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1.
Nat Commun ; 11(1): 4955, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009385

RESUMO

The light-harvesting-reaction center complex (LH1-RC) from the purple phototrophic bacterium Thiorhodovibrio strain 970 exhibits an LH1 absorption maximum at 960 nm, the most red-shifted absorption for any bacteriochlorophyll (BChl) a-containing species. Here we present a cryo-EM structure of the strain 970 LH1-RC complex at 2.82 Å resolution. The LH1 forms a closed ring structure composed of sixteen pairs of the αß-polypeptides. Sixteen Ca ions are present in the LH1 C-terminal domain and are coordinated by residues from the αß-polypeptides that are hydrogen-bonded to BChl a. The Ca2+-facilitated hydrogen-bonding network forms the structural basis of the unusual LH1 redshift. The structure also revealed the arrangement of multiple forms of α- and ß-polypeptides in an individual LH1 ring. Such organization indicates a mechanism of interplay between the expression and assembly of the LH1 complex that is regulated through interactions with the RC subunits inside.


Assuntos
Cálcio/metabolismo , Microscopia Crioeletrônica , Complexos de Proteínas Captadores de Luz/ultraestrutura , Peptídeos/metabolismo , Fotossíntese , Sequência de Aminoácidos , Bacterioclorofila A/metabolismo , Sítios de Ligação , Chromatiaceae/metabolismo , Detergentes/química , Dimerização , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos/química , Peptídeos/química , Quinonas/química
2.
Nat Commun ; 11(1): 4614, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929069

RESUMO

The suprachiasmatic nucleus (SCN) is a complex structure dependent upon multiple mechanisms to ensure rhythmic electrical activity that varies between day and night, to determine circadian adaptation and behaviours. SCN neurons are exposed to glutamate from multiple sources including from the retino-hypothalamic tract and from astrocytes. However, the mechanism preventing inappropriate post-synaptic glutamatergic effects is unexplored and unknown. Unexpectedly we discovered that TRESK, a calcium regulated two-pore potassium channel, plays a crucial role in this system. We propose that glutamate activates TRESK through NMDA and AMPA mediated calcium influx and calcineurin activation to then oppose further membrane depolarisation and rising intracellular calcium. Hence, in the absence of TRESK, glutamatergic activity is unregulated leading to membrane depolarisation, increased nocturnal SCN firing, inverted basal calcium levels and impaired sensitivity in light induced phase delays. Our data reveals TRESK plays an essential part in SCN regulatory mechanisms and light induced adaptive behaviours.


Assuntos
Adaptação Ocular , Escuridão , Canais de Potássio/metabolismo , Núcleo Supraquiasmático/fisiologia , Animais , Comportamento Animal , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Luz , Potenciais da Membrana/efeitos da radiação , Camundongos Endogâmicos C57BL , Canais de Potássio/deficiência , Transdução de Sinais/efeitos da radiação , Núcleo Supraquiasmático/efeitos da radiação
3.
Nat Commun ; 11(1): 4581, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917893

RESUMO

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética
4.
Nat Commun ; 11(1): 4416, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887881

RESUMO

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Dilatada/metabolismo , Infarto do Miocárdio/fisiopatologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/fisiopatologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/cirurgia , Transdução de Sinais
5.
PLoS Comput Biol ; 16(9): e1008198, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32931495

RESUMO

Calcium imaging with fluorescent protein sensors is widely used to record activity in neuronal populations. The transform between neural activity and calcium-related fluorescence involves nonlinearities and low-pass filtering, but the effects of the transformation on analyses of neural populations are not well understood. We compared neuronal spikes and fluorescence in matched neural populations in behaving mice. We report multiple discrepancies between analyses performed on the two types of data, including changes in single-neuron selectivity and population decoding. These were only partially resolved by spike inference algorithms applied to fluorescence. To model the relation between spiking and fluorescence we simultaneously recorded spikes and fluorescence from individual neurons. Using these recordings we developed a model transforming spike trains to synthetic-imaging data. The model recapitulated the differences in analyses. Our analysis highlights challenges in relating electrophysiology and imaging data, and suggests forward modeling as an effective way to understand differences between these data.


Assuntos
Cálcio/metabolismo , Fenômenos Eletrofisiológicos/fisiologia , Modelos Neurológicos , Imagem Molecular/métodos , Neurônios , Potenciais de Ação/fisiologia , Animais , Lobo Frontal/citologia , Lobo Frontal/fisiologia , Camundongos , Neurônios/metabolismo , Neurônios/fisiologia , Imagem Óptica
6.
PLoS Biol ; 18(9): e3000873, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32966273

RESUMO

The inhibitory axonless olfactory bulb granule cells form reciprocal dendrodendritic synapses with mitral and tufted cells via large spines, mediating recurrent and lateral inhibition. As a case in point for dendritic transmitter release, rat granule cell dendrites are highly excitable, featuring local Na+ spine spikes and global Ca2+- and Na+-spikes. To investigate the transition from local to global signaling, we performed holographic, simultaneous 2-photon uncaging of glutamate at up to 12 granule cell spines, along with whole-cell recording and dendritic 2-photon Ca2+ imaging in acute juvenile rat brain slices. Coactivation of less than 10 reciprocal spines was sufficient to generate diverse regenerative signals that included regional dendritic Ca2+-spikes and dendritic Na+-spikes (D-spikes). Global Na+-spikes could be triggered in one third of granule cells. Individual spines and dendritic segments sensed the respective signal transitions as increments in Ca2+ entry. Dendritic integration as monitored by the somatic membrane potential was mostly linear until a threshold number of spines was activated, at which often D-spikes along with supralinear summation set in. As to the mechanisms supporting active integration, NMDA receptors (NMDARs) strongly contributed to all aspects of supralinearity, followed by dendritic voltage-gated Na+- and Ca2+-channels, whereas local Na+ spine spikes, as well as morphological variables, barely mattered. Because of the low numbers of coactive spines required to trigger dendritic Ca2+ signals and thus possibly lateral release of GABA onto mitral and tufted cells, we predict that thresholds for granule cell-mediated bulbar lateral inhibition are low. Moreover, D-spikes could provide a plausible substrate for granule cell-mediated gamma oscillations.


Assuntos
Potenciais de Ação , Sinalização do Cálcio , Dendritos/metabolismo , Bulbo Olfatório/metabolismo , Sódio/metabolismo , Animais , Cálcio/metabolismo , Feminino , Holografia , Técnicas In Vitro , Masculino , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo
7.
Nat Commun ; 11(1): 4117, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807785

RESUMO

Strategies for eradicating cancer stem cells (CSCs) are urgently required because CSCs are resistant to anticancer drugs and cause treatment failure, relapse and metastasis. Here, we show that photoactive functional nanocarbon complexes exhibit unique characteristics, such as homogeneous particle morphology, high water dispersibility, powerful photothermal conversion, rapid photoresponsivity and excellent photothermal stability. In addition, the present biologically permeable second near-infrared (NIR-II) light-induced nanocomplexes photo-thermally trigger calcium influx into target cells overexpressing the transient receptor potential vanilloid family type 2 (TRPV2). This combination of nanomaterial design and genetic engineering effectively eliminates cancer cells and suppresses stemness of cancer cells in vitro and in vivo. Finally, in molecular analyses of mechanisms, we show that inhibition of cancer stemness involves calcium-mediated dysregulation of the Wnt/ß-catenin signalling pathway. The present technological concept may lead to innovative therapies to address the global issue of refractory cancers.


Assuntos
Raios Infravermelhos , Nanotecnologia/métodos , Células-Tronco Neoplásicas/efeitos da radiação , Animais , Apoptose/efeitos da radiação , Western Blotting , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/metabolismo , Via de Sinalização Wnt
8.
Nat Commun ; 11(1): 3984, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770009

RESUMO

The epsin family of endocytic adapter proteins are widely expressed, and interact with both proteins and lipids to regulate a variety of cell functions. However, the role of epsins in atherosclerosis is poorly understood. Here, we show that deletion of endothelial epsin proteins reduces inflammation and attenuates atherosclerosis using both cell culture and mouse models of this disease. In atherogenic cholesterol-treated murine aortic endothelial cells, epsins interact with the ubiquitinated endoplasmic reticulum protein inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), which triggers proteasomal degradation of this calcium release channel. Epsins potentiate its degradation via this interaction. Genetic reduction of endothelial IP3R1 accelerates atherosclerosis, whereas deletion of endothelial epsins stabilizes IP3R1 and mitigates inflammation. Reduction of IP3R1 in epsin-deficient mice restores atherosclerotic progression. Taken together, epsin-mediated degradation of IP3R1 represents a previously undiscovered biological role for epsin proteins and may provide new therapeutic targets for the treatment of atherosclerosis and other diseases.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aterosclerose/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteólise , Proteínas Adaptadoras de Transporte Vesicular/química , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/patologia , Cálcio/metabolismo , Colesterol/metabolismo , Células Endoteliais/metabolismo , Feminino , Deleção de Genes , Células HEK293 , Homeostase , Humanos , Inflamação/patologia , Masculino , Camundongos Knockout , Ligação Proteica , Domínios Proteicos , Ubiquitinação
9.
Nat Commun ; 11(1): 4031, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788582

RESUMO

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.


Assuntos
Canais de Cálcio/genética , Evolução Molecular , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/química , Quitridiomicetos/genética , Proteínas Fúngicas/química , Células HeLa , Humanos , Funções Verossimilhança , Filogenia , Especificidade da Espécie
10.
PLoS Genet ; 16(8): e1008963, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32780743

RESUMO

Long-term memory (LTM) formation depends on the conversed cAMP response element-binding protein (CREB)-dependent gene transcription followed by de novo protein synthesis. Thirsty fruit flies can be trained to associate an odor with water reward to form water-reward LTM (wLTM), which can last for over 24 hours without a significant decline. The role of de novo protein synthesis and CREB-regulated gene expression changes in neural circuits that contribute to wLTM remains unclear. Here, we show that acute inhibition of protein synthesis in the mushroom body (MB) αß or γ neurons during memory formation using a cold-sensitive ribosome-inactivating toxin disrupts wLTM. Furthermore, adult stage-specific expression of dCREB2b in αß or γ neurons also disrupts wLTM. The MB αß and γ neurons can be further classified into five different neuronal subsets including αß core, αß surface, αß posterior, γ main, and γ dorsal. We observed that the neurotransmission from αß surface and γ dorsal neuron subsets is required for wLTM retrieval, whereas the αß core, αß posterior, and γ main are dispensable. Adult stage-specific expression of dCREB2b in αß surface and γ dorsal neurons inhibits wLTM formation. In vivo calcium imaging revealed that αß surface and γ dorsal neurons form wLTM traces with different dynamic properties, and these memory traces are abolished by dCREB2b expression. Our results suggest that a small population of neurons within the MB circuits support long-term storage of water-reward memory in Drosophila.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Memória de Longo Prazo/fisiologia , Neurônios/metabolismo , Olfato/genética , Transativadores/genética , Animais , Animais Geneticamente Modificados , Cálcio/metabolismo , Drosophila melanogaster/fisiologia , Corpos Pedunculados/fisiologia , Neurônios/fisiologia , Biossíntese de Proteínas/genética , Recompensa , Olfato/fisiologia , Transmissão Sináptica/genética , Água
11.
Nat Commun ; 11(1): 4012, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782388

RESUMO

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Indenos/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Ribossomos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
12.
Nat Commun ; 11(1): 4082, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796832

RESUMO

The phytohormone ethylene has numerous effects on plant growth and development. Its immediate precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is a non-proteinogenic amino acid produced by ACC SYNTHASE (ACS). ACC is often used to induce ethylene responses. Here, we demonstrate that ACC exhibits ethylene-independent signaling in Arabidopsis thaliana reproduction. By analyzing an acs octuple mutant with reduced seed set, we find that ACC signaling in ovular sporophytic tissue is involved in pollen tube attraction, and promotes secretion of the pollen tube chemoattractant LURE1.2. ACC activates Ca2+-containing ion currents via GLUTAMATE RECEPTOR-LIKE (GLR) channels in root protoplasts. In COS-7 cells expressing moss PpGLR1, ACC induces the highest cytosolic Ca2+ elevation compared to all twenty proteinogenic amino acids. In ovules, ACC stimulates transient Ca2+ elevation, and Ca2+ influx in octuple mutant ovules rescues LURE1.2 secretion. These findings uncover a novel ACC function and provide insights for unraveling new physiological implications of ACC in plants.


Assuntos
Arabidopsis/metabolismo , Etilenos/metabolismo , Óvulo Vegetal/metabolismo , Tubo Polínico/metabolismo , Aminoácidos Cíclicos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Liases/metabolismo , Reguladores de Crescimento de Planta/metabolismo
13.
Chemosphere ; 258: 127408, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32782161

RESUMO

This study investigates the impacts of exposure to an environment Ca2+ challenge and the mechanism of action of dibutyl phthalate (DBP) on Ca2+ influx in the gills of Danio rerio. In vitro profile of 45Ca2+ influx in gills was verified through the basal time-course. Fish were exposed to low, normal and high Ca2+ concentrations (0.02, 0.7 and 2 mM) for 12 h. So, gills were morphologically analysed and ex vivo45Ca2+ influx at 30 and 60 min was determined. For the in vitro studies, gills were treated for 60 min with DBP (1 pM, 1 nM and 1 µM) with/without blockers/activators of ionic channels, Ca2+ chelator, inhibitors of ATPases, ionic exchangers and protein kinase C to study the mechanism of DBP-induced 45Ca2+ influx. Exposure to high environmental Ca2+ augmented 45Ca2+ influx when compared to fish exposed to normal and low Ca2+ concentrations. Additionally, histopathological changes were observed in the gills of fish maintained for 12 h in low and high Ca2+. In vitro exposure of gills to DBP (1 pM) disturbed Ca2+ homeostasis. DBP stimulated 45Ca2+ influx in gills through the transitory receptor potential vanilloid 1 (TRPV1), and reverse-mode Na+/Ca2+ exchanger (NCX) activation, protein kinase C and K+ channels and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). These data suggest that in vivo short-term exposure of gills to low and high Ca2+ leads to 45Ca2+ influx and histopathological changes. Additionally, the DBP-induced rapid 45Ca2+ influx is mediated by TRPV1, NCX activation with the involvement of PKC, K+-channels and SERCA, thereby altering Ca2+ homeostasis.


Assuntos
Radioisótopos de Cálcio/metabolismo , Cálcio/metabolismo , Dibutilftalato/toxicidade , Brânquias/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/metabolismo , Animais , Cálcio/toxicidade , Dibutilftalato/metabolismo , Retículo Endoplasmático/metabolismo , Brânquias/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Canais de Cátion TRPV/metabolismo , Poluentes Químicos da Água/metabolismo
14.
Nat Commun ; 11(1): 4243, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843625

RESUMO

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1ß release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1ß release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.


Assuntos
Artrite Reumatoide/imunologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Animais , Calcinose , Cálcio/metabolismo , Células Cultivadas , Humanos , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Fosfatos/metabolismo , Pinocitose , Receptores de Detecção de Cálcio/deficiência , Transdução de Sinais , Células THP-1 , alfa-2-Glicoproteína-HS/metabolismo
15.
PLoS Biol ; 18(8): e3000826, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776935

RESUMO

Ca2+/calmodulin-dependent kinase II (CaMKII) regulates synaptic plasticity in multiple ways, supposedly including the secretion of neuromodulators like brain-derived neurotrophic factor (BDNF). Here, we show that neuromodulator secretion is indeed reduced in mouse α- and ßCaMKII-deficient (αßCaMKII double-knockout [DKO]) hippocampal neurons. However, this was not due to reduced secretion efficiency or neuromodulator vesicle transport but to 40% reduced neuromodulator levels at synapses and 50% reduced delivery of new neuromodulator vesicles to axons. αßCaMKII depletion drastically reduced neuromodulator expression. Blocking BDNF secretion or BDNF scavenging in wild-type neurons produced a similar reduction. Reduced neuromodulator expression in αßCaMKII DKO neurons was restored by active ßCaMKII but not inactive ßCaMKII or αCaMKII, and by CaMKII downstream effectors that promote cAMP-response element binding protein (CREB) phosphorylation. These data indicate that CaMKII regulates neuromodulation in a feedback loop coupling neuromodulator secretion to ßCaMKII- and CREB-dependent neuromodulator expression and axonal targeting, but CaMKIIs are dispensable for the secretion process itself.


Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cálcio/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Animais , Astrócitos/citologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/deficiência , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Fosforilação , Cultura Primária de Células , Subunidades Proteicas/deficiência , Sinapses/fisiologia , Transmissão Sináptica , Imagem com Lapso de Tempo
16.
Nat Commun ; 11(1): 4328, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859919

RESUMO

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's anti-depressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.


Assuntos
Ketamina/efeitos adversos , Transdução de Sinais/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/genética , Proliferação de Células , Cistite/induzido quimicamente , Modelos Animais de Doenças , Humanos , Rim/efeitos dos fármacos , Rim/patologia , Camundongos , Camundongos Knockout , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Músculo Liso/patologia , Oócitos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Bexiga Urinária/patologia , Xenopus
17.
Chem Biol Interact ; 330: 109178, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32738201

RESUMO

The capsaicin (vanilloid) receptor, TRPV1, is a heat-activated cation channel modulated by inflammatory mediators and contributes to acute and chronic pain. TRPV1 channel is one of the most researched and targeted mechanisms for the development of novel analgesics. Over the years, natural products have contributed enormously to the development of important therapeutic drugs used currently in modern medicine. A literature review was conducted using Medline, Google Scholar, and PubMed. Searching the literature resulted in listing 136 natural compounds that interacted with TRPV1 channel. These compounds were phytochemicals that belong to different chemical groups including vanilloids, flavonoids, alkaloids, terpenoids, terpenyl phenols, fatty acids, cannabinoids, sulfur_containing compounds, etc. Other natural TRPV1 modulators were of animal, fungal or bacterial origin. Some natural products were small agonists or antagonists of TRPV1. Others were protein venoms. Most in vitro studies utilized electrophysiological or calcium imaging techniques to study calcium flow through the channel using primary cultures of rat dorsal root and trigeminal ganglia. Other studies used hTRPV1 or rTRPV1 expressed in HEK239, CHO cells or Xenopus oocytes. In vivo studies concentrated on different pain models conducted mainly in mice and rats. In conclusion, natural products are highly diverse in their modulatory action on TRPV1. Many gaps in natural product research are present in distinguishing modality-specific from polymodal antagonists. Species' differences in TRPV1 functionality must be taken into account in any future study. Proceeding into clinical trials needs more efforts to discover potent TRPV1 antagonists devoid of hyperthermia, the main side effect.


Assuntos
Analgésicos/farmacologia , Produtos Biológicos/farmacologia , Canais de Cátion TRPV/metabolismo , Analgésicos/efeitos adversos , Analgésicos/uso terapêutico , Animais , Produtos Biológicos/efeitos adversos , Cálcio/metabolismo , Técnicas de Cultura de Células , Febre/etiologia , Humanos , Dor/complicações , Dor/tratamento farmacológico , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/genética
18.
Life Sci ; 258: 118232, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781066

RESUMO

AIMS: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca2+ increase in androgen-independent prostate cancer (PCa) cells. MAIN METHODS: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca2+ dynamics by the fluorescent Ca2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. KEY FINDINGS: EGCG inhibited DU145 and PC3 proliferation with IC50 = 46 and 56 µM, respectively, and induced dose-dependent peaks of internal Ca2+ that were dependent on extracellular Ca2+. The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca2+ influx acts as a trigger for Ca2+-induced Ca2+ release, involving both ryanodine and IP3 receptors. Different from EGCG, ATP caused a rapid Ca2+ increase, which was independent of external Ca2+, but sensitive to U73122. SIGNIFICANCE: EGCG induces an internal Ca2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.


Assuntos
Antioxidantes/farmacologia , Cálcio/metabolismo , Catequina/análogos & derivados , Líquido Intracelular/metabolismo , Neoplasias da Próstata/metabolismo , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Líquido Intracelular/efeitos dos fármacos , Masculino , Células PC-3
19.
PLoS One ; 15(8): e0237591, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833978

RESUMO

The slow cardiac delayed rectifier current (IKs) is formed by KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Decrease of IKs currents either due to inherited mutations or pathological remodeling is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in heart disease and diabetes. Recently, we found that sustained stimulation of the calcium-dependent PKCßII isoform leads to decrease in KCNQ1 subunit membrane localization and KCNQ1/KCNE1 channel activity, although the role of KCNE1 in this regulation was not explored. Here, we show that the auxiliary KCNE1 subunit expression is necessary for channel internalization. A mutation in a KCNE1 phosphorylation site (KCNE1(S102A)) abolished channel internalization in both heterologous expression systems and cardiomyocytes. Altogether, our results suggest that KCNE1(S102) phosphorylation by PKCßII leads to KCNQ1/KCNE1 channel internalization in response to sustained PKC stimulus, while leaving KCNQ1 homomeric channels in the membrane. This preferential internalization is expected to have strong impact on cardiac repolarization. Our results suggest that KCNE1(S102) is an important anti-arrhythmic drug target to prevent IKs pathological remodeling leading to cardiac arrhythmias.


Assuntos
Cálcio/metabolismo , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteína Quinase C/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Canal de Potássio KCNQ1/genética , Mutação , Miócitos Cardíacos/citologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteína Quinase C/genética , Ratos
20.
Am Surg ; 86(8): 976-980, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32762469

RESUMO

BACKGROUND: Coronary artery disease (CAD) is a leading cause of mortality following orthotopic liver transplant, yet there is no standardized protocol for pre-liver-transplant coronary artery disease assessment. The main objective of this study was to determine the agreement between 2 methods of cardiac risk assessment: dobutamine stress echocardiogram (DSE) and coronary calcium score (CCS) and to determine which test was best able to predict coronary calcification in low-risk patients. METHODS: A retrospective study was performed using the medical records of 436 patients who received cardiac clearance for a liver transplant. A total of 152 patients' medical records were included based on the inclusion of patients who had received both DSE and CCS. A kappa coefficient was calculated to determine the agreement between the DSE and CCS results. In addition, the positive predictive values (PPVs) of both the CCS and DSE along with cardiac catheterization indicating abdominal occlusion were analyzed to compare the accuracy of the 2 tests. RESULTS: It was determined that there was a 12% agreement between DSE results and CCS. It was found that the DSE had a PPV of 56% and the CCS had a PPV of 80%. CONCLUSION: From this data, it was concluded that there was no agreement between the results of the CCS and the DSE. While neither the CCS nor the DSE presents an optimal method of risk assessment, the CCS had a much higher PPV and was therefore determined to be the more accurate test.


Assuntos
Doença da Artéria Coronariana/diagnóstico , Transplante de Fígado , Complicações Pós-Operatórias/prevenção & controle , Cuidados Pré-Operatórios/métodos , Adulto , Idoso , Biomarcadores/metabolismo , Cálcio/metabolismo , Cateterismo Cardíaco , Regras de Decisão Clínica , Doença da Artéria Coronariana/etiologia , Doença da Artéria Coronariana/metabolismo , Ecocardiografia sob Estresse , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias/diagnóstico , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Medição de Risco , Sensibilidade e Especificidade
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