RESUMEN
Ca2+ puffs are brief, localized Ca2+ signals evoked by physiological stimuli that arise from the coordinated opening of a few clustered inositol 1,4,5-trisphosphate receptors (IP3Rs). However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.
Asunto(s)
Señalización del Calcio , Calcio , Receptores de Inositol 1,4,5-Trifosfato , Inositol 1,4,5-Trifosfato , Calcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Humanos , Microscopía Fluorescente , Células HEK293RESUMEN
Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-ß5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Opsinas , Humanos , Ratones , Animales , Opsinas/genética , Opsinas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Calcio/metabolismo , Transducción de Señal , Receptores Acoplados a Proteínas G/metabolismo , Opsinas de Bastones/metabolismo , Mamíferos/metabolismoRESUMEN
Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.
Asunto(s)
Neuronas/metabolismo , Neurotransmisores/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Exocitosis , Humanos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Regulación hacia Arriba , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Calcium-mediated signaling through inositol 1,4,5-triphosphate receptors (IP3Rs) is essential for the regulation of numerous physiological processes, including fertilization, muscle contraction, apoptosis, secretion, and synaptic plasticity. Deregulation of IP3Rs leads to pathological calcium signaling and is implicated in many common diseases, including cancer and neurodegenerative, autoimmune, and metabolic diseases. Revealing the mechanism of activation and inhibition of this ion channel will be critical to an improved understanding of the biological processes that are controlled by IP3Rs. Here, we report structural findings of the human type-3 IP3R (IP3R-3) obtained by cryo-EM (at an overall resolution of 3.8 Å), revealing an unanticipated regulatory mechanism where a loop distantly located in the primary sequence occupies the IP3-binding site and competitively inhibits IP3 binding. We propose that this inhibitory mechanism must differ qualitatively among IP3R subtypes because of their diverse loop sequences, potentially serving as a key molecular determinant of subtype-specific calcium signaling in IP3Rs. In summary, our structural characterization of human IP3R-3 provides critical insights into the mechanistic function of IP3Rs and into subtype-specific regulation of these important calcium-regulatory channels.
Asunto(s)
Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Péptidos/metabolismo , Sitios de Unión , Señalización del Calcio , Microscopía por Crioelectrón , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/ultraestructura , Modelos Moleculares , Conformación ProteicaRESUMEN
Structural analyses identified the central domain of ryanodine receptor (RyR) as a transducer converting conformational changes in the cytoplasmic platform to the RyR gate. The central domain is also a regulatory hub encompassing the Ca2+-, ATP-, and caffeine-binding sites. However, the role of the central domain in RyR activation and regulation has yet to be defined. Here, we mutated five residues that form the Ca2+ activation site and 10 residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site. We also generated eight disease-associated mutations within the central domain of RyR2. We determined the effect of these mutations on Ca2+, ATP, and caffeine activation and Mg2+ inhibition of RyR2. Mutating the Ca2+ activation site markedly reduced the sensitivity of RyR2 to Ca2+ and caffeine activation. Unexpectedly, Ca2+ activation site mutation E3848A substantially enhanced the Ca2+-independent basal activity of RyR2, suggesting that E3848A may also affect the stability of the closed state of RyR2. Mutations in the Ca2+ activation site also abolished the effect of ATP/caffeine on the Ca2+-independent basal activity, suggesting that the Ca2+ activation site is also a critical determinant of ATP/caffeine action. Mutating residues with negatively charged or oxygen-containing side chains near the Ca2+ activation site significantly altered Ca2+ and caffeine activation and reduced Mg2+ inhibition. Furthermore, disease-associated RyR2 mutations within the central domain significantly enhanced Ca2+ and caffeine activation and reduced Mg2+ inhibition. Our data demonstrate that the central domain plays an important role in channel activation, channel regulation, and closed state stability.
Asunto(s)
Miocardio/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Cafeína/farmacología , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células HEK293 , Humanos , Magnesio/química , Magnesio/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/efectos de los fármacos , Estabilidad Proteica , Estructura Terciaria de Proteína , Rianodina/química , Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genéticaRESUMEN
The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which form tetrameric channels, play pivotal roles in regulating the spatiotemporal patterns of intracellular calcium signals. Mutations in IP3Rs have been increasingly associated with many debilitating human diseases such as ataxia, Gillespie syndrome, and generalized anhidrosis. However, how these mutations affect IP3R function, and how the perturbation of as-sociated calcium signals contribute to the pathogenesis and severity of these diseases remains largely uncharacterized. Moreover, many of these diseases occur as the result of autosomal dominant inheritance, suggesting that WT and mutant subunits associate in heterotetrameric channels. How the in-corporation of different numbers of mutant subunits within the tetrameric channels affects its activities and results in different disease phenotypes is also unclear. In this report, we investigated representative disease-associated missense mutations to determine their effects on IP3R channel activity. Additionally, we designed concatenated IP3R constructs to create tetrameric channels with a predefined subunit composition to explore the functionality of heteromeric channels. Using calcium imaging techniques to assess IP3R channel function, we observed that all the mutations studied resulted in severely attenuated Ca2+ release when expressed as homotetramers. However, some heterotetramers retained varied degrees of function dependent on the composition of the tetramer. Our findings suggest that the effect of mutations depends on the location of the mutation in the IP3R structure, as well as on the stoichiometry of mutant subunits assembled within the tetrameric channel. These studies provide insight into the pathogenesis and penetrance of these devastating human diseases.
Asunto(s)
Linfocitos B/metabolismo , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Activación del Canal Iónico , Mutación , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Señalización del Calcio , Pollos , Receptores de Inositol 1,4,5-Trifosfato/genética , Multimerización de Proteína , Homología de SecuenciaRESUMEN
Phospholipase Cε (PLCε) is activated downstream of G protein-coupled receptors and receptor tyrosine kinases through direct interactions with small GTPases, including Rap1A and Ras. Although Ras has been reported to allosterically activate the lipase, it is not known whether Rap1A has the same ability or what its molecular mechanism might be. Rap1A activates PLCε in response to the stimulation of ß-adrenergic receptors, translocating the complex to the perinuclear membrane. Because the C-terminal Ras association (RA2) domain of PLCε was proposed to the primary binding site for Rap1A, we first confirmed using purified proteins that the RA2 domain is indeed essential for activation by Rap1A. However, we also showed that the PLCε pleckstrin homology (PH) domain and first two EF hands (EF1/2) are required for Rap1A activation and identified hydrophobic residues on the surface of the RA2 domain that are also necessary. Small-angle X-ray scattering showed that Rap1A binding induces and stabilizes discrete conformational states in PLCε variants that can be activated by the GTPase. These data, together with the recent structure of a catalytically active fragment of PLCε, provide the first evidence that Rap1A, and by extension Ras, allosterically activate the lipase by promoting and stabilizing interactions between the RA2 domain and the PLCε core.
Asunto(s)
Fosfoinositido Fosfolipasa C/metabolismo , Proteínas de Unión al GTP rap1/metabolismo , Regulación Alostérica , GTP Fosfohidrolasas/metabolismo , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfoinositido Fosfolipasa C/química , Fosfoinositido Fosfolipasa C/genética , Dominios Homólogos a Pleckstrina , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genéticaRESUMEN
Sonic hedgehog (SHH) is important for organogenesis during development. Recent studies have indicated that SHH is also involved in the proliferation and transformation of astrocytes to the reactive phenotype. However, the mechanisms underlying these are unknown. Involvement of SHH signaling in calcium (Ca) signaling has not been extensively studied. Here, we report that SHH and Smoothened agonist (SAG), an activator of the signaling receptor Smoothened (SMO) in the SHH pathway, activate Ca oscillations in cultured murine hippocampal astrocytes. The response was rapid, on a minute time scale, indicating a noncanonical pathway activity. Pertussis toxin blocked the SAG effect, indicating an involvement of a Gi coupled to SMO. Depletion of extracellular ATP by apyrase, an ATP-degrading enzyme, inhibited the SAG-mediated activation of Ca oscillations. These results indicate that SAG increases extracellular ATP levels by activating ATP release from astrocytes, resulting in Ca oscillation activation. We hypothesize that SHH activates SMO-coupled Gi in astrocytes, causing ATP release and activation of Gq/11-coupled P2 receptors on the same cell or surrounding astrocytes. Transcription factor activities are often modulated by Ca patterns; therefore, SHH signaling may trigger changes in astrocytes by activating Ca oscillations. This enhancement of Ca oscillations by SHH signaling may occur in astrocytes in the brain in vivo because we also observed it in hippocampal brain slices. In summary, SHH and SAG enhance Ca oscillations in hippocampal astrocytes, Gi mediates SAG-induced Ca oscillations downstream of SMO, and ATP-permeable channels may promote the ATP release that activates Ca oscillations in astrocytes.
Asunto(s)
Astrocitos/metabolismo , Señalización del Calcio , Proteínas Hedgehog/metabolismo , Hipocampo/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células Cultivadas , Hipocampo/citología , Ratones , Ratones Endogámicos ICR , Receptor Smoothened/metabolismoRESUMEN
The CD38 molecule (CD38) catalyzes biogenesis of the calcium-mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, and type III CD38, with its catalytic domain facing the cytosol, has low abundance but is efficient in cyclizing cytosolic NAD to produce cADPR. The role of cell surface type II CD38 in cellular cADPR production is unknown. Here we modulated type II CD38 expression and assessed the effects of this modulation on cADPR levels. We developed a photoactivatable cross-linking probe based on a CD38 nanobody, and, combining it with MS analysis, we discovered that cell surface CD38 interacts with CD71. CD71 knockdown increased CD38 levels, and CD38 knockout reciprocally increased CD71, and both could be cocapped and coimmunoprecipitated. We constructed a chimera comprising the N-terminal segment of CD71 and a CD38 nanobody to mimic CD71's ligand property. Overexpression of this chimera induced a dramatically large decrease in CD38 via lysosomes. Remarkably, cellular cADPR levels did not decrease correspondingly. Bafilomycin-mediated blockade of lysosomal degradation greatly elevated active type II CD38 by trapping it in the lysosomes but also did not increase cADPR levels. Retention of type II CD38 in the endoplasmic reticulum (ER) by expressing an ER construct that prevented its transport to the cell surface likewise did not change cADPR levels. These results provide first and direct evidence that cADPR biogenesis occurs in the cytosol and is catalyzed mainly by type III CD38 and that type II CD38, compartmentalized in the ER or lysosomes or on the cell surface, contributes only minimally to cADPR biogenesis.
Asunto(s)
Antígenos CD/metabolismo , ADP-Ribosa Cíclica/metabolismo , Receptores de Transferrina/metabolismo , ADP-Ribosil Ciclasa 1/genética , ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD/genética , Calcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Citosol/metabolismo , Células HEK293 , Humanos , Unión Proteica , Receptores de Transferrina/genéticaRESUMEN
NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.
Asunto(s)
Citocinas/metabolismo , Estrés del Retículo Endoplásmico , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Peptidoglicano/sangre , Calcimicina/química , Calcimicina/farmacología , Calcio/química , Calcio/metabolismo , Quimiocina CXCL1/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Técnicas de Inactivación de Genes , Células HCT116 , Humanos , Interleucina-8/metabolismo , Proteína Adaptadora de Señalización NOD1/deficiencia , Proteína Adaptadora de Señalización NOD1/genética , Proteína Adaptadora de Señalización NOD2/deficiencia , Proteína Adaptadora de Señalización NOD2/genética , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Sitios de Unión , Femenino , Humanos , Unión ProteicaRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Ecdisterona/farmacología , Agregado de Proteínas/efectos de los fármacos , Molécula de Interacción Estromal 1/metabolismo , Animales , Apoptosis/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/metabolismo , Lepidópteros/efectos de los fármacos , Lepidópteros/crecimiento & desarrollo , Lepidópteros/metabolismo , Metamorfosis Biológica/efectos de los fármacos , Fosforilación/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Molécula de Interacción Estromal 1/deficiencia , Molécula de Interacción Estromal 1/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Most members of the family of proteins containing a transmembrane BAX inhibitor motif (TMBIM) have anti-apoptotic activity, but their in vivo functions and intracellular mechanisms remain obscure. Here, we report that zebrafish Tmbim3a/Grinaa functions in the prevention of cold-induced endoplasmic reticulum (ER) stress and apoptosis. Using a gene-trapping approach, we obtained a mutant zebrafish line in which the expression of the tmbim3a/grinaa gene is disrupted by a Tol2 transposon insertion. Homozygous tmbim3a/grinaa mutant larvae exhibited time-dependently increased mortality and apoptosis under cold exposure (at 16 °C). Mechanistically, using immunofluorescence, fluorescence-based assessments of intracellular/mitochondrial Ca2+ levels, mitochondrial membrane potential measurements, and Ca2+-ATPase assays, we found that cold exposure suppresses sarcoplasmic/ER Ca2+-ATPase (SERCA) activity and induces the unfolded protein response (UPR) and ER stress. We also found that the cold-induced ER stress is increased in homozygous tmbim3a/grinaa mutant embryos. The cold-stress hypersensitivity of the tmbim3a/grinaa mutants was tightly associated with disrupted intracellular Ca2+ homeostasis, followed by mitochondrial Ca2+ overload and cytochrome c release, leading to the activation of caspase 9- and caspase-3-mediated intrinsic apoptotic pathways. Treatment of zebrafish larvae with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM) or with 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of the calcium-releasing protein inositol 1,4,5-trisphosphate receptor (IP3R), alleviated cold-induced cell death. Together, these findings unveil a key role of Tmbim3a/Grinaa in relieving cold-induced ER stress and in protecting cells against caspase 9- and caspase 3-mediated apoptosis during zebrafish development.
Asunto(s)
Apoptosis , Frío , Estrés del Retículo Endoplásmico , Proteínas de la Membrana/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Apoptosis/efectos de los fármacos , Compuestos de Boro/farmacología , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión no Mamífero/efectos de los fármacos , Proteínas de la Membrana/genética , Mutación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Fracciones Subcelulares/metabolismo , Respuesta de Proteína Desplegada , Pez Cebra/embriología , Pez Cebra/crecimiento & desarrollo , Proteínas de Pez Cebra/genéticaRESUMEN
Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca2+ stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca2+ stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca2+-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca2+-signaling enzyme like CD38.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Señalización del Calcio , ADP-Ribosa Cíclica/metabolismo , NADP/análogos & derivados , ADP-Ribosil Ciclasa 1/química , ADP-Ribosil Ciclasa 1/genética , Animales , Proteínas de Unión al Calcio/antagonistas & inhibidores , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas del Citoesqueleto/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , NADP/metabolismo , ARN Guía de Kinetoplastida/metabolismoRESUMEN
Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.
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Señalización del Calcio/efectos de los fármacos , Núcleo Celular/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Fosfolipasa C delta/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Cadenas Pesadas de Clatrina/antagonistas & inhibidores , Cadenas Pesadas de Clatrina/genética , Cadenas Pesadas de Clatrina/metabolismo , Ciclina A/metabolismo , Ciclina B1/metabolismo , Receptores ErbB/metabolismo , Humanos , Hidrólisis , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipasa C delta/antagonistas & inhibidores , Fosfolipasa C delta/genética , Fosfolipasa C gamma/antagonistas & inhibidores , Fosfolipasa C gamma/genética , Fosfolipasa C gamma/metabolismo , Proteína Quinasa C/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismoRESUMEN
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.
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Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Compuestos de Boro/química , Compuestos de Boro/farmacología , Calcio/química , Retículo Endoplásmico/química , Retículo Endoplásmico/genética , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dominios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/química , Molécula de Interacción Estromal 2/genéticaRESUMEN
Fine-tuning of the activity of inositol 1,4,5-trisphosphate receptors (IP3R) by a diverse array of regulatory inputs results in intracellular Ca2+ signals with distinct characteristics. These events allow the activation of specific downstream effectors. We reported previously that region-specific proteolysis represents a novel regulatory event for type 1 IP3R (R1). Specifically, caspase-fragmented R1 display a marked increase in single-channel open probability. More importantly, the distinct characteristics of the Ca2+ signals elicited via fragmented R1 can activate alternate downstream effectors. In this report, we expand these studies to investigate whether all IP3R subtypes are regulated by proteolysis. We now show that type 2 and type 3 IP3R (R2 and R3, respectively) are proteolytically cleaved in rodent models of acute pancreatitis. Surprisingly, fragmented IP3R retained tetrameric architecture, remained embedded in endoplasmic reticulum membranes and were not functionally disabled. Proteolysis was associated with a marked attenuation of the frequency of Ca2+ signals in pancreatic lobules. Consistent with these data, expression of DNAs encoding complementary R2 and R3 peptides mimicking fragmented receptors at particular sites, resulted in a significant decrease in the frequency of agonist-stimulated Ca2+ oscillations. Further, proteolysis of R2 resulted in a marked decrease in single-channel open probability. Taken together, proteolytic fragmentation modulates R2 and R3 activity in a region-specific manner, and this event may contribute to the altered Ca2+ signals in pancreatic acinar cells during acute pancreatitis.
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Señalización del Calcio , Modelos Animales de Enfermedad , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Pancreatitis/fisiopatología , Enfermedad Aguda , Animales , Receptores de Inositol 1,4,5-Trifosfato/genética , Activación del Canal Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , Proteolisis , Ratas , Ratas WistarRESUMEN
The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.
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Señalización del Calcio , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , ARN Interferente Pequeño/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
A sensitization of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release is associated with oxidative stress in multiple cell types. These effects are thought to be mediated by alterations in the redox state of critical thiols in the IP3R, but this has not been directly demonstrated in intact cells. Here, we utilized a combination of gel-shift assays with MPEG-maleimides and LC-MS/MS to monitor the redox state of recombinant IP3R1 expressed in HEK293 cells. We found that under basal conditions, â¼5 of the 60 cysteines are oxidized in IP3R1. Cell treatment with 50 µm thimerosal altered gel shifts, indicating oxidation of â¼20 cysteines. By contrast, the shifts induced by 0.5 mm H2O2 or other oxidants were much smaller. Monitoring of biotin-maleimide attachment to IP3R1 by LC-MS/MS with 71% coverage of the receptor sequence revealed modification of two cytosolic (Cys-292 and Cys-1415) and two intraluminal cysteines (Cys-2496 and Cys-2533) under basal conditions. The thimerosal treatment modified an additional eleven cysteines, but only three (Cys-206, Cys-767, and Cys-1459) were consistently oxidized in multiple experiments. H2O2 also oxidized Cys-206 and additionally oxidized two residues not modified by thimerosal (Cys-214 and Cys-1397). Potentiation of IP3R channel function by oxidants was measured with cysteine variants transfected into a HEK293 IP3R triple-knockout cell line, indicating that the functionally relevant redox-sensitive cysteines are predominantly clustered within the N-terminal suppressor domain of IP3R. To our knowledge, this study is the first that has used proteomic methods to assess the redox state of individual thiols in IP3R in intact cells.
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Peróxido de Hidrógeno/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Sustitución de Aminoácidos , Señalización del Calcio , Cisteína/química , Cisteína/genética , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Receptores de Inositol 1,4,5-Trifosfato/genética , Oxidación-ReducciónRESUMEN
Sarcoplasmic reticulum (SR) Ca2+ cycling is governed by the cardiac ryanodine receptor (RyR2) and SR Ca2+-ATPase (SERCA2a). Abnormal SR Ca2+ cycling is thought to be the primary cause of Ca2+ alternans that can elicit ventricular arrhythmias and sudden cardiac arrest. Although alterations in either RyR2 or SERCA2a function are expected to affect SR Ca2+ cycling, whether and to what extent altered RyR2 or SERCA2a function affects Ca2+ alternans is unclear. Here, we employed a gain-of-function RyR2 variant (R4496C) and the phospholamban-knockout (PLB-KO) mouse model to assess the effect of genetically enhanced RyR2 or SERCA2a function on Ca2+ alternans. Confocal Ca2+ imaging revealed that RyR2-R4496C shortened SR Ca2+ release refractoriness and markedly suppressed rapid pacing-induced Ca2+ alternans. Interestingly, despite enhancing RyR2 function, intact RyR2-R4496C hearts exhibited no detectable spontaneous SR Ca2+ release events during pacing. Unlike for RyR2, enhancing SERCA2a function by ablating PLB exerted a relatively minor effect on Ca2+ alternans in intact hearts expressing RyR2 WT or a loss-of-function RyR2 variant, E4872Q, that promotes Ca2+ alternans. Furthermore, partial SERCA2a inhibition with 3 µm 2,5-di-tert-butylhydroquinone (tBHQ) also had little impact on Ca2+ alternans, whereas strong SERCA2a inhibition with 10 µm tBHQ markedly reduced the amplitude of Ca2+ transients and suppressed Ca2+ alternans in intact hearts. Our results demonstrate that enhanced RyR2 function suppresses Ca2+ alternans in the absence of spontaneous Ca2+ release and that RyR2, but not SERCA2a, is a key determinant of Ca2+ alternans in intact working hearts, making RyR2 an important therapeutic target for cardiac alternans.