Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
1.
FASEB J ; 38(15): e23853, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-39120544

RESUMO

Sodium butyrate (NaB) improves ß-cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been fully elucidated. In this study, we investigated the impact of NaB on ß-cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 ß cells. Consistently, NaB improved glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the ß cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1ß-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent ß-cell death in response to IL-1ß treatment. Mechanistic experiments revealed that NaB mediated these beneficial effects in the ß-cell through histone deacetylase (HDAC) inhibition, iNOS suppression, and modulation of AKT-GSK-3 signaling. Taken together, these data support a model whereby NaB treatment promotes ß-cell function and Ca2+ homeostasis under proinflammatory conditions through pleiotropic effects that are linked with maintenance of SOCE. These results also suggest a relationship between ß-cell SOCE and gut microbiome-derived butyrate that may be relevant in the treatment and prevention of diabetes.


Assuntos
Ácido Butírico , Cálcio , Células Secretoras de Insulina , Molécula 1 de Interação Estromal , Animais , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Camundongos , Humanos , Ácido Butírico/farmacologia , Cálcio/metabolismo , Citocinas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Retículo Endoplasmático/metabolismo , Diabetes Mellitus Tipo 2/metabolismo
2.
J Biol Chem ; 299(8): 104970, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37380078

RESUMO

Intracellular calcium signaling is essential for many cellular processes, including store-operated Ca2+ entry (SOCE), which is initiated by stromal interaction molecule 1 (STIM1) detecting endoplasmic reticulum (ER) Ca2+ depletion. STIM1 is also activated by temperature independent of ER Ca2+ depletion. Here we provide evidence, from advanced molecular dynamics simulations, that EF-SAM may act as a true temperature sensor for STIM1, with the prompt and extended unfolding of the hidden EF-hand subdomain (hEF) even at slightly elevated temperatures, exposing a highly conserved hydrophobic Phe108. Our study also suggests an interplay between Ca2+ and temperature sensing, as both, the canonical EF-hand subdomain (cEF) and the hidden EF-hand subdomain (hEF), exhibit much higher thermal stability in the Ca2+-loaded form compared to the Ca2+-free form. The SAM domain, surprisingly, displays high thermal stability compared to the EF-hands and may act as a stabilizer for the latter. We propose a modular architecture for the EF-hand-SAM domain of STIM1 composed of a thermal sensor (hEF), a Ca2+ sensor (cEF), and a stabilizing domain (SAM). Our findings provide important insights into the mechanism of temperature-dependent regulation of STIM1, which has broad implications for understanding the role of temperature in cellular physiology.


Assuntos
Retículo Endoplasmático , Simulação de Dinâmica Molecular , Cálcio/metabolismo , Sinalização do Cálcio , Retículo Endoplasmático/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Temperatura , Humanos
3.
Int J Mol Sci ; 25(5)2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38474242

RESUMO

Ceramides regulate phagocytosis; however, their exact function remains poorly understood. Here, we sought (1) to develop genetically encoded fluorescent tools for imaging ceramides, and (2) to use them to examine ceramide dynamics during phagocytosis. Fourteen enhanced green fluorescent protein (EGFP) fusion constructs based on four known ceramide-binding domains were generated and screened. While most constructs localized to the nucleus or cytosol, three based on the CA3 ceramide-binding domain of kinase suppressor of ras 1 (KSR1) localized to the plasma membrane or autolysosomes. C-terminally tagged CA3 with a vector-based (C-KSR) or glycine-serine linker (C-KSR-GS) responded sensitively and similarly to ceramide depletion and accumulation using a panel of ceramide modifying drugs, whereas N-terminally tagged CA3 (N-KSR) responded differently to a subset of treatments. Lipidomic and liposome microarray analysis suggested that, instead, N-KSR may preferentially bind glucosyl-ceramide. Additionally, the three probes showed distinct dynamics during phagocytosis. Despite partial autolysosomal degradation, C-KSR and C-KSR-GS accumulated at the plasma membrane during phagocytosis, whereas N-KSR did not. Moreover, the weak recruitment of C-KSR-GS to the endoplasmic reticulum and phagosomes was enhanced through overexpression of the endoplasmic reticulum proteins stromal interaction molecule 1 (STIM1) and Sec22b, and was more salient in dendritic cells. The data suggest these novel probes can be used to analyze sphingolipid dynamics and function in living cells.


Assuntos
Ceramidas , Corantes Fluorescentes , Proteínas Quinases , Ceramidas/metabolismo , Transdução de Sinais/fisiologia , Fagocitose
4.
Int J Mol Sci ; 24(24)2023 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-38139137

RESUMO

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai-a naturally growing species in South Korea-and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca2+ release-activated Ca2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4+ T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.


Assuntos
Agrimonia , Humanos , Linfócitos T , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Anti-Inflamatórios/farmacologia
5.
Am J Physiol Cell Physiol ; 322(1): C38-C48, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34788146

RESUMO

The gaseous signaling molecule hydrogen sulfide (H2S) physiologically regulates store-operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane-localized Orai channels (Orai1-3) and endoplasmic reticulum-localized stromal interaction molecule (STIM)1 and STIM2 proteins. H2S inhibits Orai3- but not Orai1- or Orai2-mediated SOCE. The current objective was to define the mechanism by which H2S selectively modifies Orai3. We measured SOCE and STIM1/Orai3 dynamics and interactions in HEK293 cells exogenously expressing fluorescently tagged human STIM1 and Orai3 in the presence and absence of the H2S donor GYY4137. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2. When we mutated either of these cysteines to serine, alone or in combination, SOCE inhibition by H2S was abolished. We also established that inhibition was dependent on an interaction with STIM1. To further define the effects of H2S on STIM1/Orai3 interaction, we performed a series of fluorescence recovery after photobleaching (FRAP), colocalization, and fluorescence resonance energy transfer (FRET) experiments. Treatment with H2S did not affect the mobility of Orai3 in the membrane, nor did it influence STIM1/Orai3 puncta formation or STIM1-Orai3 protein-protein interactions. These data support a model in which H2S modification of Orai3 at cysteines 226 and 232 limits SOCE evoked upon store depletion and STIM1 engagement, by a mechanism independent of the interaction between Orai3 and STIM1.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cisteína/metabolismo , Sulfeto de Hidrogênio/toxicidade , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas Sensoras de Cálcio Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores
6.
Cell Mol Life Sci ; 78(15): 5827-5846, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34155535

RESUMO

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.


Assuntos
Proliferação de Células/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Células Epiteliais da Tireoide/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Fase G1/efeitos dos fármacos , Fase G1/genética , Humanos , Indóis/administração & dosagem , Masculino , Pessoa de Meia-Idade , Nanopartículas/administração & dosagem , Proteína ORAI1/genética , Polímeros/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Células Epiteliais da Tireoide/efeitos dos fármacos , Glândula Tireoide/efeitos dos fármacos , Neoplasias da Glândula Tireoide/tratamento farmacológico , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Adulto Jovem , Peixe-Zebra
7.
J Biol Chem ; 295(50): 17071-17082, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33023909

RESUMO

Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 WT (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621A-HEK and STIM1-KO-T626A-HEK cells was lower than that in STIM1-KO-WT-HEK cells because of reduced phosphorylation at Ser621 Treatment with the O-GlcNAcase inhibitor Thiamet G or O-GlcNAc transferase (OGT) transfection, which increases O-GlcNAcylation, reduced SOCE activity, whereas treatment with the OGT inhibitor ST045849 or siOGT transfection, which decreases O-GlcNAcylation, also reduced SOCE activity. Decrease in SOCE activity due to increase and decrease in O-GlcNAcylation was attributable to reduced phosphorylation at Ser621 These data suggest that both decrease in O-GlcNAcylation at Thr626 and increase in O-GlcNAcylation at Ser621 in STIM1 lead to impairment of SOCE activity through decrease in Ser621 phosphorylation. Targeting STIM1 O-GlcNAcylation could provide a promising treatment option for the related diseases, such as neurodegenerative diseases.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Acilação , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Serina , Molécula 1 de Interação Estromal/genética
8.
Pflugers Arch ; 473(3): 417-434, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33638008

RESUMO

Store-operated Ca2+ entry (SOCE) is an ancient and ubiquitous Ca2+ signaling pathway that is present in virtually every cell type. Over the last two decades, many studies have implicated this non-voltage dependent Ca2+ entry pathway in cardiac physiology. The relevance of the SOCE pathway in cardiomyocytes is often questioned given the well-established role for excitation contraction coupling. In this review, we consider the evidence that STIM1 and SOCE contribute to Ca2+ dynamics in cardiomyocytes. We discuss the relevance of this pathway to cardiac growth in response to developmental and pathologic cues. We also address whether STIM1 contributes to Ca2+ store refilling that likely impacts cardiac pacemaking and arrhythmogenesis in cardiomyocytes.


Assuntos
Sinalização do Cálcio/fisiologia , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Miócitos Cardíacos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Acoplamento Excitação-Contração/fisiologia , Humanos
9.
J Biol Chem ; 294(41): 14922-14936, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31413111

RESUMO

Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.


Assuntos
Cálcio/metabolismo , Ecdisterona/farmacologia , Agregados Proteicos/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Interação Estromal/deficiência , Molécula 1 de Interação Estromal/genética , Regulação para Cima/efeitos dos fármacos
10.
J Biol Chem ; 294(16): 6318-6332, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30824535

RESUMO

Store-operated Ca2+ entry (SOCE) is a ubiquitous pathway for Ca2+ influx across the plasma membrane (PM). SOCE is mediated by the endoplasmic reticulum (ER)-associated Ca2+-sensing proteins stromal interaction molecule 1 (STIM1) and STIM2, which transition into an active conformation in response to ER Ca2+ store depletion, thereby interacting with and gating PM-associated ORAI1 channels. Although structurally homologous, STIM1 and STIM2 generate distinct Ca2+ signatures in response to varying strengths of agonist stimulation. The physiological functions of these Ca2+ signatures, particularly under native conditions, remain unclear. To investigate the structural properties distinguishing STIM1 and STIM2 activation of ORAI1 channels under native conditions, here we used CRISPR/Cas9 to generate STIM1-/-, STIM2-/-, and STIM1/2-/- knockouts in HEK293 and colorectal HCT116 cells. We show that depending on cell type, STIM2 can significantly sustain SOCE in response to maximal store depletion. Utilizing the SOCE modifier 2-aminoethoxydiphenyl borate (2-APB), we demonstrate that 2-APB-activated store-independent Ca2+ entry is mediated exclusively by endogenous STIM2. Using variants that either stabilize or disrupt intramolecular interactions of STIM C termini, we show that the increased flexibility of the STIM2 C terminus contributes to its selective store-independent activation by 2-APB. However, STIM1 variants with enhanced flexibility in the C terminus failed to support its store-independent activation. STIM1/STIM2 chimeric constructs indicated that coordination between N-terminal sensitivity and C-terminal flexibility is required for specific store-independent STIM2 activation. Our results clarify the structural determinants underlying activation of specific STIM isoforms, insights that are potentially useful for isoform-selective drug targeting.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Compostos de Boro/química , Compostos de Boro/farmacologia , Cálcio/química , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Técnicas de Silenciamento de Genes , Células HCT116 , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/genética
11.
J Biol Chem ; 294(32): 12054-12065, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31213528

RESUMO

Charcot-Marie-Tooth (CMT) disease is a peripheral neuropathy associated with gene duplication and point mutations in the peripheral myelin protein 22 (PMP22) gene. However, the role of PMP22 in Schwann cell physiology and the mechanisms by which PMP22 mutations cause CMT are not well-understood. On the basis of homology between PMP22 and proteins associated with modulation of ion channels, we hypothesized that PMP22 alters ion channel activity. Using whole-cell electrophysiology, we show here that heterologous PMP22 expression increases the amplitude of currents similar to those ascribed to store-operated calcium (SOC) channels, particularly those involving transient receptor canonical channel 1 (TrpC1). These channels help replenish Ca2+ in the endoplasmic reticulum (ER) following stimulus-induced depletion. Currents with similar properties were recorded in WT but not pmp22-/- mouse Schwann cells. Heterologous expression of the CMT-associated PMP22_L16P variant, which fails to reach the plasma membrane and localizes to the ER, led to larger currents than WT PMP22. Similarly, Schwann cells isolated from Trembler J (TrJ; PMP22_L16P) mice had larger currents than WT littermates. Calcium imaging in live nerves and cultured Schwann cells revealed elevated intracellular Ca2+ in TrJ mice compared with WT. Moreover, we found that PMP22 co-immunoprecipitated with stromal interaction molecule 1 (STIM1), the Ca2+ sensor SOC channel subunit in the ER. These results suggest that in the ER, PMP22 interacts with STIM1 and increases Ca2+ influx through SOC channels. Excess or mutant PMP22 in the ER may elevate intracellular Ca2+ levels, which could contribute to CMT pathology.


Assuntos
Canais de Cálcio/metabolismo , Proteínas da Mielina/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/química , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Retículo Endoplasmático/metabolismo , Potenciais Evocados/efeitos dos fármacos , Gadolínio/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Proteínas da Mielina/deficiência , Proteínas da Mielina/genética , Células de Schwann/citologia , Células de Schwann/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/metabolismo
12.
Int J Mol Sci ; 21(4)2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32098295

RESUMO

Glioblastoma multiforme (GBM) is an aggressive adult brain tumour with poor prognosis. Roles for peptidylarginine deiminases (PADs) in GBM have recently been highlighted. Here, two GBM cell lines were treated with PAD2, PAD3 and PAD4 isozyme-specific inhibitors. Effects were assessed on extracellular vesicle (EV) signatures, including EV-microRNA cargo (miR21, miR126 and miR210), and on changes in cellular protein expression relevant for mitochondrial housekeeping (prohibitin (PHB)) and cancer progression (stromal interaction molecule 1 (STIM-1) and moesin), as well as assessing cell invasion. Overall, GBM cell-line specific differences for the three PAD isozyme-specific inhibitors were observed on modulation of EV-signatures, PHB, STIM-1 and moesin protein levels, as well as on cell invasion. The PAD3 inhibitor was most effective in modulating EVs to anti-oncogenic signatures (reduced miR21 and miR210, and elevated miR126), to reduce cell invasion and to modulate protein expression of pro-GBM proteins in LN229 cells, while the PAD2 and PAD4 inhibitors were more effective in LN18 cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for deiminated proteins relating to cancer, metabolism and inflammation differed between the two GBM cell lines. Our findings highlight roles for the different PAD isozymes in the heterogeneity of GBM tumours and the potential for tailored PAD-isozyme specific treatment.


Assuntos
Inibidores Enzimáticos/farmacologia , Vesículas Extracelulares/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Proteína-Arginina Desiminase do Tipo 2/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 3/antagonistas & inibidores , Proteína-Arginina Desiminase do Tipo 4/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Vesículas Extracelulares/metabolismo , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proibitinas , Proteína-Arginina Desiminase do Tipo 2/metabolismo , Proteína-Arginina Desiminase do Tipo 3/metabolismo , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
13.
J Biol Chem ; 293(23): 8900-8911, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29661937

RESUMO

Store-operated Ca2+ entry (SOCE) is a major Ca2+ signaling pathway facilitating extracellular Ca2+ influx in response to the initial release of intracellular endo/sarcoplasmic reticulum (ER/SR) Ca2+ stores. Stromal interaction molecule 1 (STIM1) is the Ca2+ sensor that activates SOCE following ER/SR Ca2+ depletion. The EF-hand and the adjacent sterile α-motif (EFSAM) domains of STIM1 are essential for detecting changes in luminal Ca2+ concentrations. Low ER Ca2+ levels trigger STIM1 destabilization and oligomerization, culminating in the opening of Orai1-composed Ca2+ channels on the plasma membrane. NO-mediated S-nitrosylation of cysteine thiols regulates myriad protein functions, but its effects on the structural mechanisms that regulate SOCE are unclear. Here, we demonstrate that S-nitrosylation of Cys49 and Cys56 in STIM1 enhances the thermodynamic stability of its luminal domain, resulting in suppressed hydrophobic exposure and diminished Ca2+ depletion-dependent oligomerization. Using solution NMR spectroscopy, we pinpointed a structural mechanism for STIM1 stabilization driven by complementary charge interactions between an electropositive patch on the core EFSAM domain and the S-nitrosylated nonconserved region of STIM1. Finally, using live cells, we found that the enhanced luminal domain stability conferred by either Cys49 and Cys56S-nitrosylation or incorporation of negatively charged residues into the EFSAM electropositive patch in the full-length STIM1 context significantly suppresses SOCE. Collectively, our results suggest that S-nitrosylation of STIM1 inhibits SOCE by interacting with an electropositive patch on the EFSAM core, which modulates the thermodynamic stability of the STIM1 luminal domain.


Assuntos
Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sequência de Aminoácidos , Sinalização do Cálcio , Cisteína/química , Cisteína/metabolismo , Motivos EF Hand , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Retículo Sarcoplasmático/metabolismo , Alinhamento de Sequência , Molécula 1 de Interação Estromal/química , Termodinâmica
14.
J Biol Chem ; 293(4): 1271-1285, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29237733

RESUMO

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.


Assuntos
Canais de Cálcio/química , Proteína ORAI1/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
15.
J Biol Chem ; 293(4): 1259-1270, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29237734

RESUMO

Calcium (Ca2+) is an essential second messenger required for diverse signaling processes in immune cells. Ca2+ release-activated Ca2+ (CRAC) channels represent one main Ca2+ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum, and the Ca2+ ion channel Orai in the plasma membrane. After Ca2+ store depletion, STIM1 and Orai couple to each other, allowing Ca2+ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca2+ selectivity, an increase in current density when switching from a Ca2+-containing solution to a divalent-free Na+ one, and fast Ca2+-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.


Assuntos
Canais de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Ativação do Canal Iônico , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Molécula 1 de Interação Estromal/genética
16.
J Biol Chem ; 293(9): 3145-3155, 2018 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-29326165

RESUMO

Low-conductance, highly calcium-selective channels formed by the Orai proteins exist as store-operated CRAC channels and store-independent, arachidonic acid-activated ARC channels. Both are activated by stromal interaction molecule 1 (STIM1), but CRAC channels are activated by STIM1 located in the endoplasmic reticulum membrane, whereas ARC channels are activated by the minor plasma membrane-associated pool of STIM1. Critically, maximally activated CRAC channel and ARC channel currents are completely additive within the same cell, and their selective activation results in their ability to each induce distinct cellular responses. We have previously shown that specific ARC channel activation requires a PKA-mediated phosphorylation of a single threonine residue (Thr389) within the cytoplasmic region of STIM1. Here, examination of the molecular basis of this phosphorylation-dependent activation revealed that phosphorylation of the Thr389 residue induces a significant structural change in the STIM1-Orai-activating region (SOAR) that interacts with the Orai proteins, and it is this change that determines the selective activation of the store-independent ARC channels versus the store-operated CRAC channels. In conclusion, our findings reveal the structural changes underlying the selective activation of STIM1-induced CRAC or ARC channels that determine the specific stimulation of these two functionally distinct Ca2+ entry pathways.


Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Transporte Biológico , Cálcio/metabolismo , Células HEK293 , Humanos , Fosforilação , Domínios Proteicos
17.
J Biol Chem ; 292(48): 19656-19673, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28972171

RESUMO

Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 µm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2-4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.


Assuntos
Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/antagonistas & inibidores , Tapsigargina/análogos & derivados , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Tapsigargina/farmacologia
18.
Int J Mol Sci ; 19(11)2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30366379

RESUMO

Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2ß splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2ß insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2ß functional assessment to provide insights into the molecular mechanism by which the 2ß insert precludes SOCE activation. We find that the 2ß insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2ß insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2ß insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.


Assuntos
Cálcio/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Fluorometria , Humanos , Estabilidade Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
19.
J Biol Chem ; 291(1): 334-41, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26546674

RESUMO

Calcium flux through store-operated calcium entry is a central regulator of intracellular calcium signaling. The two key components of the store-operated calcium release-activated calcium channel are the Ca(2+)-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. During store-operated calcium entry activation, calcium depletion from the endoplasmic reticulum triggers a series of conformational changes in STIM1 that unmask a minimal Orai1-activating domain (CRAC activation region (CAD)). To gate Orai1 channels, the exposed STIM1-activating domain binds to two sites in Orai1, one in the N terminus and one in the C terminus. Whether the two sites operate as distinct binding domains or cooperate in CAD binding is unknown. In this study, we show that the N and C-terminal domains of Orai1 synergistically contribute to the interaction with STIM1 and couple STIM1 binding with channel gating and modulation of ion selectivity.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Células HEK293 , Humanos , Ativação do Canal Iônico , Íons , Proteínas Mutantes/metabolismo , Proteína ORAI1 , Ligação Proteica , Estrutura Terciária de Proteína , Molécula 1 de Interação Estromal
20.
J Biol Chem ; 291(16): 8440-52, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26826124

RESUMO

T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca(2+) to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca(2+) has been determined. However, the role of Ca(2+) in controlling NF-κB signaling is poorly understood, and the source of Ca(2+) required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca(2+) via STIM1-dependent Ca(2+) release-activated Ca(2+)/Orai channels. We further show that Ca(2+) influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca(2+) is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca(2+)-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca(2+)-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca(2+) required for TCR-induced NF-κB activation and define a new distal Ca(2+)-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.


Assuntos
Canais de Cálcio/biossíntese , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Fator de Transcrição RelA/metabolismo , Ativação Transcricional/fisiologia , Canais de Cálcio/genética , Humanos , Células Jurkat , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Fosforilação/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Molécula 1 de Interação Estromal , Fator de Transcrição RelA/genética
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa