia , Timidilato Sintase/metabolismo , Biocatálise , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Domínio Catalítico , Ativação Enzimática , Flavinas/metabolismo , Metilação , Eletricidade Estática , Timidilato Sintase/química
16.
Int Heart J ; 62(4): 910-918, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34276002

RESUMO

Calcium antagonists are used for coronary spastic angina (CSA) treatment. We previously identified a phospholipase C (PLC) -δ1 gene variant that results in enhanced PLC activity in patients with CSA and developed a CSA animal model by generating vascular smooth muscle cell-specific human variant PLC-δ1 overexpression (PLC-TG) mice. In this study, we investigated the molecular mechanism of CSA using the PLC-TG mice and the inhibitory effect of a calcium antagonist, diltiazem hydrochloride (DL).We treated the PLC-TG and wild-type (WT) mice with oral DL or trichlormethiazide (TM) (control) for 2 weeks. Ergometrine injection-induced coronary spasm was observed on the electrocardiogram in all 5 PLC-TG mice treated with TM, but only in 1 of 5 PLC-TG mice treated with DL. Voltage-dependent calcium channel (Cav1.2) phosphorylation and protein kinase C (PKC) activity were enhanced in the aortas of PLC-TG mice treated with TM. DL treatment significantly inhibited Cav1.2 phosphorylation and PKC activity. Although total Cav1.2 expression was similar between WT and PLC-TG mice treated with TM, DL treatment significantly increased its expression in PLC-TG mice. Furthermore, its expression remained high after DL discontinuation. DL and PKC inhibitor suppressed intracellular calcium response to acetylcholine in cultured rat aortic smooth muscle cells transfected with variant PLC-δ1.These results indicate that enhanced PLC activity causes coronary spasm, presumably via enhanced Cav1.2 phosphorylation and PKC activity, both of which were inhibited by DL. Enhanced total Cav1.2 expression after DL discontinuation and high PKC activity may be an important mechanism underlying the calcium antagonist withdrawal syndrome.


Assuntos
Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo L/metabolismo , Vasoespasmo Coronário/tratamento farmacológico , Diltiazem/uso terapêutico , Proteína Quinase C/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Vasoespasmo Coronário/metabolismo , Diltiazem/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Masculino , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos
17.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204297

RESUMO

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)-mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR-SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.


Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Quinases da Família src/química , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Arrestinas/química , Arrestinas/metabolismo , Ativação Enzimática , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
18.
Phys Chem Chem Phys ; 23(26): 14422-14432, 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34180917

RESUMO

Experimental studies relevant to the nonthermal effects of electric fields on biological systems are emerging. However, these effects are poorly understood at the molecular level. The present study investigates pectin methylesterase, a cell wall modifying enzyme in plants, exposed to various electric field strengths. Molecular dynamics (MD) of the enzyme were studied with and without (thermal-only) electric field applications. The measurements were interpreted on the basis of equivalent energy input to gain insights into the effect of electric field treatment time at a constant temperature (50 °C). Results reveal that electric fields exert nonthermal effects on both local and global protein structure. In 1 µs simulations, the results show significant (P ≤ 0.05) shrinkage of the catalytic domain and shortening of enzyme-water hydrogen bond lifetime by a 50 V cm-1 electric field. Unwinding of the helical segments, altered intra- and intermolecular hydrogen bond patterns, and increased hydration are also caused by the 50 V cm-1 electric field. This study serves to understand the electric field influence on the functional role of proteins.


Assuntos
Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Campos Eletromagnéticos , Ativação Enzimática , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Temperatura , Água
20.
Cell Syst ; 12(8): 780-794.e7, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34139154

RESUMO

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease.


Assuntos
Biomarcadores/análise , COVID-19/patologia , Progressão da Doença , Proteoma/fisiologia , Fatores Etários , Contagem de Células Sanguíneas , Gasometria , Ativação Enzimática , Humanos , Inflamação/patologia , Aprendizado de Máquina , Prognóstico , Proteômica , SARS-CoV-2/imunologia
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