RESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 disease. Through its viral spike (S) protein, the virus enters and infects epithelial cells by utilizing angiotensin-converting enzyme 2 as a host cell's receptor protein. The COVID-19 pandemic had a profound impact on global public health and economies. Although various effective vaccinations and medications are now available to prevent and treat COVID-19, natural compounds derived from medicinal plants, particularly flavonoids, demonstrated therapeutic potential to treat COVID-19 disease. Flavonoids exhibit dual antiviral mechanisms: direct interference with viral invasion and inhibition of replication. Specifically, they target key viral molecules, particularly viral proteases, involved in infection. These compounds showcase significant immunomodulatory and anti-inflammatory properties, effectively inhibiting various inflammatory cytokines. Additionally, emerging evidence supports the potential of flavonoids to mitigate the progression of COVID-19 in individuals with obesity by positively influencing lipid metabolism. This review aims to elucidate the molecular structure of SARS-CoV-2 and the underlying mechanism of action of flavonoids on the virus. This study evaluates the potential anti-SARS-CoV-2 properties exhibited by flavonoid compounds, with a specific interest in their structure and mechanisms of action, as therapeutic applications for the prevention and treatment of COVID-19. Nevertheless, a significant portion of existing knowledge is based on theoretical frameworks and findings derived from in vitro investigations. Further research is required to better assess the effectiveness of flavonoids in combating SARS-CoV-2, with a particular emphasis on in vivo and clinical investigations.
Assuntos
COVID-19 , Plantas Medicinais , Humanos , SARS-CoV-2 , Plantas Medicinais/metabolismo , Flavonoides/química , Pandemias , Tratamento Farmacológico da COVID-19 , Antivirais/farmacologia , Peptidil Dipeptidase A/metabolismoRESUMO
Medicinal plants exert therapeutic effects or have beneficial healing functions on the human or animal body. Medicinal plants are widely used in traditional medicine as an interesting alternative and/or complementary to science-based medicine. Compared to chemical drugs, medicinal plants have a lower risk of side effects, are eco-friendly, and have cost-effective production. This encouraged researchers to extensively exploit them for their therapeutic use. One of the most well-known medicinal plants is Vitex agnus-castus L., which belongs to the Verbenaceae family. This shrub tree is mainly grown in tropical and sub-tropical regions. The parts of VAC, especially berries and leaves, contain essential oils, flavonoids, and diterpenes. Many medical benefits of VAC have already been reported, including mastalgia, regulating menstrual cycles and premenstrual complaints, and infertility. Respiratory and cardiovascular effects are also reported. In this review, we will analyze and characterize the known roles of VAC in mastalgia, as well as the mechanism of action reported in in vitro and/or in vivo studies, and show the potential for alternative therapeutic uses in mastalgia, also known as breast pain (Fig. 2, Ref. 40). Keywords: mastalgia, Vitex agnus-castus, therapy, traditional medicine.
Assuntos
Mastodinia , Plantas Medicinais , Vitex , Feminino , Animais , Humanos , Vitex/química , Mastodinia/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Folhas de PlantaRESUMO
The antiaging protein Klotho is encoded by the Klotho gene first identified as an 'aging suppressor', in mice. Klotho deficiency is involved in premature aging and early death, while its overexpression is related to longevity. Klotho is mostly expressed in the kidney, but also in the brain, and in other organs. Two forms of Klotho, the cell membrane and secreted form, have pleiotropic activities that include regulation of general metabolism, oxidative stress, and mineral metabolism that correlates with its effect on accelerating aging. Membrane Klotho serves as an obligate co-receptor for the fibroblast growth factor (FGF), while secreted Klotho plays its role as a humoral factor. Klotho protein participates in the regulation of several biological activities, including regulation of calcium-phosphate homeostasis and PTH as well as vitamin D metabolism. The active form of vitamin D, 1,25(OH)2D3 (1,25-dihydroxy-vitamin D3 = calcitriol), acts as a neurosteroid that participates in the regulation of multiple brain functions. It provides neuroprotection and suppresses oxidative stress, inhibits inflammation and inflammatory mediators, and stimulates various neurotrophins. Calcitriol is involved in many brain-related diseases, including multiple sclerosis, Alzheimer´s disease, Parkinson´s disease, and schizophrenia. This review covers the most recent advances in Klotho research and discusses Klotho-dependent roles of calcitriol in neuro-psycho-pathophysiology.
Assuntos
Calcitriol , Glucuronidase , Animais , Encéfalo/metabolismo , Cálcio da Dieta , Glucuronidase/metabolismo , Proteínas Klotho , CamundongosRESUMO
BACKGROUND/AIMS: The transmembrane Klotho protein contributes to inhibition of 1,25(OH)2D3 formation. The extracellular domain of Klotho protein could function as an enzyme with e.g. ß-glucuronidase activity, be cleaved off and be released into blood and cerebrospinal fluid. Klotho regulates several cellular transporters. Klotho protein deficiency accelerates the appearance of age related disorders including neurodegeneration and muscle wasting and eventually leads to premature death. The main site of Klotho protein expression is the kidney. Klotho protein is also appreciably expressed in other tissues including chorioid plexus. The present study explored the effect of Klotho protein on the creatine transporter CreaT (Slc6A8), which participates in the maintenance of neuronal function and survival. METHODS: To this end cRNA encoding Slc6A8 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho protein. Creatine transporter CreaT (Slc6A8) activity was estimated from creatine induced current determined by two-electrode voltage-clamp. RESULTS: Coexpression of Klotho protein significantly increased creatine-induced current in Slc6A8 expressing Xenopus oocytes. Coexpression of Klotho protein delayed the decline of creatine induced current following inhibition of carrier insertion into the cell membrane by brefeldin A (5 µM). The increase of creatine induced current by coexpression of Klotho protein in Slc6A8 expressing Xenopus oocytes was reversed by ß-glucuronidase inhibitor (DSAL). Similarly, treatment of Slc6A8 expressing Xenopus oocytes with recombinant human alpha Klotho protein significantly increased creatine induced current. CONCLUSION: Klotho protein up-regulates the activity of creatine transporter CreaT (Slc6A8) by stabilizing the carrier protein in the cell membrane, an effect requiring ß-glucuronidase activity of Klotho protein.
Assuntos
Glucuronidase/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/biossíntese , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/genética , Inibidores Enzimáticos/uso terapêutico , Glucuronidase/antagonistas & inibidores , Glucuronidase/genética , Glicoproteínas , Humanos , Proteínas Klotho , Proteínas do Tecido Nervoso/genética , Neurônios , Oócitos , Técnicas de Patch-Clamp , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , RNA Complementar/biossíntese , RNA Complementar/genética , Regulação para Cima , XenopusRESUMO
The Klotho gene was identified as an 'aging suppressor' in mice. Overexpression of the Klotho gene extends lifespan and defective Klotho results in rapid aging and early death. Both the membrane and secreted forms of Klotho have biological activity that include regulatory effects on general metabolism and a more specific effect on mineral metabolism that correlates with its effect on aging. Klotho serves as a co-receptor for fibroblast growth factor (FGF), but it also functions as a humoral factor that regulates cell survival and proliferation, vitamin D metabolism, and calcium and phosphate homeostasis and may serve as a potential tumor suppressor. Moreover, Klotho protects against several pathogenic processes in a FGF23-independent manner. These processes include cancer metastasis, vascular calcification, and renal fibrosis. This review covers the recent advances in Klotho research and discusses novel Klotho-dependent mechanisms that are clinically relevant in aging and age-related diseases.
Assuntos
Envelhecimento/fisiologia , Glucuronidase/fisiologia , Animais , Cálcio/metabolismo , Proliferação de Células , Sobrevivência Celular , Fator de Crescimento de Fibroblastos 23 , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Glucuronidase/química , Glucuronidase/genética , Glucuronidase/metabolismo , Homeostase , Humanos , Nefropatias/fisiopatologia , Proteínas Klotho , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Fosfatos/metabolismo , Transdução de Sinais , Calcificação Vascular/metabolismo , Calcificação Vascular/fisiopatologiaRESUMO
BACKGROUND/AIMS: The Na(+)-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na(+) gradient across the apical cell membrane, which is maintained by Na(+) extrusion across the basolateral cell membrane through the Na(+)/K(+) ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na(+) accumulation and K(+) loss with eventual decrease of cell membrane potential, Cl(-) entry and cell swelling. Upon energy depletion, early inhibition of Na(+)-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK), a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. METHODS: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPK(γ1)-HA), of inactive AMPK(αK45R) (AMPK(α1K45R)+AMPK(ß1)-Flag+AMPK(γ1)-HA) or of constitutively active AMPK(γR70Q) (AMPK(α1)-HA+AMPK(ß1)-Flag+AMPKγ1(R70Q)). NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. RESULTS: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM) to the extracellular bath solution generated a current (Ip), which was significantly decreased by coexpression of wild-type AMPK and of AMPK(γR70Q) but not of AMPK(αK45R). The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM). Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. CONCLUSION: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport. © 2013 S. Karger AG, Basel.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Regulação para Baixo/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Animais , Domínio Catalítico/genética , Feminino , Luminescência , Mutação , Oócitos/enzimologia , Oócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/biossíntese , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Xenopus laevisRESUMO
The Tau-tubulin-kinase 2 (TTBK2) is a serine/threonine kinase expressed in various tissues including tumors. Up-regulation of TTBK2 increases resistance of tumor cells against antiangiogenic treatment and confers cell survival. Tumor cell survival critically depends on cellular uptake of glucose, which is partially accomplished by SGLT1 (SLC5A1) mediated Na(+)-coupled glucose transport. The present study explored whether TTBK2 participates in the regulation of SGLT1 activity. To this end, electrogenic glucose transport was determined in Xenopus oocytes expressing SGLT1 with or without wild-type TTBK2, truncated TTBK2([1-450]) or kinase inactive mutants TTBK2-KD and TTBK2-KD([1-450]). TTBK2, but not TTBK2([1-450]), TTBK2-KD or TTBK2-KD([1-450]), increased membrane carrier protein abundance and electrogenic glucose transport capacity in SGLT1-expressing Xenopus oocytes. Thus TTBK2 is a completely novel regulator of Na(+)-coupled glucose transport.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Transportador 1 de Glucose-Sódio/metabolismo , Regulação para Cima , Animais , Feminino , Glucose/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Oócitos/metabolismo , Técnicas de Patch-Clamp , Proteínas Serina-Treonina Quinases/genética , Transportador 1 de Glucose-Sódio/genética , Xenopus/crescimento & desenvolvimentoRESUMO
The myoinositol transporter SMIT (SLC5A3) and the betaine/γ-aminobutyric acid (GABA) transporter BGT1 (SLC6A12) accomplish cellular accumulation of organic osmolytes and thus contribute to cell volume regulation. Challenges of cell volume constancy include energy depletion, which compromises the function of the Na(+)/K(+) ATPase leading to cellular Na(+) accumulation and subsequent cell swelling. Energy depletion is sensed by AMP-activated protein kinase (AMPK). The present study explored whether AMPK influences the activity of SMIT and BGT1. To this end, cRNA encoding SMIT or BGT1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKß1+AMPKγ1), of constitutively active (γR70Q)AMPK (AMPKα1+AMPKß1+(R70Q)AMPKγ1) or of catalytically inactive (αK45R)AMPK ((K45R)AMPKα1+AMPKß1+AMPKγ1). Substrate-induced current in dual electrode voltage-clamp experiments was taken as measure of osmolyte transport. As a result, in SMIT-expressing, but not in water-injected Xenopus oocytes, myoinositol, added to the extracellular bath, generated a current (I(SMIT)), which was half maximal (K(M)) at ≈7.2µM myoinositol concentration. Furthermore, in BGT1-expressing, but not in water-injected Xenopus oocytes, GABA added to the bath generated a current (I(GABA)), which was half maximal (K(M)) at ≈0.5mM GABA concentration. Coexpression of AMPK and of (γR70Q)AMPK but not of (αK45R)AMPK significantly decreased I(SMIT) and I(GABA). AMPK decreased the respective maximal currents without significantly modifying the respective K(M). In conclusion, the AMP-activated kinase AMPK is a powerful regulator of the organic osmolyte transporters SMIT and BGT1 and thus interacts with cell volume regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Transporte/metabolismo , Simportadores/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/antagonistas & inibidores , Regulação para Baixo , Proteínas da Membrana Plasmática de Transporte de GABA , Inositol/metabolismo , Oócitos , Simportadores/antagonistas & inibidores , Xenopus , Ácido gama-Aminobutírico/metabolismoRESUMO
The heterotetrameric K(+)-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na(+) channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKß1 + AMPKγ1), of the constitutively active (γR70Q)AMPK (α1ß1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)ß1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canal de Potássio KCNQ1/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Western Blotting , Membrana Celular/metabolismo , Imunofluorescência , Humanos , Ativação do Canal Iônico , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Túbulos Renais Proximais/metabolismo , Microscopia Confocal , Mutagênese Sítio-Dirigida , Ubiquitina-Proteína Ligases Nedd4 , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , RNA Complementar , Ribonucleotídeos/farmacologia , Xenopus , Proteínas de XenopusRESUMO
This study was undertaken to describe and characterize the relaxing effects of the medicinal plant Vitex agnus-castus (VAC) extract on isolated rabbit arterial rings. The VAC extracts (VACE) were extracted with ethanol and tested in aorta rings (3-4 mm) of rabbits suspended in an organ bath (Krebs, 37°C, 95% O2/5% CO2) under a resting tension of 1 g to record isometric contractions. After the stabilization period (1-2 hours), contractions were induced by the addition of phenylephrine (0.5 µM) or high KCl (80 mM) and VACE was added on the plateau of the contractions. Experiments were performed to determine the effects and to get insights into the potential mechanism involved in VACE-induced relaxations. The cumulative addition of VACE (0.15-0.75 mg/mL) relaxed, in a concentration-dependent manner, the rabbit aorta rings precontracted either with phenylephrine- or with high KCl thus suggesting calcium channel blocking activities. The VACE effect appeared to be endothelium-dependent. The preincubation with L-NAME (the inhibitor of nitric oxide synthases (NOS)), ODQ (the selective inhibitor of guanylyl cyclase), and indomethacin (the cyclooxygenase inhibitor), downregulated VACE-induced relaxation of aorta rings precontracted with phenylephrine, whereas the bradykinin (stimulator of NOS) and zaprinast (phosphodiesterase inhibitor) further upregulated relaxant effects induced by VACE. These results revealed that the aorta relaxation effect of VACE was mainly endothelium-dependent and mediated by NO/cGMP and prostaglandins synthesis. This vasodilator effect of VACE may be useful to treat cardiovascular disorders, including hypertensive diseases.
RESUMO
The Janus-activated kinase-2 JAK2 is involved in the signaling of leptin and erythropoietin receptors and mediates neuroprotective effects of the hormones. In theory, JAK2 could be effective through modulation of the glutamate transporters, carriers accounting for the clearance of glutamate released during neurotransmission. The present study thus elucidated the effect of JAK2 on the glutamate transporters EAAT1, EAAT2, EAAT3 and EAAT4. To this end, cRNA encoding the carriers was injected into Xenopus oocytes with or without cRNA encoding JAK2 and glutamate transport was estimated from glutamate induced current (I(glu)). I(glu) was observed in Xenopus oocytes expressing EAAT1 or EAAT2 or EAAT3 or EAAT4, but not in water injected oocytes. Coexpression of JAK2 resulted in an increase of I(glu) by 83% (EAAT1), 67% (EAAT2), 42% (EAAT3) and 126% (EAAT4). As shown for EAAT4 expressing Xenopus oocytes, the effect of JAK2 was mimicked by gain of function mutation (V617F)JAK2 but not by the inactive mutant (K882E)JAK2. Incubation with JAK2 inhibitor AG490 (40 µM) resulted in a gradual decrease of I(glu) by 53%, 79% and 92% within 3, 6 and 24 hours. Confocal microscopy and chemiluminescence analysis revealed that JAK2 coexpression increased EAAT4 protein abundance in the cell membrane. Disruption of transcription did not appreciably modify the up-regulation of I(glu) in EAAT4 expressing oocytes. The decay of I(glu) following inhibition of carrier insertion with brefeldin A was similar in oocytes expressing EAAT4 + JAK2 and oocytes expressing EAAT4 alone, indicating that JAK2 did not appreciably affect carrier retrieval from the membrane. In conclusion, JAK2 is a novel powerful regulator of glutamate transporters and thus participates in the protection against excitotoxicity.
Assuntos
Proteínas de Transporte de Glutamato da Membrana Plasmática/metabolismo , Janus Quinase 2/metabolismo , Substituição de Aminoácidos , Animais , Transportador 1 de Aminoácido Excitatório/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Transportador 4 de Aminoácido Excitatório/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Janus Quinase 2/genética , Oócitos/metabolismo , Técnicas de Patch-Clamp , Regulação para Cima , Xenopus laevis/genéticaRESUMO
Klotho, a transmembrane protein, protease and hormone has been shown to exert a profound effect on phosphate metabolism. Klotho overexpression lowers and Klotho deficiency increases the plasma phosphate concentration, effects in part attributed to an inhibitory effect of Klotho on the formation of 1,25-dihydroxycholecalciferol (1,25(OH) (2)D(3)), the active form of Vitamin D. Beyond that Klotho has been shown to decrease renal tubular phosphate transport more directly. The influence of Klotho on the plasma phosphate concentration contributes to the profound effect of Klotho on ageing and life span. The present study explored whether Klotho influences the major renal tubular (NaPi-IIa) and the major intestinal (NaPi-IIb) phosphate transporters. For functional analysis NaPi-IIa or NaPi-IIb were expressed in Xenopus oocytes both, without or with additional coexpression of Klotho and electrogenic phosphate transport was estimated from the phosphate-induced current (Ip). According to RT-PCR Klotho is expressed in the murine kidney and intestine. Coexpression of Klotho decreased Ip in both NaPi-IIa- and NaPi-IIb-expressing oocytes. Klotho decreased the maximal Ip without appreciably affecting the concentration required for halfmaximal Ip. Treatment of NaPi-IIa- or NaPi-IIb-expressing oocytes with Klotho protein similarly decreased Ip. In conclusion, Klotho down regulates both, renal (NaPi-IIa) and intestinal (NaPi-IIb) phosphate transporters.
Assuntos
Regulação para Baixo , Glucuronidase/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Animais , Calcifediol/farmacologia , Regulação para Baixo/efeitos dos fármacos , Glucuronidase/genética , Mucosa Intestinal/metabolismo , Intestinos/efeitos dos fármacos , Rim/efeitos dos fármacos , Rim/metabolismo , Proteínas Klotho , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Oócitos/fisiologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa/fisiologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/fisiologia , Xenopus laevisRESUMO
The inward rectifier K(+) channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen-Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKß1+AMPKγ1), of the constitutively active (γR70Q)AMPK (α1ß1γ1(R70Q)), of the kinase dead mutant (αK45R)AMPK (α1(K45R)ß1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2(S795A) lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2(S795A) was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Animais , Regulação para Baixo , Humanos , Mutação , Oócitos , Fosforilação , XenopusRESUMO
The coronavirus disease 2019 (COVID-19) is currently a new public health crisis threatening the world. This pandemic disease is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus has been reported to be originated in bats, and by yet unknown intermediary animals were transmitted to humans in China 2019. The SARS-CoV-2 spreads faster than its two ancestors, the SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) but has reduced fatality. At present, the SARS-CoV-2 has caused about 1.16 million deaths with more than 43.4 million confirmed cases worldwide, resulting in a serious threat to public health globally with yet uncertain impact. The disease is transmitted by inhalation or direct contact with an infected person. The incubation period ranges from 1 to 14 days. COVID-19 is accompanied by various symptoms, including cough and fatigue. In most people, the disease is mild, but in some other people, such as in the elderly and people with chronic diseases, it may progress from pneumonia to a multi-organ dysfunction. Many people are reported asymptomatic. The virus genome is sequenced, but new variants are reported. Numerous biochemical aspects of its structure and function are revealed. To date, no clinically approved vaccines and/or specific therapeutic drugs are available to prevent or treat COVID-19. However, there are reported intensive researches on the SARS-CoV-2 to potentially identify vaccines and/or drug targets, which may help to overcome the disease. In this review, we discuss recent advances in understanding the molecular structure of SARS-CoV-2 and its biochemical characteristics.
Assuntos
COVID-19/diagnóstico , Genoma Viral , SARS-CoV-2/fisiologia , SARS-CoV-2/patogenicidade , Proteínas Virais/metabolismo , COVID-19/etiologia , COVID-19/virologia , Coronavirus/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , SARS-CoV-2/genética , Proteínas Virais/genética , Internalização do Vírus , Replicação ViralRESUMO
Angiotensin-converting enzyme (ACE) shares some homologies with ACE2. However, they are not inhibited by the same inhibitors, but both are associated primarily with the hypertensive disorder through the renin-angiotensin system (RAS). The principal activity of ACE2 is to metabolize Ang II into the vasodilatory Ang-(1-7). The ongoing COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought the ACE2 to the center of attention. This coronavirus uses the host cell ACE2 protein to enter and infect the epithelial cells. In light of the virus's entrance into human cells, the differences in the molecular basis of ACE2 among affected patients may cause their different responses to the virus. Many details about the specific interaction between the viral S protein and ACE2 are already reported. To date, some effective clinically approved vaccines are in use globally, and many others are under development, but no effective specific therapeutic drugs are available against COVID-19. Inhibitors, especially peptide inhibitors, have a great potential to be used for the treatment of COVID-19 and other possible emerging diseases caused by viral pathogens. As a result of the well-known viral protein structures and their host cell targets such as ACE2, antiviral peptides could be appropriately designed and optimized for therapeutic purposes. A better understanding of the structure and pathophysiology of the ACE2 receptor and the interplay between the viral S protein and ACE2 may help to find the solution for the virus treatment. This review summarizes the current understanding of S protein interaction with the ACE2 protein as a potential specific target against SARS-CoV-2 and strategies using peptides against COVID-19.
Assuntos
Enzima de Conversão de Angiotensina 2 , Glicoproteína da Espícula de Coronavírus , COVID-19 , PandemiasRESUMO
Janus kinase-2 (JAK2) is a non-receptor tyrosine kinase that serves key roles as the intracellular signaling effector of the cytokine receptor, such as mediating effects of leptin, erythropoietin, interferon, and growth hormone. A lot of molecular underlying mechanisms of JAK2 participation are known, however, additional signaling mechanisms of its activation, regulation, and pleiotropic signaling roles are still being explored. Here, we review the current knowledge of JAK2-mediated cellular signaling at the molecular level. In the beginning, we will focus on the recent advances in JAK2 activation and regulation. A part of our review focuses on the JAK2 involvement in various diseases/conditions. Recent advances highlight the molecular regulatory mechanisms utilized by the JAK2 signaling, thus, enabling to consider alternative therapeutic strategies to treat various diseases/conditions mediated by JAK2 by using it as a therapeutic target.
Assuntos
Janus Quinase 2/metabolismo , Transdução de Sinais/fisiologia , HumanosRESUMO
Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.
Assuntos
Células Dendríticas/efeitos dos fármacos , Imunossupressores/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Sirolimo/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/fisiologia , Células Dendríticas/imunologia , Feminino , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
The glutamate transporters EAAT3 and EAAT4 are expressed in neurons. They contribute to the cellular uptake of glutamate and aspartate and thus to the clearance of the excitatory transmitters from the extracellular space. During ischemia, extracellular accumulation of glutamate may trigger excitotoxicity. Energy depletion leads to activation of the AMP-activated protein kinase (AMPK), a kinase enhancing energy production and limiting energy expenditure. The present study thus explored the possibility that AMPK regulates EAAT3 and/or EAAT4. To this end, EAAT3 or EAAT4 were expressed in Xenopus oocytes with or without AMPK and electrogenic glutamate transport determined by dual electrode voltage clamp. In EAAT3- and in EAAT4-expressing oocytes glutamate generated a current (I(g)), which was half maximal (K(M)) at 74 microM (EAAT3) or at 4 microM (EAAT4) glutamate. Co-expression of constitutively active (gammaR70Q)AMPK or of wild type AMPK did not affect K(M) but significantly decreased the maximal I(g) in both EAAT3- (by 34%) and EAAT4- (by 49%) expressing oocytes. Co-expression of the inactive mutant (alphaK45R)AMPK [alpha1(K45R)beta1gamma1] did not appreciably affect I(g). According to confocal microscopy and chemiluminescence co-expression of (gammaR70Q)AMPK or of wild type AMPK reduced the membrane abundance of EAAT3 and EAAT4. The observations show that AMPK down-regulates Na(+)-coupled glutamate transport.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Regulação para Baixo/fisiologia , Transportador 3 de Aminoácido Excitatório/metabolismo , Transportador 4 de Aminoácido Excitatório/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Arginina/genética , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/farmacologia , Transportador 3 de Aminoácido Excitatório/genética , Transportador 4 de Aminoácido Excitatório/genética , Ácido Glutâmico/farmacologia , Glutamina/genética , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Microscopia Confocal/métodos , Mutação/genética , Ubiquitina-Proteína Ligases Nedd4 , Óxidos de Nitrogênio/farmacologia , Oócitos , Técnicas de Patch-Clamp/métodos , Transdução Genética , Tirosina/genética , Ubiquitina-Proteína Ligases/farmacologia , XenopusRESUMO
Glycogen synthase kinase 3 GSK3ß participates in a wide variety of functions including regulation of glucose metabolism. It is ubiquitously expressed including epithelial tissues. However, whether GSK3ß participates in the regulation of epithelial transport is not known. The present study thus explored whether GSK3ß influences the Na(+)-coupled transport of glucose. To this end, SGLT1 was expressed in Xenopus oocytes with or without GSK3ß and glucose-induced current (I(g)) determined by dual electrode voltage clamp. In Xenopus oocytes expressing SGLT1 but not in water-injected oocytes glucose induced an inwardly directed I(g), which was significantly enhanced by coexpression of GSK3ß. According to chemiluminescence and confocal microscopy, GSK3ß increased the SGLT1 protein abundance in the oocyte cell membrane. To explore whether GSK3ß sensitivity of SGLT1 participates in the regulation of electrogenic intestinal glucose transport, Ussing chamber experiments were performed in intestinal segments from gene-targeted knockin mice with mutated and thus PKB/SGK-resistant GSK3α,ß (gsk3(KI)), in which the serine of the PKB/SGK phosphorylation site was replaced by alanine, and from wild type mice (gsk3(WT)). The glucose-induced current was significantly larger in gsk3(KI) than in gsk3(WT) mice. The present observations reveal a novel function of GSK3, i.e. the stimulation of Na(+)-coupled glucose transport.
Assuntos
Glucose/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Feminino , Técnicas de Introdução de Genes , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Masculino , Camundongos , Mutação , Oócitos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Xenopus/crescimento & desenvolvimentoRESUMO
ß-Catenin is a multifunctional protein stimulating as oncogenic transcription factor several genes important for cell proliferation. ß-Catenin-regulated genes include the serum- and glucocorticoid-inducible kinase SGK1, which is known to stimulate a variety of transport systems. The present study explored the possibility that ß-catenin influences membrane transport. To this end, ß-catenin was expressed in Xenopus oocytes with or without SGLT1 and electrogenic transport determined by dual electrode voltage clamp. As a result, expression of ß-catenin significantly enhanced the ouabain-sensitive current of the endogeneous Na(+)/K(+)-ATPase. Inhibition of vesicle trafficking by brefeldin A revealed that the stimulatory effect of ß-catenin on the endogenous Na(+)/K(+)-ATPase was not due to enhanced stability of the pump protein in the cell membrane. Expression of ß-catenin further enhanced glucose-induced current (Ig) in SGLT1-expressing oocytes. In the absence of SGLT1 Ig was negligible irrespective of ß-catenin expression. The stimulating effect of ß-catenin on both Na(+)/K(+) ATPase and SGLT1 activity was observed even in the presence of actinomycin D, an inhibitor of transcription. The experiments disclose a completely novel function of ß-catenin, i.e. the regulation of transport.