RESUMO
The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.
Assuntos
Proteínas de Neoplasias , Multimerização Proteica , Molécula 1 de Interação Estromal , Humanos , Sítios de Ligação , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/química , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/química , Ligação Proteica , Domínios Proteicos , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/químicaRESUMO
BACKGROUND: Bone metastasis is a principal cause of mortality in patients with prostate cancer (PCa). Increasing evidence indicates that high expression of stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) significantly activates the calcium (Ca2+) signaling pathway and is involved in multiple steps of bone metastasis in PCa. However, the regulatory mechanism and target therapy of STIM1 is poorly defined. METHODS: Liquid chromatography-mass spectrometry analysis was performed to identify tetraspanin 18 (TSPAN18) as a binding protein of STIM1. Co-IP assay was carried out to explore the mechanism by which TSPAN18 inhibits STIM1 degradation. The biological function of TSPAN18 in bone metastasis of PCa was further investigated in vitro and in vivo models. RESULT: We identified that STIM1 directly interacted with TSPAN18, and TSPAN18 competitively inhibited E3 ligase tripartite motif containing 32 (TRIM32)-mediated STIM1 ubiquitination and degradation, leading to increasing STIM1 protein stability. Furthermore, TSPAN18 significantly stimulated Ca2+ influx in an STIM1-dependent manner, and then markedly accelerated PCa cells migration and invasion in vitro and bone metastasis in vivo. Clinically, overexpression of TSPAN18 was positively associated with STIM1 protein expression, bone metastasis and poor prognosis in PCa. CONCLUSION: Taken together, this work discovers a novel STIM1 regulative mechanism that TSPAN18 protects STIM1 from TRIM32-mediated ubiquitination, and enhances bone metastasis of PCa by activating the STIM1-Ca2+ signaling axis, suggesting that TSPAN18 may be an attractive therapeutic target for blocking bone metastasis in PCa.
Assuntos
Cálcio , Neoplasias da Próstata , Masculino , Humanos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Neoplasias da Próstata/genética , Ubiquitinação , Sinalização do Cálcio , Proteína ORAI1/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Tetraspaninas/genética , Tetraspaninas/metabolismoRESUMO
Stromal interaction molecule 1 (STIM1) is an endo/sarcoplasmic reticulum (ER/SR) calcium (Ca2+) sensing protein that regulates store-operated calcium entry (SOCE). In SOCE, STIM1 activates Orai1-composed Ca2+ channels in the plasma membrane (PM) after ER stored Ca2+ depletion. S-Glutathionylation of STIM1 at Cys56 evokes constitutive SOCE in DT40 cells; however, the structural and biophysical mechanisms underlying the regulation of STIM1 by this modification are poorly defined. By establishing a protocol for site-specific STIM1 S-glutathionylation using reduced glutathione and diamide, we have revealed that modification of STIM1 at either Cys49 or Cys56 induces thermodynamic destabilization and conformational changes that result in increased solvent-exposed hydrophobicity. Further, S-glutathionylation or point-mutation of Cys56 reduces Ca2+ binding affinity, as measured by intrinsic fluorescence and far-UV circular dichroism spectroscopies. Solution NMR showed S-glutathionylated-induced perturbations in STIM1 are localized to the α1 helix of the canonical EF-hand, the α3 and α4 helices of the non-canonical EF-hand and α6 and α8 helices of the SAM domain. Finally, we designed an S-glutathiomimetic mutation that strongly recapitulates the structural, biophysical and functional effects within the STIM1 luminal domain and we envision to be another tool for understanding the effects of protein S-glutathionylation in vitro, in cellulo and in vivo.
Assuntos
Glutationa , Molécula 1 de Interação Estromal , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Motivos EF Hand , Retículo Sarcoplasmático/metabolismo , Molécula 1 de Interação Estromal/química , Glutationa/química , Domínios Proteicos , Humanos , AnimaisRESUMO
Molecular steps that activate store-operated calcium entry (SOCE) via Orai channel supramolecular complex remain incompletely defined. We have earlier shown that α-SNAP regulates the on-site functional assembly and calcium selectivity of Orai1 channels. Here we investigate the molecular basis of its association with Orai, Stim and find that the affinity of α-SNAP for Orai and Stim is substantially higher than previously reported affinities between Stim and Orai sub-domains. α-SNAP binds the coiled-coil 3 (CC3) sub-domain of Stim1. Mutations of Tryptophan 430 in Stim1-CC3 disrupted α-SNAP association and SOCE, demonstrating a novel α-SNAP dependent function for this crucial subdomain. Further, α-SNAP binds the hinge region near the C-terminus of Orai1 and an additional broad region near the N-terminus and Valine 262 and Leucine 74 were necessary for these respective interactions, but not Orai, Stim co-clustering. Thus, high affinity interactions with α-SNAP are necessary for imparting functionality to Stim, Orai clusters and induction of SOCE.
Assuntos
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/química , Proteína ORAI1/química , Ligação Proteica , Molécula 1 de Interação Estromal/químicaRESUMO
Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the "classical" Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Sinalização do Cálcio , Cálcio , Retículo Endoplasmático , Animais , Cálcio/química , Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/química , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Retículo Endoplasmático/química , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , 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 , Relação Estrutura-AtividadeRESUMO
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Ativação do Canal Iônico/genética , Proteínas de Neoplasias/química , Proteína ORAI1/química , Molécula 1 de Interação Estromal/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Técnicas de Patch-Clamp , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.
Assuntos
Autofagia , Miopatias Congênitas Estruturais/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Motivos EF Hand , Humanos , Simulação de Dinâmica Molecular , Mutação , Miopatias Congênitas Estruturais/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Conformação Proteica em alfa-Hélice , Desdobramento de Proteína , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismoRESUMO
BACKGROUND/AIMS: Store-operated Ca2+ entry (SOCE) through plasma membrane Ca2+ channel Orai1 is essential for many cellular processes. SOCE, activated by ER Ca2+ store-depletion, relies on the gating function of STIM1 Orai1-activating region SOAR of the ER-anchored Ca2+-sensing protein STIM1. Electrophysiologically, SOCE is characterized as Ca2+ release-activated Ca2+ current (ICRAC). A major regulatory mechanism that prevents deleterious Ca2+ overload is the slow Ca2+-dependent inactivation (SCDI) of ICRAC. Several studies have suggested a role of Ca2+/calmodulin (Ca2+/CaM) in triggering SCDI. However, a direct contribution of STIM1 in regulating Ca2+/CaM-mediated SCDI of ICRAC is as yet unclear. METHODS: The Ca2+/CaM binding to STIM1 was tested by pulling down recombinant GFP-tagged human STIM1 C-terminal fragments on CaM sepharose beads. STIM1 was knocked out by CRISPR/Cas9 technique in HEK293 cells stably overexpressing human Orai1. Store-operated Ca2+ influx was measured using Fluorometric Imaging Plate Reader and whole-cell patch clamp in cells transfected with STIM1 CaM binding mutants. The involvement of Ca2+/CaM in SCDI was investigated by including recombinant human CaM in patch pipette in electrophysiology. RESULTS: Here we identified residues Leu374/Val375 (H1) and Leu390/Phe391 (H2) within SOAR that serve as hydrophobic anchor sites for Ca2+/CaM binding. The bifunctional H2 site is critical for both Orai1 activation and Ca2+/CaM binding. Single residue mutations of Phe391 to less hydrophobic residues significantly diminished SOCE and ICRAC, independent of Ca2+/CaM. Hence, the role of H2 residues in Ca2+/CaM-mediated SCDI cannot be precisely evaluated. In contrast, the H1 site controls exclusively Ca2+/CaM binding and subsequently SCDI, but not Orai1 activation. V375A but not V375W substitution eliminated SCDI of ICRAC caused by Ca2+/CaM, proving a direct role of STIM1 in coordinating SCDI. CONCLUSION: Taken together, we propose a mechanistic model, wherein binding of Ca2+/CaM to STIM1 hydrophobic anchor residues, H1 and H2, triggers SCDI by disrupting the functional interaction between STIM1 and Orai1. Our findings reveal how STIM1, Orai1, and Ca2+/CaM are functionally coordinated to control ICRAC.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/fisiologia , Sistemas CRISPR-Cas , Canais de Cálcio/genética , Sinalização do Cálcio , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Modelos Químicos , Modelos Moleculares , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/genética , Ligação Proteica , Domínios Proteicos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Regulação para CimaRESUMO
Genetically encoded photoswitches have enabled spatial and temporal control of cellular events to achieve tailored functions in living cells, but their applications to probe the structure-function relations of signaling proteins are still underexplored. We illustrate herein the incorporation of various blue light-responsive photoreceptors into modular domains of the stromal interaction molecule 1 (STIM1) to manipulate protein activity and faithfully recapitulate STIM1-mediated signaling events. Capitalizing on these optogenetic tools, we identify the molecular determinants required to mediate protein oligomerization, intramolecular conformational switch, and protein-target interactions. In parallel, we have applied these synthetic devices to enable light-inducible gating of calcium channels, conformational switch, dynamic protein-microtubule interactions and assembly of membrane contact sites in a reversible manner. Our optogenetic engineering approach can be broadly applied to aid the mechanistic dissection of cell signaling, as well as non-invasive interrogation of physiological processes with high precision.
Assuntos
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Optogenética/métodos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Cálcio/metabolismo , Criptocromos/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ativação do Canal Iônico , Proteínas Luminescentes/genética , Mutação , Proteínas de Neoplasias/química , Neoplasias/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Molécula 1 de Interação Estromal/química , Relação Estrutura-AtividadeRESUMO
Esophageal squamous cell carcinoma (ESCC) is a disease with poor prognosis which urgently is in need of effective prognostic marker. To discover novel prognostic protein marker for ESCC, we applied a high-throughput monoclonal antibody microarray to compare tumor and adjacent non-tumor tissues from ESCC patients. Antibody #ESmAb270 was consistent higher expressed in tumors and it was identified via mass spectrometry to be stromal interaction molecule 1 (STIM1). STIM1 H scores in tumor tissues were significantly up-regulated in esophageal tumor tissues compared to non-tumor tissues in 105 ESCC patients. We also observed that high STIM1 expression was correlated with advanced tumor grade and poor prognosis of ESCC. In addition, attenuation of STIM1 by siRNA or chemical inhibitors significantly inhibited cell viability and migration of ESCC cells. Evidence from high-throughput monoclonal antibody microarray, IHC microarray with associated survival data and functional analysis show that STIM1 is an unfavorable prognostic biomarker in ESCC.
Assuntos
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Esofágicas/imunologia , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/imunologia , Carcinoma de Células Escamosas do Esôfago/mortalidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/química , Proteínas de Neoplasias/imunologia , Prognóstico , Análise Serial de Proteínas , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/imunologiaRESUMO
Stromal interaction molecule 1 (STIM1) mediates extracellular Ca2+ entry into the cytosol through a store-operated Ca2+ entry (SOCE) mechanism, which is involved in the physiological functions of various tissues, including skeletal muscle. STIM1 is also associated with skeletal muscle diseases, but its pathological mechanisms have not been well addressed. The present study focused on examining the pathological mechanism(s) of a mutant STIM1 (R429C) that causes human muscular hypotonia. R429C was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-cell Ca2+ imaging of myotubes and transmission electron microscopy (TEM) along with biochemical approaches. R429C did not interfere with the terminal differentiation of myoblasts to myotubes. Unlike wild-type STIM1, there was no further increase of SOCE by R429C. R429C bound to endogenous STIM1 and slowed down the initial rate of SOCE that were mediated by endogenous STIM1. Moreover, R429C increased intracellular Ca2+ movement in response to membrane depolarization by eliminating the attenuation on dihydropyridine receptor-ryanodine receptor (DHPR-RyR1) coupling by endogenous STIM1. The cytosolic Ca2+ level was also increased due to the reduction in SR Ca2+ level. In addition, R429C-expressing myotubes showed abnormalities in mitochondrial shape, a significant decrease in ATP levels, and the higher expression levels of mitochondrial fission-mediating proteins. Therefore, serial defects in SOCE, intracellular Ca2+ movement, and cytosolic Ca2+ level along with mitochondrial abnormalities in shape and ATP level could be a pathological mechanism of R429C for human skeletal muscular hypotonia. This study also suggests a novel clue that STIM1 in skeletal muscle could be related to mitochondria via regulating intra and extracellular Ca2+ movements.
Assuntos
Cálcio/metabolismo , Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Hipotonia Muscular/genética , Músculo Esquelético/patologia , Mutação/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Canais de Cálcio Tipo L/metabolismo , Citosol/metabolismo , Humanos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Biológicos , Fibras Musculares Esqueléticas/metabolismo , Hipotonia Muscular/patologia , Proteínas de Neoplasias/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação Estromal/químicaRESUMO
The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.
Assuntos
Cálcio/metabolismo , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/química , Domínios Proteicos , Desdobramento de Proteína , Molécula 1 de Interação Estromal/química , Algoritmos , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Motivos EF Hand , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Ratos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.
Assuntos
Cálcio/metabolismo , Proteínas Sensoras de Cálcio Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Células Mieloides/imunologia , Proteínas de Neoplasias/química , Osteogênese/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Molécula 1 de Interação Estromal/químicaRESUMO
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/genéticaRESUMO
Several signaling proteins require self-association of individual monomer units to be activated for triggering downstream signaling cascades in cells. Methods that allow visualizing their underlying molecular mechanisms will immensely benefit cell biology. Using enhanced Green Fluorescent Protein (eGFP) complementation, here I present a functional imaging approach for visualizing the protein-protein interaction in cells. Activation mechanism of an ER (endoplasmic reticulum) resident Ca2+ sensor, STIM1 (Stromal Interaction Molecule 1) that regulates store-operated Ca2+ entry in cells is considered as a model system. Co-expression of engineered full-length human STIM1 (ehSTIM1) with N-terminal complementary split eGFP pairs in mammalian cells fluoresces to form 'puncta' upon a drop in ER lumen Ca2+ concentration. Quantization of discrete fluorescent intensities of ehSTIM1 molecules at a diffraction-limited resolution revealed a diverse set of intensity levels not exceeding six-fold. Detailed screening of the ehSTIM1 molecular entities characterized by one to six fluorescent emitters across various in-plane sections shows a greater probability of occurrence for entities with six emitters in the vicinity of the plasma membrane (PM) than at the interior sections. However, the number density of entities with six emitters was lesser than that of others localized close to the PM. This finding led to hypothesize that activated ehSTIM1 dimers perhaps oligomerize in bundles ranging from 1-6 with an increased propensity for the occurrence of hexamers of ehSTIM1 dimer units close to PM even when its partner protein, ORAI1 (PM resident Ca2+ channel) is not sufficiently over-expressed in cells. The experimental data presented here provide direct evidence for luminal domain association of ehSTIM1 monomer units to trigger activation and allow enumerating various oligomers of ehSTIM1 in cells.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Neoplasias/química , Imagem Óptica/métodos , Engenharia de Proteínas , Molécula 1 de Interação Estromal/química , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Microscopia/métodos , Distribuição Normal , Óptica e Fotônica , Probabilidade , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Transporte Proteico , Transdução de Sinais , Difração de Raios XRESUMO
Substantial progress has been made in the past several years in establishing the stoichiometries of STIM and Orai proteins and understanding their influence on store-operated calcium entry. Depletion of ER Ca2+ triggers STIM1 to accumulate at ER-plasma membrane junctions where it binds and opens Ca2+ release-activated Ca2+ (CRAC) channels. STIM1 is a dimer, and release of Ca2+ from its two luminal domains is reported to promote their association as well as drive formation of higher-order STIM1 oligomers. The CRAC channel, originally thought to be tetrameric, is now considered to be a hexamer of Orai1 subunits based on crystallographic and electrophysiological studies. STIM1 binding activates CRAC channels in a highly nonlinear way, such that all six Orai1 binding sites must be occupied to account for the activation and signature properties of native channels. The structural basis of STIM1 engagement with the channel is currently unclear, with evidence suggesting that STIM1 dimers bind to individual or pairs of Orai1 subunits. This review examines evidence that has led to points of consensus and debate about STIM1 and Orai1 stoichiometries, and explains the importance of STIM-Orai complex stoichiometry for the regulation of store-operated calcium entry.
Assuntos
Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Cálcio/química , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteína ORAI1/química , Molécula 1 de Interação Estromal/químicaRESUMO
Highly Ca2+ selective channels trigger a large variety of cellular signaling processes in both excitable and non-excitable cells. Among these channels, the Orai channel is unique in its activation mechanism and its structure. It mediates Ca2+ influx into the cytosol with an extremely small unitary conductance over longer time-scales, ranging from minutes up to several hours. Its activation is regulated by the Ca2+ content of the endoplasmic reticulum (ER). Depletion of luminal [Ca2+]ER is sensed by the STIM1 single transmembrane protein that directly binds and gates the Orai1 channel. Orai mediated Ca2+ influx increases cytosolic Ca2+ from 100 nM up to low micromolar range close to the pore and thereby forms Ca2+ microdomains. Hence, these features of the Orai channel can trigger long-term signaling processes without affecting the overall Ca2+ content of a single living cell. Here we focus on the architecture and dynamic conformational changes within the Orai channel. This review summarizes current achievements of molecular dynamics simulations in combination with live cell recordings to address gating and permeation of the Orai channel with molecular precision.
Assuntos
Cálcio/metabolismo , Simulação de Dinâmica Molecular , Proteína ORAI1/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/química , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismoRESUMO
Stromal interaction molecule 1 (STIM1) monitors ER-luminal Ca2+ levels to maintain cellular Ca2+ balance and to support Ca2+ signalling. The prevailing view has been that STIM1 senses reduced ER Ca2+ through dissociation of bound Ca2+ from a single EF-hand site, which triggers a dramatic loss of secondary structure and dimerization of the STIM1 luminal domain. Here we find that the STIM1 luminal domain has 5-6 Ca2+-binding sites, that binding at these sites is energetically coupled to binding at the EF-hand site, and that Ca2+ dissociation controls a switch to a second structured conformation of the luminal domain rather than protein unfolding. Importantly, the other luminal-domain Ca2+-binding sites interact with the EF-hand site to control physiological activation of STIM1 in cells. These findings fundamentally revise our understanding of physiological Ca2+ sensing by STIM1, and highlight molecular mechanisms that govern the Ca2+ threshold for activation and the steep Ca2+ concentration dependence.
Assuntos
Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismo , Animais , Sítios de Ligação , Calorimetria , Cisteína/metabolismo , Medição da Troca de Deutério , Fluorescência , Células HeLa , Humanos , Camundongos , Mutação/genética , Domínios Proteicos , Estrutura Secundária de Proteína , Solubilidade , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. OBJECTIVE AND METHODS: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE. RESULTS: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. CONCLUSIONS: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors.
Assuntos
Sinalização do Cálcio , Cálcio , Digitoxina , Proteína ORAI1 , Substituição de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Digitoxina/química , Digitoxina/farmacologia , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/química , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
The involvement of inositol trisphosphate receptor (IP3R) in modulating store-operated calcium entry (SOCE) was established many years ago. Nevertheless, the molecular mechanism responsible for this observation has not been elucidated to this date. In the present study we show that IP3R associates to STIM1 upon depletion of the endoplasmic reticulum (ER) by activation of the inositol trisphosphate signaling cascade via G-protein coupled receptors. IP3R-STIM1 association results in enhanced STIM1 puncta formation and larger Orai-mediated whole-cell currents as well as increased calcium influx. Depleting the ER with a calcium ATPase inhibitor (thapsigargin, TG) does not induce IP3R-STIM1 association, indicating that this association requires an active IP3R. The IP3R-STIM1 association is only observed after IP3R activation, as evidenced by FRET experiments and co-immunoprecipitation assays. ER intraluminal calcium measurements using Mag-Fluo-4 showed enhanced calcium depletion when IP3R is overexpressed. A STIM1-GCaMP fusion protein indicates that STIM1 detects lower calcium concentrations near its EF-hand domain when IP3R is overexpressed when compared with the fluorescence reported by a GCaMP homogenously distributed in the ER lumen (ER-GCaMP). All these data together strongly suggest that activation of inositol trisphosphate signaling cascade induces the formation of the IP3R-STIM1 complex. The activated IP3R provides a reduced intraluminal calcium microenvironment near STIM1, resulting in enhanced activation of Orai currents and SOCE.