RESUMO
Genetically encoded calcium indicators (GECIs) are indispensable tools for real-time monitoring of intracellular calcium signals and cellular activities in living organisms. Current GECIs face the challenge of suboptimal peak signal-to-baseline ratio (SBR) with limited resolution for reporting subtle calcium transients. We report herein the development of a suite of calcium sensors, designated NEMO, with fast kinetics and wide dynamic ranges (>100-fold). NEMO indicators report Ca2+ transients with peak SBRs around 20-fold larger than the top-of-the-range GCaMP6 series. NEMO sensors further enable the quantification of absolution calcium concentration with ratiometric or photochromic imaging. Compared with GCaMP6s, NEMOs could detect single action potentials in neurons with a peak SBR two times higher and a median peak SBR four times larger in vivo, thereby outperforming most existing state-of-the-art GECIs. Given their high sensitivity and resolution to report intracellular Ca2+ signals, NEMO sensors may find broad applications in monitoring neuronal activities and other Ca2+-modulated physiological processes in both mammals and plants.
Assuntos
Cálcio , Neurônios , Animais , Cálcio/metabolismo , Neurônios/fisiologia , Sinalização do Cálcio/fisiologia , Indicadores e Reagentes , Mamíferos/metabolismoRESUMO
Ferroptosis is iron-dependent oxidative cell death. Labile iron and polyunsaturated fatty acid (PUFA)-containing lipids are two critical factors for ferroptosis execution. Many processes regulating iron homeostasis and lipid synthesis are critically involved in ferroptosis. However, it remains unclear whether biological processes other than iron homeostasis and lipid synthesis are associated with ferroptosis. Using kinase inhibitor library screening, we discovered a small molecule named CGI1746 that potently blocks ferroptosis. Further studies demonstrate that CGI1746 acts through sigma-1 receptor (σ1R), a chaperone primarily located at mitochondria-associated membranes (MAMs), to inhibit ferroptosis. Suppression of σ1R protects mice from cisplatin-induced acute kidney injury hallmarked by ferroptosis. Mechanistically, CGI1746 treatment or genetic disruption of MAMs leads to defective Ca2+ transfer, mitochondrial reactive oxygen species (ROS) production and PUFA-containing triacylglycerol accumulation. Therefore, we propose a critical role for MAMs in ferroptosis execution.
Assuntos
Ferroptose , Membranas Associadas à Mitocôndria , Espécies Reativas de Oxigênio , Receptores sigma , Receptor Sigma-1 , Animais , Camundongos , Ferroptose/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Membranas Associadas à Mitocôndria/efeitos dos fármacos , Membranas Associadas à Mitocôndria/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores sigma/metabolismoRESUMO
The MICU1-MICU2 heterodimer regulates the mitochondrial calcium uniporter (MCU) and mitochondrial calcium uptake. Herein, we present two crystal structures of the MICU1-MICU2 heterodimer, in which Ca2+ -free and Ca2+ -bound EF-hands are observed in both proteins, revealing both electrostatic and hydrophobic interfaces. Furthermore, we show that MICU1 interacts with EMRE, another regulator of MCU, through a Ca2+ -dependent alkaline groove. Ca2+ binding strengthens the MICU1-EMRE interaction, which in turn facilitates Ca2+ uptake. Conversely, the MICU1-MCU interaction is favored in the absence of Ca2+ , thus inhibiting the channel activity. This Ca2+ -dependent switch illuminates how calcium signals are transmitted from regulatory subunits to the calcium channel and the transition between gatekeeping and activation channel functions. Furthermore, competition with an EMRE peptide alters the uniporter threshold in resting conditions and elevates Ca2+ accumulation in stimulated mitochondria, confirming the gatekeeper role of the MICU1-MICU2 heterodimer. Taken together, these structural and functional data provide new insights into the regulation of mitochondrial calcium uptake.
Assuntos
Canais de Cálcio/química , Proteínas de Ligação ao Cálcio/química , Cálcio/química , Proteínas de Transporte de Cátions/química , Proteínas de Transporte da Membrana Mitocondrial/química , Complexos Multiproteicos/química , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Cristalografia por Raios X , Humanos , Transporte de Íons , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismoRESUMO
OBJECTIVE: Tuberculum sellae meningiomas (TSMs) usually compress the optic nerve and optic chiasma, thus affecting vision. Surgery is an effective means to remove tumors and improve visual outcomes. On a larger scale, this study attempted to further explore and confirm the factors related to postoperative visual outcomes to guide the treatment of TSMs. METHODS: Data were obtained from 208 patients with TSMs who underwent surgery at our institution between January 2010 and August 2022. Demographics, ophthalmologic examination results, imaging data, extent of resection, radiotherapy status, and surgical approaches were included in the analysis. Univariate and multivariate logistic regressions were used to assess the factors that could lead to favorable visual outcomes. RESULTS: The median follow-up duration was 63 months, and gross total resection (GTR) was achieved in 174 (83.7%) patients. According to our multivariate logistic regression analysis, age < 60 years (odds ratio [OR] = 0.310; P = 0.007), duration of preoperative visual symptoms (DPVS) < 10 months (OR = 0.495; P = 0.039), tumor size ≤ 27 mm (OR = 0.337; P = 0.002), GTR (OR = 3.834; P = 0.006), and a tumor vertical-to-horizontal dimensional ratio < 1 (OR = 2.593; P = 0.006) were found to be significant independent predictors of favorable visual outcomes. CONCLUSION: Age, DPVS, tumor size, GTR, and the tumor vertical-to-horizontal dimensional ratio were found to be powerful predictors of favorable visual outcomes. This study may help guide decisions regarding the treatment of TSMs.
Assuntos
Neoplasias Meníngeas , Meningioma , Neoplasias da Base do Crânio , Humanos , Pessoa de Meia-Idade , Meningioma/complicações , Meningioma/diagnóstico por imagem , Meningioma/cirurgia , Neoplasias Meníngeas/complicações , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/cirurgia , Resultado do Tratamento , Sela Túrcica/diagnóstico por imagem , Sela Túrcica/cirurgia , Sela Túrcica/patologia , Procedimentos Neurocirúrgicos/métodos , Neoplasias da Base do Crânio/cirurgia , Estudos RetrospectivosRESUMO
Sensing the lowering of endoplasmic reticulum (ER) calcium (Ca2+), STIM1 mediates a ubiquitous Ca2+ influx process called the store-operated Ca2+ entry (SOCE). Dysregulated STIM1 function or abnormal SOCE is strongly associated with autoimmune disorders, atherosclerosis, and various forms of cancers. Therefore, uncovering the molecular intricacies of post-translational modifications, such as oxidation, on STIM1 function is of paramount importance. In a recent proteomic screening, we identified three protein disulfide isomerases (PDIs)-Prolyl 4-hydroxylase subunit beta (P4HB), protein disulfide-isomerase A3 (PDIA3), and thioredoxin domain-containing protein 5 (TXNDC5)-as the ER-luminal interactors of STIM1. Here, we demonstrated that these PDIs dynamically associate with STIM1 and STIM2. The mutation of the two conserved cysteine residues of STIM1 (STIM1-2CA) decreased its Ca2+ affinity both in cellulo and in situ. Knockdown of PDIA3 or P4HB increased the Ca2+ affinity of wild-type STIM1 while showing no impact on the STIM1-2CA mutant, indicating that PDIA3 and P4HB regulate STIM1's Ca2+ affinity by acting on ER-luminal cysteine residues. This modulation of STIM1's Ca2+ sensitivity was further confirmed by Ca2+ imaging experiments, which showed that knockdown of these two PDIs does not affect STIM1-mediated SOCE upon full store depletion but leads to enhanced SOCE amplitudes upon partial store depletion. Thus, P4HB and PDIA3 dynamically modulate STIM1 activation by fine-tuning its Ca2+ binding affinity, adjusting the level of activated STIM1 in response to physiological cues. The coordination between STIM1-mediated Ca2+ signaling and redox responses reported herein may have implications for cell physiology and pathology.
Assuntos
Cálcio , Proteínas de Neoplasias , Oxirredução , Pró-Colágeno-Prolina Dioxigenase , Isomerases de Dissulfetos de Proteínas , Molécula 1 de Interação Estromal , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Humanos , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Cálcio/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno-Prolina Dioxigenase/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Ligação Proteica , Sinalização do Cálcio , Molécula 2 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/genéticaRESUMO
Membrane contact sites (MCSs) mediate crucial physiological processes in eukaryotic cells, including ion signaling, lipid metabolism, and autophagy. Dysregulation of MCSs is closely related to various diseases, such as type 2 diabetes mellitus (T2DM), neurodegenerative diseases, and cancers. Visualization, proteomic mapping and manipulation of MCSs may help the dissection of the physiology and pathology MCSs. Recent technical advances have enabled better understanding of the dynamics and functions of MCSs. Here we present a summary of currently known functions of MCSs, with a focus on optical approaches to visualize and manipulate MCSs, as well as proteomic mapping within MCSs.
Assuntos
Diabetes Mellitus Tipo 2 , Retículo Endoplasmático , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Membranas Mitocondriais/metabolismo , Optogenética , ProteômicaRESUMO
The heart requires a variety of energy substrates to maintain proper contractile function. Glucose and long-chain fatty acids (FA) are the major cardiac metabolic substrates under physiological conditions. Upon stress, a shift of cardiac substrate preference toward either glucose or FA is associated with cardiac diseases. For example, in pressure-overloaded hypertrophic hearts, there is a long-lasting substrate shift toward glucose, while in hearts with diabetic cardiomyopathy, the fuel is switched toward FA. Stromal interaction molecule 1 (STIM1), a well-established calcium (Ca2+) sensor of endoplasmic reticulum (ER) Ca2+ store, is increasingly recognized as a critical player in mediating both cardiac hypertrophy and diabetic cardiomyopathy. However, the cause-effect relationship between STIM1 and glucose/FA metabolism and the possible mechanisms by which STIM1 is involved in these cardiac metabolic diseases are poorly understood. In this review, we first discussed STIM1-dependent signaling in cardiomyocytes and metabolic changes in cardiac hypertrophy and diabetic cardiomyopathy. Second, we provided examples of the involvement of STIM1 in energy metabolism to discuss the emerging role of STIM1 in the regulation of energy substrate preference in metabolic cardiac diseases and speculated the corresponding underlying molecular mechanisms of the crosstalk between STIM1 and cardiac energy substrate preference. Finally, we briefly discussed and presented future perspectives on the possibility of targeting STIM1 to rescue cardiac metabolic diseases. Taken together, STIM1 emerges as a key player in regulating cardiac energy substrate preference, and revealing the underlying molecular mechanisms by which STIM1 mediates cardiac energy metabolism could be helpful to find novel targets to prevent or treat cardiac metabolic diseases.
Assuntos
Cardiomiopatias Diabéticas , Cardiopatias , Molécula 1 de Interação Estromal , Humanos , Cardiomegalia , Glucose , Miócitos Cardíacos , Proteínas de NeoplasiasRESUMO
STIM1 (stromal interaction molecule 1) is one of the key components of the store operated Ca2+ entry channel (SOCE), which is located on the endoplasmic reticulum membrane and highly expressed in most kinds of tumors. STIM1 promotes tumorigenesis and metastasis by modulating the formation of invadopodia, promoting angiogenesis, mediating inflammatory response, altering the cytoskeleton and cell dynamics. However, the roles and mechanism of STIM1 in different tumors have not been fully elucidated. In this review, we summarize the latest progress and mechanisms of STIM1 in tumorigenesis and metastasis, thereby providing insights and references for the study on STIM1 in the field of cancer biology in the future.
Assuntos
Cálcio , Carcinogênese , Humanos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Carcinogênese/genética , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Neoplasias/genéticaRESUMO
The endoplasmic reticulum (ER) Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2, which connect ER Ca2+ depletion with extracellular Ca2+ influx, are crucial for the maintenance of Ca2+ homeostasis in mammalian cells. Despite the recent progress in unraveling the role of STIM2 in Ca2+ signaling, the mechanistic underpinnings of its activation remain underexplored. We use an engineering approach to direct ER-resident STIMs to the plasma membrane (PM) while maintaining their correct membrane topology, as well as Förster resonance energy transfer (FRET) sensors that enabled in cellulo real-time monitoring of STIM activities. This allowed us to determine the calcium affinities of STIM1 and STIM2 both in cellulo and in situ, explaining the current discrepancies in the literature. We also identified the key structural determinants, especially the corresponding G residue in STIM1, which define the distinct activation dynamics of STIM2. The chimeric E470G mutation could switch STIM2 from a slow and weak Orai channel activator into a fast and potent one like STIM1 and vice versa. The systemic dissection of STIM2 activation by protein engineering sets the stage for the elucidation of the regulation and function of STIM2-mediated signaling in mammals.
Assuntos
Proteínas de Neoplasias/fisiologia , Molécula 1 de Interação Estromal/fisiologia , Molécula 2 de Interação Estromal/genética , Molécula 2 de Interação Estromal/fisiologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/fisiologia , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Células HEK293 , Células HeLa , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismoRESUMO
The transmembrane docking of endoplasmic reticulum (ER) Ca2+-sensing STIM proteins with plasma membrane (PM) Orai Ca2+ channels is a critical but poorly understood step in Ca2+ signal generation. STIM1 protein dimers unfold to expose a discrete STIM-Orai activating region (SOAR1) that tethers and activates Orai1 channels within discrete ER-PM junctions. We reveal that each monomer within the SOAR dimer interacts independently with single Orai1 subunits to mediate cross-linking between Orai1 channels. Superresolution imaging and mobility measured by fluorescence recovery after photobleaching reveal that SOAR dimer cross-linking leads to substantial Orai1 channel clustering, resulting in increased efficacy and cooperativity of Orai1 channel function. A concatenated SOAR1 heterodimer containing one monomer point mutated at its critical Orai1 binding residue (F394H), although fully activating Orai channels, is completely defective in cross-linking Orai1 channels. Importantly, the naturally occurring STIM2 variant, STIM2.1, has an eight-amino acid insert in its SOAR unit that renders it functionally identical to the F394H mutant in SOAR1. Contrary to earlier predictions, the SOAR1-SOAR2.1 heterodimer fully activates Orai1 channels but prevents cross-linking and clustering of channels. Interestingly, combined expression of full-length STIM1 with STIM2.1 in a 5:1 ratio causes suppression of sustained agonist-induced Ca2+ oscillations and protects cells from Ca2+ overload, resulting from high agonist-induced Ca2+ release. Thus, STIM2.1 exerts a powerful regulatory effect on signal generation likely through preventing Orai1 channel cross-linking. Overall, STIM-mediated cross-linking of Orai1 channels is a hitherto unrecognized functional paradigm that likely provides an organizational microenvironment within ER-PM junctions with important functional impact on Ca2+ signal generation.
Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/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 , Cálcio/metabolismo , Dimerização , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
Underlying topography plays an important role in the national economic construction, military security, resource exploration and investigation. Since synthetic aperture radar tomography (TomoSAR) can achieve the three-dimensional imaging of forests, it has been widely used in underlying topography estimation. At present, there are two kinds of TomoSAR based on the applied datasets: single polarimetric TomoSAR (SP-TomoSAR) and fully polarimetric TomoSAR (FP-TomoSAR). However, SP-TomoSAR cannot obtain the underlying topography accurately due to the lack of enough observations. FP-TomoSAR can improve the estimation accuracy of underlying topography. However, it requires high-cost data acquisition for the large-scale application. Thus, this paper proposes the dual polarimetric TomoSAR (DP-TomoSAR) as another suitable candidate to estimate the underlying topography because of its wide swath and multiple polarimetric observations. Moreover, three frequently used spectral estimation algorithms, namely, Beamforming, Capon and MUSIC, are used in DP-TomoSAR. For validation, a series of simulated experiments was carried out, and the airborne P-band multiple polarimetric SAR data over the Lope, Gabon was also acquired to estimate the underlying topography. The results suggest that DP-TomoSAR in HH & HV combination is more suitable to estimate underlying topography over forest areas than other DP combinations. Moreover, the estimation accuracy of DP-TomoSAR is slightly lower than that of FP-TomoSAR but is higher than that of SP-TomoSAR.
Assuntos
Monitoramento Ambiental , Radar , Florestas , Menogaril , TomografiaRESUMO
The Ca2+ release-activated Ca2+ (CRAC) channels control many Ca2+-modulated physiological processes in mammals. Hyperactivating CRAC channels are known to cause several human diseases, including Stormorken syndrome. Here, we show the design of azopyrazole-derived photoswitchable CRAC channel inhibitors (designated piCRACs), which enable optical inhibition of store-operated Ca2+ influx and downstream signaling. Moreover, piCRAC-1 has been applied in vivo to alleviate thrombocytopenia and hemorrhage in a zebrafish model of Stormorken syndrome in a light-dependent manner.
RESUMO
Store-operated Ca2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel.
Assuntos
Retículo Endoplasmático/metabolismo , Ativação do Canal Iônico , Mutação de Sentido Incorreto , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Multimerização Proteica , Molécula 1 de Interação Estromal/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Animais , Drosophila melanogaster , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Molécula 1 de Interação Estromal/genéticaRESUMO
Near-IR-emitting and/or efficiently photodynamic water-soluble Ru(II) complexes that hold great application potentials as photodynamic therapy and/or photodetection agents for cancers have been poorly explored. In this paper, the solvatochromism, calf thymus DNA binding, and singlet oxygen generation properties of a known ruthenium(II) complex of visible-emitting [Ru(bpy)2(dtdpq)](ClO4)2 (Ru1) and a new homoleptic complex of near-IR-emitting [Ru(dtdpq)3](ClO4)2 (Ru2) (bpy = 2,2'-bipyridine, dtdpq = 2,3-bis(thiophen-2-yl)pyrazino[2,3-f][1,10]phenanothroline) in water are reported. Moreover, DNA photocleavage, singlet oxygen generation in HeLa cells, cellular uptake/localization, and in vitro photodynamic therapy for cancer cells of water-soluble Ru1 are described in detail. The results show that Ru1 acted as potent photodynamic cancer therapy and mitochondrial imaging agents. Ru2 exhibited very strong solvatochromism from a visible emission maximum at 588 nm in CH2Cl2 to the near-IR region at 700 nm in water and singlet oxygen generation yield in water (23%) and DNA binding properties (intercalative DNA binding constant on the order of 106 M-1) comparable to those of Ru1, which should make Ru2 attractive for the aforementioned applications of Ru1 if the water solubility of Ru2 can be improved enough for the studies above.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , DNA de Neoplasias/efeitos dos fármacos , Luz , Fotoquimioterapia , Rutênio/farmacologia , Tiofenos/farmacologia , Células A549 , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Células HeLa , Humanos , Raios Infravermelhos , Células MCF-7 , Estrutura Molecular , Oxigênio/análise , Oxigênio/metabolismo , Rutênio/química , Tiofenos/químicaRESUMO
OBJECTIVE: To detect the disease-causing mutation in a family with hereditary spherocytosis type â . METHODS: Genomic DNA was extracted from peripheral blood samples of the proband and his relatives. Next-generation sequencing was used to detect the mutations of relevant genes. Suspected pathogenic mutation was verified by Sanger sequencing. RESULTS: The proband was found to harbor a novel frameshifting mutation in the coding region of ANK1 gene, which has resulted in abnormal structure or function of the protein. The mutation was confirmed by Sanger sequencing, with both his father and brother found to have carried the same mutation. CONCLUSION: The c.247delG mutation of proband hereditary spherocytosis typeâ in this family due to mutation of the ANK1 geneï¼.
Assuntos
Anquirinas/genética , Esferocitose Hereditária/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Fases de Leitura AbertaRESUMO
Mediated through the combined action of STIM proteins and Orai channels, store-operated Ca2+ entry (SOCE) functions ubiquitously among different cell types. The existence of multiple STIM and Orai genes has made it difficult to assign specific roles of each STIM and Orai homolog in mediating Ca2+ signals. Using CRISPR/Cas9 gene editing tools, we generated cells with both STIM or all three Orai homologs deleted and directly monitored store Ca2+ and Ca2+ signals. We found that unstimulated, SOCE null KO cells still retain 50~70% of ER Ca2+ stores of wildtype (wt) cells. After brief exposure to store-emptying conditions, acute refilling of ER Ca2+ stores was totally blocked in KO cells. However, after 24 h in culture, stores were eventually refilled. Thus, SOCE is critical for immediate refilling of ER Ca2+ but is dispensable for the maintenance of long-term ER Ca2+ homeostasis. Using the Orai null background triple Orai-KO cells, we examined the plasma membrane translocation properties of a series of truncated STIM1 variants. FRET analysis reveals that, even though PM tethering of STIM1 expedites the activation of STIM1 by facilitating its oligomerization, migration, and accumulation in ER-PM junctions, it is not required for the conformational switch, oligomerization, and clustering of STIM1. Even without overt puncta formation at ER-PM junctions, STIM11-491 and STIM11-666 could still rescue SOCE when expressed in STIM KO cells. Thus, ER-PM trapping and clustering of STIM molecules only facilitates the process of SOCE activation, but is not essential for the activation of Orai channels.
Assuntos
Sinalização do Cálcio , Proteína ORAI1/deficiência , Molécula 1 de Interação Estromal/deficiência , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteína ORAI1/genética , Multimerização Proteica , Transporte Proteico , Molécula 1 de Interação Estromal/genéticaRESUMO
Voltage-gated Ca2+ (CaV ) channels mediate Ca2+ entry into excitable cells to regulate a myriad of cellular events following membrane depolarization. We report the engineering of RGK GTPases, a class of genetically encoded CaV channel modulators, to enable photo-tunable modulation of CaV channel activity in excitable mammalian cells. This optogenetic tool (designated optoRGK) tailored for CaV channels could find broad applications in interrogating a wide range of CaV -mediated physiological processes.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Optogenética/métodos , Animais , Canais de Cálcio/genética , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Engenharia de Proteínas/métodosRESUMO
BACKGROUND: Because phylogenetic inference is an important basis for answering many evolutionary problems, a large number of algorithms have been developed. Some of these algorithms have been improved by integrating gene evolution models with the expectation of accommodating the hierarchy of evolutionary processes. To the best of our knowledge, however, there still is no single unifying model or algorithm that can take all evolutionary processes into account through a stepwise or simultaneous method. RESULTS: On the basis of three existing phylogenetic inference algorithms, we built an integrated pipeline for inferring the evolutionary history of a given gene family; this pipeline can model gene sequence evolution, gene duplication-loss, gene transfer and multispecies coalescent processes. As a case study, we applied this pipeline to the STIMATE (TMEM110) gene family, which has recently been reported to play an important role in store-operated Ca2+ entry (SOCE) mediated by ORAI and STIM proteins. We inferred their phylogenetic trees in 69 sequenced chordate genomes. CONCLUSIONS: By integrating three tree reconstruction algorithms with diverse evolutionary models, a pipeline for inferring the evolutionary history of a gene family was developed, and its application was demonstrated.
Assuntos
Evolução Molecular , Proteínas de Membrana/genética , Família Multigênica , Filogenia , Algoritmos , Animais , Duplicação Gênica , Humanos , Funções Verossimilhança , Modelos GenéticosRESUMO
Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.
Assuntos
Cálcio , Proteína ORAI1/metabolismo , Multimerização Proteica/fisiologia , Substituição de Aminoácidos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteína ORAI1/genéticaRESUMO
Store-operated Ca2+ entry fulfills a crucial role in controlling Ca2+ signals in almost all cells. The Ca2+-sensing stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER) undergo complex conformational changes in response to depleted ER luminal Ca2+, allowing them to unfold and become trapped in ER-plasma membrane (PM) junctions. Dimers of STIM proteins trap and gate the plasma membrane Orai Ca2+ channels within these junctions to generate discrete zones of high Ca2+ and regulate sensitive Ca2+-dependent intracellular signaling pathways. The STIM-Orai activating region (SOAR) of STIM1 becomes exposed upon store depletion and promotes trapping of Orai1 at the PM. Residue Phe-394 within SOAR forms an integral part of the high-affinity Orai1-interacting site. Our results demonstrate that only a single active site within the dimeric SOAR domain of STIM1 is required for the activation of Orai1 channel activity. This unimolecular model is strongly supported by evidence of variable STIM1:Orai1 stoichiometry reported in many studies. We hypothesize that unimolecular coupling promotes cross-linking of channels, localizing Ca2+ signals, and regulating channel activity. We have also identified a key "nexus" region in Orai1 near the C-terminal STIM1-binding site that can be mutated to constitutively activate Ca2+ entry, mimicking STIM1 activated channels. This suggests that STIM1 mediates gating of Orai1 in an allosteric manner via interaction with the Orai1 C-terminus alone. This model suggests the dual role of STIM1 in regulating both localization and gating of Orai1 channels and has important implications for the regulation of SOCE-mediated downstream signaling and the kinetics of channel activation.