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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34156466

RESUMO

Store-operated Ca2+ entry is a central component of intracellular Ca2+ signaling pathways. The Ca2+ release-activated channel (CRAC) mediates store-operated Ca2+ entry in many different cell types. The CRAC channel is composed of the plasma membrane (PM)-localized Orai1 channel and endoplasmic reticulum (ER)-localized STIM1 Ca2+ sensor. Upon ER Ca2+ store depletion, Orai1 and STIM1 form complexes at ER-PM junctions, leading to the formation of activated CRAC channels. Although the importance of CRAC channels is well described, the underlying mechanisms that regulate the recruitment of Orai1 to ER-PM junctions are not fully understood. Here, we describe the rapid and transient S-acylation of Orai1. Using biochemical approaches, we show that Orai1 is rapidly S-acylated at cysteine 143 upon ER Ca2+ store depletion. Importantly, S-acylation of cysteine 143 is required for Orai1-mediated Ca2+ entry and recruitment to STIM1 puncta. We conclude that store depletion-induced S-acylation of Orai1 is necessary for recruitment to ER-PM junctions, subsequent binding to STIM1 and channel activation.


Assuntos
Canais de Cálcio , Cálcio , Acilação , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
2.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205207

RESUMO

Since dysregulation of intracellular calcium (Ca2+) levels is a common occurrence in neurodegenerative diseases, including Alzheimer's disease (AD), the study of proteins that can correct neuronal Ca2+ dysregulation is of great interest. In previous work, we have shown that plasma membrane Ca2+-ATPase (PMCA), a high-affinity Ca2+ pump, is functionally impaired in AD and is inhibited by amyloid-ß peptide (Aß) and tau, two key components of pathological AD hallmarks. On the other hand, sorcin is a Ca2+-binding protein highly expressed in the brain, although its mechanism of action is far from being clear. Sorcin has been shown to interact with the intracellular sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), and other modulators of intracellular Ca2+ signaling, such as the ryanodine receptor or presenilin 2, which is closely associated with AD. The present work focuses on sorcin in search of new regulators of PMCA and antagonists of Aß and tau toxicity. Results show sorcin as an activator of PMCA, which also prevents the inhibitory effects of Aß and tau on the pump, and counteracts the neurotoxicity of Aß and tau by interacting with them.


Assuntos
Doença de Alzheimer/genética , Proteínas de Ligação ao Cálcio/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Encéfalo/patologia , Cálcio/metabolismo , Sinalização do Cálcio/genética , Humanos , Neurônios/metabolismo , Neurônios/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Presenilina-2/genética , Ligação Proteica/genética , Mapas de Interação de Proteínas/genética , Proteínas tau/genética
3.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199520

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl- channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1-/-, was larger in male when compared to female Pkd1-/- cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1-/- males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.


Assuntos
Anoctamina-1/genética , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proliferação de Células/genética , Cloretos/metabolismo , Di-Hidrotestosterona/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Caracteres Sexuais
4.
FASEB J ; 35(8): e21723, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34224609

RESUMO

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.


Assuntos
Canais de Cálcio/metabolismo , Espermatozoides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/deficiência , Canais de Cálcio/genética , Sinalização do Cálcio , AMP Cíclico/metabolismo , Feminino , Fertilização In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Transdução de Sinais , Capacitação Espermática/fisiologia , Motilidade Espermática/fisiologia , Cauda do Espermatozoide/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/ultraestrutura , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores
5.
Nat Commun ; 12(1): 3321, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059669

RESUMO

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder. The mechanisms underlying ASD are unclear. Astrocyte alterations are noted in ASD patients and animal models. However, whether astrocyte dysfunction is causal or consequential to ASD-like phenotypes in mice is unresolved. Type 2 inositol 1,4,5-trisphosphate 6 receptors (IP3R2)-mediated Ca2+ release from intracellular Ca2+ stores results in the activation of astrocytes. Mutations of the IP3R2 gene are associated with ASD. Here, we show that both IP3R2-null mutant mice and astrocyte-specific IP3R2 conditional knockout mice display ASD-like behaviors, such as atypical social interaction and repetitive behavior. Furthermore, we show that astrocyte-derived ATP modulates ASD-like behavior through the P2X2 receptors in the prefrontal cortex and possibly through GABAergic synaptic transmission. These findings identify astrocyte-derived ATP as a potential molecular player in the pathophysiology of ASD.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/patologia , Transtorno do Espectro Autista/patologia , Sinalização do Cálcio/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Comportamento Animal , Cálcio/metabolismo , Modelos Animais de Doenças , Neurônios GABAérgicos/fisiologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Knockout , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Transmissão Sináptica/fisiologia
6.
Methodist Debakey Cardiovasc J ; 17(1): 43-47, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-34104319

RESUMO

Atrial fibrillation (AF) is the most common cardiac arrhythmia and is associated with an increased risk of all-cause mortality and complications. The autonomic nervous system (ANS) plays a central role in AF, with the heart regulated by both extrinsic and intrinsic properties. In the extrinsic ANS, the sympathetic fibers are derived from the major paravertebral ganglia, especially the stellate ganglion (SG), which is a source of cardiac sympathetic innervation since it connects with multiple intrathoracic nerves and structures. The major intrinsic ANS is a network of axons and ganglionated plexi that contains a variety of sympathetic and parasympathetic neurons, which communicate with the extrinsic ANS. Simultaneous sympathovagal activation contributes to the development of AF because it increases calcium entry and shortens the atrial action potential duration. In animal and human studies, neuromodulation methods such as electrical stimulation and renal denervation have indicated potential benefits in controlling AF in patients as they cause SG remodeling and reduce sympathetic outflow. This review focuses on the neural mechanisms relevant to AF and the recent developments of neuromodulation methods for AF control.


Assuntos
Fibrilação Atrial/fisiopatologia , Sistema Nervoso Autônomo/fisiopatologia , Átrios do Coração/inervação , Frequência Cardíaca , Técnicas de Ablação , Potenciais de Ação , Animais , Fibrilação Atrial/diagnóstico , Fibrilação Atrial/terapia , Sistema Nervoso Autônomo/cirurgia , Sinalização do Cálcio , Terapia por Estimulação Elétrica , Humanos , Simpatectomia , Resultado do Tratamento
7.
Methods Enzymol ; 654: 255-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34120716

RESUMO

Optogenetics combines optics and genetics to enable non-invasive interrogation of cell physiology at an unprecedented high spatiotemporal resolution. Here, we introduce Opto-CRAC as a set of genetically-encoded calcium actuators (GECAs) engineered from the calcium release-activated calcium (CRAC) channel, which has been tailored for optical control of calcium entry and calcium-dependent physiological responses in non-excitable cells and tissues. We describe a detailed protocol for applying Opto-CRAC as an optogenetic tool to achieve photo-tunable control over intracellular calcium signals and calcium-dependent gene expression in mammalian cells.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Optogenética
8.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34072954

RESUMO

During cardiac excitation contraction coupling, the arrival of an action potential at the ventricular myocardium triggers voltage-dependent L-type Ca2+ (CaV1.2) channels in individual myocytes to open briefly. The level of this Ca2+ influx tunes the amplitude of Ca2+-induced Ca2+ release from ryanodine receptors (RyR2) on the junctional sarcoplasmic reticulum and thus the magnitude of the elevation in intracellular Ca2+ concentration and ultimately the downstream contraction. The number and activity of functional CaV1.2 channels at the t-tubule dyads dictates the amplitude of the Ca2+ influx. Trafficking of these channels and their auxiliary subunits to the cell surface is thus tightly controlled and regulated to ensure adequate sarcolemmal expression to sustain this critical process. To that end, recent discoveries have revealed the existence of internal reservoirs of preformed CaV1.2 channels that can be rapidly mobilized to enhance sarcolemmal expression in times of acute stress when hemodynamic and metabolic demand increases. In this review, we provide an overview of the current thinking on CaV1.2 channel trafficking dynamics in the heart. We highlight the numerous points of control including the biosynthetic pathway, the endosomal recycling pathway, ubiquitination, and lysosomal and proteasomal degradation pathways, and discuss the effects of ß-adrenergic and angiotensin receptor signaling cascades on this process.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio , Cálcio/metabolismo , Ventrículos do Coração/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Animais , Linhagem Celular , Ventrículos do Coração/citologia , Humanos , Miócitos Cardíacos/citologia
9.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065973

RESUMO

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)-dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors' interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5-15% vs. 30-80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.


Assuntos
Adipócitos Brancos/metabolismo , Sinalização do Cálcio , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/efeitos dos fármacos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Epididimo , Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Obesidade/induzido quimicamente , Palmitoilcarnitina/farmacologia , Periodicidade , Cultura Primária de Células , Fosfolipases Tipo C/metabolismo
10.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070742

RESUMO

Nephrogenesis is driven by complex signaling pathways that control cell growth and differentiation. The endoplasmic reticulum chaperone calreticulin (Calr) is well known for its function in calcium storage and in the folding of glycoproteins. Its role in kidney development is still not understood. We provide evidence for a pivotal role of Calr in nephrogenesis in this investigation. We show that Calr deficiency results in the disrupted formation of an intact nephrogenic zone and in retardation of nephrogenesis, as evidenced by the disturbance in the formation of comma-shaped and s-shaped bodies. Using proteomics and transcriptomics approaches, we demonstrated that in addition to an alteration in Wnt-signaling key proteins, embryonic kidneys from Calr-/- showed an overall impairment in expression of ribosomal proteins which reveals disturbances in protein synthesis and nephrogenesis. CRISPR/cas9 mediated knockout confirmed that Calr deficiency is associated with a deficiency of several ribosomal proteins and key proteins in ribosome biogenesis. Our data highlights a direct link between Calr expression and the ribosome biogenesis.


Assuntos
Cálcio/metabolismo , Calreticulina/genética , Rim/metabolismo , Biogênese de Organelas , Proteínas Ribossômicas/genética , Ribossomos/genética , Animais , Sinalização do Cálcio , Calreticulina/deficiência , Embrião de Mamíferos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Glicoproteínas/classificação , Glicoproteínas/genética , Glicoproteínas/metabolismo , Rim/crescimento & desenvolvimento , Rim/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese/genética , Dobramento de Proteína , Proteômica/métodos , Proteínas Ribossômicas/deficiência , Ribossomos/metabolismo , Ribossomos/patologia , Via de Sinalização Wnt
11.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34074030

RESUMO

The ability of organisms to quickly sense and transduce signals of environmental stresses is critical for their survival. Ca2+ is a versatile intracellular messenger involved in sensing a wide variety of stresses and regulating the subsequent cellular responses. So far, our understanding for calcium signaling was mostly obtained from ex vivo tissues and cultured cell lines, and the in vivo spatiotemporal dynamics of stress-triggered calcium signaling in a vertebrate remains to be characterized. Here, we describe the generation and characterization of a transgenic zebrafish line with ubiquitous expression of GCaMP6s, a genetically encoded calcium indicator (GECI). We developed a method to investigate the spatiotemporal patterns of Ca2+ events induced by heat stress. Exposure to heat stress elicited immediate and transient calcium signaling in developing zebrafish. Cells extensively distributed in the integument of the head and body trunk were the first batch of responders and different cell populations demonstrated distinct response patterns upon heat stress. Activity of the heat stress-induced calcium signaling peaked at 30 s and swiftly decreased to near the basal level at 120 s after the beginning of exposure. Inhibition of the heat-induced calcium signaling by LaCl3 and capsazepine and treatment with the inhibitors for CaMKII (Ca²2/calmodulin-dependent protein kinase II) and HSF1 (Heat shock factor 1) all significantly depressed the enhanced heat shock response (HSR). Together, we delineated the spatiotemporal dynamics of heat-induced calcium signaling and confirmed functions of the Ca2+-CaMKII-HSF1 pathway in regulating the HSR in zebrafish.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Resposta ao Choque Térmico/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/genética , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Proteínas de Fluorescência Verde/genética , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Hibridização In Situ , Lantânio/farmacologia , Microscopia de Fluorescência , Análise Espaço-Temporal , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
13.
Neuron ; 109(12): 2009-2024.e6, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-33957065

RESUMO

Making flexible decisions based on prior knowledge about causal environmental structures is a hallmark of goal-directed cognition in mammalian brains. Although several association brain regions, including the orbitofrontal cortex (OFC), have been implicated, the precise neuronal circuit mechanisms underlying knowledge-based decision-making remain elusive. Here, we established an inference-based auditory categorization task where mice performed within-session flexible stimulus re-categorization by inferring the changing task rules. We constructed a reinforcement learning model to recapitulate the inference-based flexible behavior and quantify the hidden variables associated with task structural knowledge. Combining two-photon population imaging and projection-specific optogenetics, we found that auditory cortex (ACx) neurons encoded the hidden task rule variable, which requires feedback input from the OFC. Silencing OFC-ACx input specifically disrupted re-categorization behavior. Direct imaging from OFC axons in the ACx revealed task state-related feedback signals, supporting the knowledge-based updating mechanism. Our data reveal a cortical circuit mechanism underlying structural knowledge-based flexible decision-making.


Assuntos
Córtex Auditivo/fisiologia , Tomada de Decisões/fisiologia , Aprendizagem/fisiologia , Neurônios/fisiologia , Córtex Pré-Frontal/fisiologia , Animais , Sinalização do Cálcio , Cognição/fisiologia , Retroalimentação Fisiológica/fisiologia , Camundongos , Vias Neurais/fisiologia , Imagem Óptica , Optogenética , Desempenho Psicomotor , Reforço Psicológico
14.
FASEB J ; 35(6): e21478, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33991146

RESUMO

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.


Assuntos
Reação Acrossômica , Sinalização do Cálcio , Exocitose , Potenciais da Membrana , Capacitação Espermática , Espermatozoides/fisiologia , Humanos , Masculino , Fosforilação
15.
Nat Commun ; 12(1): 3175, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039988

RESUMO

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.


Assuntos
Envelhecimento/fisiologia , Evolução Biológica , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Edição de Genes , Técnicas de Introdução de Genes , Masculino , Camundongos , Modelos Animais , Oxirredução , Filogenia , Aptidão Física/fisiologia , Mutação Puntual
16.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946854

RESUMO

Hexokinases are a family of ubiquitous exose-phosphorylating enzymes that prime glucose for intracellular utilization. Hexokinase 2 (HK2) is the most active isozyme of the family, mainly expressed in insulin-sensitive tissues. HK2 induction in most neoplastic cells contributes to their metabolic rewiring towards aerobic glycolysis, and its genetic ablation inhibits malignant growth in mouse models. HK2 can dock to mitochondria, where it performs additional functions in autophagy regulation and cell death inhibition that are independent of its enzymatic activity. The recent definition of HK2 localization to contact points between mitochondria and endoplasmic reticulum called Mitochondria Associated Membranes (MAMs) has unveiled a novel HK2 role in regulating intracellular Ca2+ fluxes. Here, we propose that HK2 localization in MAMs of tumor cells is key in sustaining neoplastic progression, as it acts as an intersection node between metabolic and survival pathways. Disrupting these functions by targeting HK2 subcellular localization can constitute a promising anti-tumor strategy.


Assuntos
Hexoquinase/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Autofagia/fisiologia , Sinalização do Cálcio/fisiologia , Hipóxia Celular , Peptídeos Penetradores de Células/uso terapêutico , Indução Enzimática , Regulação Neoplásica da Expressão Gênica , Glicólise/fisiologia , Hexoquinase/antagonistas & inibidores , Humanos , Membranas Intracelulares/enzimologia , Camundongos , MicroRNAs/genética , Mitocôndrias/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias/terapia , Neoplasias Experimentais/enzimologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Processamento de Proteína Pós-Traducional , Ratos , Ubiquitinação
17.
J Vis Exp ; (170)2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33938876

RESUMO

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Pâncreas/metabolismo , Animais , Camundongos , Pâncreas/citologia
18.
Adv Exp Med Biol ; 1304: 147-164, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34019268

RESUMO

Inflammatory signaling is a major component in the development and progression of many lung diseases, including asthma, chronic obstructive pulmonary disorder (COPD), and pulmonary hypertension (PH). This chapter will provide a brief overview of asthma, COPD, and PH and how inflammation plays a vital role in these diseases. Specifically, we will discuss the role of reactive oxygen species (ROS) and Ca2+ signaling in inflammatory cellular responses and how these interactive signaling pathways mediate the development of asthma, COPD, and PH. We will also deliberate the key cellular responses of pulmonary arterial (PA) smooth muscle cells (SMCs) and airway SMCs (ASMCs) in these devastating lung diseases. The analysis of the importance of inflammation will shed light on the key questions remaining in this field and highlight molecular targets that are worth exploring. The crucial findings will not only demonstrate the novel roles of essential signaling molecules such as Rieske iron-sulfur protein and ryanodine receptor in the development and progress of asthma, COPD, and PH but also offer advanced insight for creating more effective and new therapeutic targets for these devastating inflammatory lung diseases.


Assuntos
Asma , Hipertensão Pulmonar , Doença Pulmonar Obstrutiva Crônica , Sinalização do Cálcio , Humanos , Inflamação , Espécies Reativas de Oxigênio
19.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946271

RESUMO

Mitochondria are key regulators of cell survival and are involved in a plethora of mechanisms, such as metabolism, Ca2+ signaling, reactive oxygen species (ROS) production, mitophagy and mitochondrial transfer, fusion, and fission (known as mitochondrial dynamics). The tuning of these processes in pathophysiological conditions is fundamental to the balance between cell death and survival. Indeed, ROS overproduction and mitochondrial Ca2+ overload are linked to the induction of apoptosis, while the impairment of mitochondrial dynamics and metabolism can have a double-faceted role in the decision between cell survival and death. Tumorigenesis involves an intricate series of cellular impairments not yet completely clarified, and a further level of complexity is added by the onset of apoptosis resistance mechanisms in cancer cells. In the majority of cases, cancer relapse or lack of responsiveness is related to the emergence of chemoresistance, which may be due to the cooperation of several cellular protection mechanisms, often mitochondria-related. With this review, we aim to critically report the current evidence on the relationship between mitochondria and cancer chemoresistance with a particular focus on the involvement of mitochondrial dynamics, mitochondrial Ca2+ signaling, oxidative stress, and metabolism to possibly identify new approaches or targets for overcoming cancer resistance.


Assuntos
Antineoplásicos/farmacologia , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas Mitocondriais/metabolismo , Mitofagia/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Estresse Oxidativo/efeitos dos fármacos
20.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946838

RESUMO

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.


Assuntos
Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Retículo Endoplasmático/metabolismo , Animais , Sítios de Ligação , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Humanos , Microdomínios da Membrana/metabolismo , Modelos Biológicos , Sistemas do Segundo Mensageiro/fisiologia , Análise Espaço-Temporal , Moléculas de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo
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