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1.
Protein Sci ; 33(8): e5108, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38989547

RESUMO

Mitochondrial magnesium (Mg2+) is a crucial modulator of protein stability, enzymatic activity, ATP synthesis, and cell death. Mitochondrial RNA splicing protein 2 (MRS2) is the main Mg2+ channel in the inner mitochondrial membrane that mediates influx into the matrix. Recent cryo-electron microscopy (cryo-EM) human MRS2 structures exhibit minimal conformational changes at high and low Mg2+, yet the regulation of human MRS2 and orthologues by Mg2+ binding to analogous matrix domains has been well established. Further, a missense variation at D216 has been identified associated with malignant melanoma and MRS2 expression and activity is implicated in gastric cancer. Thus, to gain more mechanistic and functional insight into Mg2+ sensing by the human MRS2 matrix domain and the association with proliferative disease, we assessed the structural, biophysical, and functional effects of a D216Q mutant. We show that the D216Q mutation is sufficient to abrogate Mg2+-binding and associated conformational changes including increased α-helicity, stability, and monomerization. Further, we reveal that the MRS2 matrix domains interact with ~µM affinity, which is weakened by up to two orders of magnitude in the presence of Mg2+ for wild-type but unaffected for D216Q. Finally, we demonstrate the importance of Mg2+ sensing by MRS2 to prevent matrix Mg2+ overload as HeLa cells overexpressing MRS2 show enhanced Mg2+ uptake, cell migration, and resistance to apoptosis while MRS2 D216Q robustly potentiates these cancer phenotypes. Collectively, our findings further define the MRS2 matrix domain as a critical Mg2+ sensor that undergoes conformational and assembly changes upon Mg2+ interactions dependent on D216 to temper matrix Mg2+ overload.


Assuntos
Apoptose , Proteínas de Transporte de Cátions , Movimento Celular , Mutação de Sentido Incorreto , Humanos , Células HeLa , Magnésio/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Ligação Proteica , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo
2.
J Physiol ; 602(14): 3315-3339, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38857425

RESUMO

Increased malignancy and poor treatability associated with solid tumour cancers have commonly been attributed to mitochondrial calcium (Ca2+) dysregulation. The mitochondrial Ca2+ uniporter complex (mtCU) is the predominant mode of Ca2+ uptake into the mitochondrial matrix. The main components of mtCU are the pore-forming mitochondrial Ca2+ uniporter (MCU) subunit, MCU dominant-negative beta (MCUb) subunit, essential MCU regulator (EMRE) and the gatekeeping mitochondrial Ca2+ uptake 1 and 2 (MICU1 and MICU2) proteins. In this review, we describe mtCU-mediated mitochondrial Ca2+ dysregulation in solid tumour cancer types, finding enhanced mtCU activity observed in colorectal cancer, breast cancer, oral squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma and embryonal rhabdomyosarcoma. By contrast, decreased mtCU activity is associated with melanoma, whereas the nature of mtCU dysregulation remains unclear in glioblastoma. Furthermore, we show that numerous polymorphisms associated with cancer may alter phosphorylation sites on the pore forming MCU and MCUb subunits, which cluster at interfaces with EMRE. We highlight downstream/upstream biomolecular modulators of MCU and MCUb that alter mtCU-mediated mitochondrial Ca2+ uptake and may be used as biomarkers or to aid in the development of novel cancer therapeutics. Additionally, we provide an overview of the current small molecule inhibitors of mtCU that interact with the Asp residue of the critical Asp-Ile-Met-Glu motif or through other allosteric regulatory mechanisms to block Ca2+ permeation. Finally, we describe the relationship between MCU- and MCUb-mediating microRNAs and mitochondrial Ca2+ uptake that should be considered in the discovery of new treatment approaches for cancer.


Assuntos
Canais de Cálcio , Neoplasias , Humanos , Canais de Cálcio/metabolismo , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Animais , Cálcio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos
3.
Proc Natl Acad Sci U S A ; 121(21): e2318874121, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38753510

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/química
4.
Mol Cell ; 84(7): 1321-1337.e11, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38513662

RESUMO

Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.


Assuntos
Adenosina Trifosfatases , ATPases do Tipo-P , Animais , Camundongos , Humanos , Adenosina Trifosfatases/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Transporte Biológico , ATPases do Tipo-P/metabolismo , Cálcio/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático
5.
Biochim Biophys Acta Mol Cell Res ; 1871(2): 119629, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37981034

RESUMO

The migratory and invasive potential of tumour cells relies on the actin cytoskeleton. We previously demonstrated that the tricyclic compound, TBE-31, inhibits actin polymerization and here we further examine the precise interaction between TBE-31 and actin. We demonstrate that iodoacetamide, a cysteine (Cys) alkylating agent, interferes with the ability of TBE-31 to interact with actin. In addition, in silico analysis identified Cys 217, Cys 272, Cys 285 and Cys 374 as potential binding sites for TBE-31. Using mass spectrometry analysis, we determined that TBE-31 associates with actin with a stoichiometric ratio of 1:1. We mutated the identified cysteines of actin to alanine and performed a pull-down analysis with a biotin labeled TBE-31 and demonstrated that by mutating Cys 374 to alanine the association between TBE-31 and actin was significantly reduced, suggesting that TBE-31 binds to Cys 374. A characterization of the NIH3T3 cells overexpressing eGFP-actin-C374A showed reduced stress fiber formation, suggesting Cys 374 is necessary for efficient incorporation into filamentous actin. Furthermore, migration of eGFP-Actin-WT expressing cells were observed to be inhibited by TBE-31, however fewer eGFP-Actin-C374A expressing cells were observed to migrate compared to the cells expressing eGFP-Actin-WT in the presence or absence of TBE-31. Taken together, our results suggest that TBE-31 binds to Cys 374 of actin to inhibit actin stress fiber formation and may potentially be a mechanism through which TBE-31 inhibits cell migration.


Assuntos
Actinas , Cisteína , Fenantrenos , Camundongos , Animais , Actinas/genética , Actinas/metabolismo , Cisteína/genética , Cisteína/metabolismo , Acetileno , Alcinos , Fibras de Estresse , Células NIH 3T3 , Movimento Celular , Alanina
6.
Biochem J ; 480(14): 1051-1077, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37395717

RESUMO

Connexins form intercellular communication channels, known as gap junctions (GJs), in many tissues/organs. Mutations in connexin genes are found to be linked to various inherited diseases, but the mechanisms are not fully clear. The Arg76 (R76) in Cx50 is fully conserved across the entire connexin family and is a hotspot for five connexin-linked inherited diseases, including Cx50 and Cx46-linked congenital cataract, Cx43-linked oculodentodigital dysplasia, and Cx45-linked cardiac arrhythmias. To better understand the molecular and cellular mechanism of dysfunction caused by R76/75 mutations, we examined the functional status and properties of GJs containing R76 mutations in Cx50 (R76H/C), Cx43 (R76H/S/C), and Cx45 (R75H) with an emphasis on heterotypic GJs in connexin-deficient model cells. All tested mutants showed an impairment of homotypic GJ function reflected by a decreased coupling% and conductance, except for Cx43 R76H/S. These connexin mutants also showed impaired GJ function when paired with a docking-compatible connexin, such as Cx50/Cx46 or Cx45/Cx43, except for all mutants on Cx43 which formed functional heterotypic GJs with Cx45. Localization studies on fluorescent protein tagged connexin mutants revealed that Cx45 R75H and Cx43 R76C showed impaired localization. Our homology structure models indicated that mutations of R76/75 in these GJs led to a loss of intra- and/or inter-connexin non-covalent interactions (salt bridges) at the sidechain of this residue, which could contribute to the observed GJ impairments underlying diseases. It is interesting that unlike those disease-linked variants in Cx50 and Cx45, Cx43 can tolerate some variations at R76.


Assuntos
Junções Comunicantes , Ativação do Canal Iônico , Junções Comunicantes/genética , Junções Comunicantes/metabolismo , Conexinas/genética , Conexinas/metabolismo , Cinética
7.
J Mol Biol ; 434(24): 167874, 2022 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-36332662

RESUMO

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 , Animais
8.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360596

RESUMO

Twenty-one human genes encode connexins, a family of homologous proteins making gap junction (GJ) channels, which mediate direct intercellular communication to synchronize tissue/organ activities. Genetic variants in more than half of the connexin genes are associated with dozens of different Mendelian inherited diseases. With rapid advances in DNA sequencing technology, more variants are being identified not only in families and individuals with diseases but also in people in the general population without any apparent linkage to Mendelian inherited diseases. Nevertheless, it remains challenging to classify the pathogenicity of a newly identified connexin variant. Here, we analyzed the disease- and Genome Aggregation Database (gnomAD, as a proxy of the general population)-linked variants in the coding region of the four disease-linked α connexin genes. We found that the most abundant and position-sensitive missense variants showed distinct domain distribution preference between disease- and gnomAD-linked variants. Plotting missense variants on topological and structural models revealed that disease-linked missense variants are highly enriched on the structurally stable/resolved domains, especially the pore-lining domains, while the gnomAD-linked missense variants are highly enriched in the structurally unstable/unresolved domains, especially the carboxyl terminus. In addition, disease-linked variants tend to be on highly conserved residues and those positions show evolutionary co-variation, while the gnomAD-linked missense variants are likely on less conserved residue positions and on positions without co-variation. Collectively, the revealed distribution patterns of disease- and gnomAD-linked missense variants further our understanding of the GJ structure-biological function relationship, which is valuable for classifying the pathogenicity of newly identified connexin variants.


Assuntos
Conexinas/genética , Bases de Dados Genéticas , Junções Comunicantes/genética , Doenças Genéticas Inatas/patologia , Genética Populacional , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Doenças Genéticas Inatas/genética , Humanos , Domínios Proteicos , Homologia de Sequência
9.
J Cell Sci ; 134(3)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33468626

RESUMO

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.This article has an associated First Person interview with the first author of the paper.


Assuntos
Sinalização do Cálcio , Proteínas de Neoplasias , Pancreatite Crônica , Molécula 1 de Interação Estromal , Cálcio/metabolismo , Sinalização do Cálcio/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Pancreatite Crônica/genética , Pancreatite Crônica/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo
10.
Cell ; 183(2): 474-489.e17, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33035451

RESUMO

Mg2+ is the most abundant divalent cation in metazoans and an essential cofactor for ATP, nucleic acids, and countless metabolic enzymes. To understand how the spatio-temporal dynamics of intracellular Mg2+ (iMg2+) are integrated into cellular signaling, we implemented a comprehensive screen to discover regulators of iMg2+ dynamics. Lactate emerged as an activator of rapid release of Mg2+ from endoplasmic reticulum (ER) stores, which facilitates mitochondrial Mg2+ (mMg2+) uptake in multiple cell types. We demonstrate that this process is remarkably temperature sensitive and mediated through intracellular but not extracellular signals. The ER-mitochondrial Mg2+ dynamics is selectively stimulated by L-lactate. Further, we show that lactate-mediated mMg2+ entry is facilitated by Mrs2, and point mutations in the intermembrane space loop limits mMg2+ uptake. Intriguingly, suppression of mMg2+ surge alleviates inflammation-induced multi-organ failure. Together, these findings reveal that lactate mobilizes iMg2+ and links the mMg2+ transport machinery with major metabolic feedback circuits and mitochondrial bioenergetics.


Assuntos
Retículo Endoplasmático/metabolismo , Ácido Láctico/metabolismo , Magnésio/metabolismo , Animais , Células COS , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Feminino , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo
11.
EMBO Mol Med ; 12(8): e11592, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609955

RESUMO

Immunity to fungal infections is mediated by cells of the innate and adaptive immune system including Th17 cells. Ca2+ influx in immune cells is regulated by stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. We here identify patients with a novel mutation in STIM1 (p.L374P) that abolished Ca2+ influx and resulted in increased susceptibility to fungal and other infections. In mice, deletion of STIM1 in all immune cells enhanced susceptibility to mucosal C. albicans infection, whereas T cell-specific deletion of STIM1 impaired immunity to systemic C. albicans infection. STIM1 deletion impaired the production of Th17 cytokines essential for antifungal immunity and compromised the expression of genes in several metabolic pathways including Foxo and HIF1α signaling that regulate glycolysis and oxidative phosphorylation (OXPHOS). Our study further revealed distinct roles of STIM1 in regulating transcription and metabolic programs in non-pathogenic Th17 cells compared to pathogenic, proinflammatory Th17 cells, a finding that may potentially be exploited for the treatment of Th17 cell-mediated inflammatory diseases.


Assuntos
Cálcio , Células Th17 , Animais , Antifúngicos , Cálcio/metabolismo , Canais de Cálcio/genética , Humanos , Camundongos , Proteínas de Neoplasias , Proteína ORAI1 , Molécula 1 de Interação Estromal/genética , Células Th17/metabolismo
12.
Sci Rep ; 10(1): 10177, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576932

RESUMO

Stromal interaction molecule-1 and -2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca2+) sensing proteins that, together with Orai1-composed Ca2+ channels on the plasma membrane (PM), regulate intracellular Ca2+ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca2+ and SOCE regulation.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Glutationa/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Compostos de Sulfidrila/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Proteína ORAI1/metabolismo , Ligação Proteica/fisiologia , Molécula 1 de Interação Estromal/metabolismo
13.
Int J Mol Sci ; 21(10)2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32455637

RESUMO

Calcium (Ca2+) is a universal signaling ion that is essential for the life and death processes of all eukaryotes. In humans, numerous cell stimulation pathways lead to the mobilization of sarco/endoplasmic reticulum (S/ER) stored Ca2+, resulting in the propagation of Ca2+ signals through the activation of processes, such as store-operated Ca2+ entry (SOCE). SOCE provides a sustained Ca2+ entry into the cytosol; moreover, the uptake of SOCE-mediated Ca2+ by mitochondria can shape cytosolic Ca2+ signals, function as a feedback signal for the SOCE molecular machinery, and drive numerous mitochondrial processes, including adenosine triphosphate (ATP) production and distinct cell death pathways. In recent years, tremendous progress has been made in identifying the proteins mediating these signaling pathways and elucidating molecular structures, invaluable for understanding the underlying mechanisms of function. Nevertheless, there remains a disconnect between using this accumulating protein structural knowledge and the design of new research tools and therapies. In this review, we provide an overview of the Ca2+ signaling pathways that are involved in mediating S/ER stored Ca2+ release, SOCE, and mitochondrial Ca2+ uptake, as well as pinpoint multiple levels of crosstalk between these pathways. Further, we highlight the significant protein structures elucidated in recent years controlling these Ca2+ signaling pathways. Finally, we describe a simple strategy that aimed at applying the protein structural data to initiating drug design.


Assuntos
Sinalização do Cálcio , Descoberta de Drogas/métodos , Animais , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Ligação Proteica
14.
J Biol Chem ; 295(8): 2520-2540, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-31892516

RESUMO

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.


Assuntos
Receptores de Trombina/metabolismo , Transdução de Sinais , Trombina/metabolismo , Alanina/genética , Substituição de Aminoácidos , Cálcio/metabolismo , Sinalização do Cálcio , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Isomerismo , Sistema de Sinalização das MAP Quinases , Metilação , Simulação de Acoplamento Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Peptídeos/metabolismo , Fosforilação , Agregação Plaquetária , Receptores de Trombina/agonistas , Homologia Estrutural de Proteína , beta-Arrestinas/metabolismo
15.
Biochim Biophys Acta Mol Cell Res ; 1867(1): 118567, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676354

RESUMO

Acinar cell exocytosis requires spatiotemporal Ca2+ signals regulated through endoplasmic reticulum (ER) stores, Ca2+ATPases, and store-operated Ca2+ entry (SOCE). The secretory pathway Ca2+ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca2+ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca2+ homeostasis in a similar fashion to the full-length SPCA2. Using epitope-tagged SPCA2C (SPCA2CFLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca2+] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca2+], Ca2+ release in response to carbachol, ER Ca2+ stores, and store-mediated and independent Ca2+ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca2+ entry were independent of Orai1. These findings indicate SPCA2C influences Ca2+ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca2+ regulation.


Assuntos
Sinalização do Cálcio/fisiologia , ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Pâncreas/metabolismo , Canais de Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Homeostase , Humanos , Proteína ORAI2/genética , Proteína ORAI2/metabolismo , Especificidade de Órgãos/genética , Isoformas de Proteínas/fisiologia , Via Secretória/fisiologia
16.
Sci Signal ; 12(608)2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31744929

RESUMO

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/metabolismo
17.
Int J Mol Sci ; 20(2)2019 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-30642051

RESUMO

Mitochondrial calcium (Ca2+) uptake shapes cytosolic Ca2+ signals involved in countless cellular processes and more directly regulates numerous mitochondrial functions including ATP production, autophagy and apoptosis. Given the intimate link to both life and death processes, it is imperative that mitochondria tightly regulate intramitochondrial Ca2+ levels with a high degree of precision. Among the Ca2+ handling tools of mitochondria, the leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is a transporter protein localized to the inner mitochondrial membrane shown to constitute a Ca2+/H⁺ exchanger activity. The significance of LETM1 to mitochondrial Ca2+ regulation is evident from Wolf-Hirschhorn syndrome patients that harbor a haplodeficiency in LETM1 expression, leading to dysfunctional mitochondrial Ca2+ handling and from numerous types of cancer cells that show an upregulation of LETM1 expression. Despite the significance of LETM1 to cell physiology and pathophysiology, the molecular mechanisms of LETM1 function remain poorly defined. In this review, we aim to provide an overview of the current understanding of LETM1 structure and function and pinpoint the knowledge gaps that need to be filled in order to unravel the underlying mechanistic basis for LETM1 function.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias/genética , Síndrome de Wolf-Hirschhorn/genética , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Haploinsuficiência , Células HeLa , Humanos , Proteínas de Membrana/química , Mitocôndrias/metabolismo , Modelos Moleculares , Neoplasias/metabolismo , Conformação Proteica , Regulação para Cima , Síndrome de Wolf-Hirschhorn/metabolismo
18.
J Biol Chem ; 293(23): 8900-8911, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29661937

RESUMO

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


Assuntos
Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Sequência de Aminoácidos , Sinalização do Cálcio , Cisteína/química , Cisteína/metabolismo , Motivos EF Hand , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Domínios Proteicos , Estabilidade Proteica , Retículo Sarcoplasmático/metabolismo , Alinhamento de Sequência , Molécula 1 de Interação Estromal/química , Termodinâmica
19.
J Mol Biol ; 430(12): 1773-1785, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29705071

RESUMO

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+ depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.


Assuntos
Cálcio/metabolismo , Proteínas de Neoplasias/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico/farmacologia , Molécula 1 de Interação Estromal/metabolismo , Animais , Células Cultivadas , Cisteína/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neurônios/citologia , Neurônios/enzimologia , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética
20.
Cell Calcium ; 73: 88-94, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29698850

RESUMO

Stromal interaction molecule (STIM)-1 and -2 are multi-domain, single-pass transmembrane proteins involved in sensing changes in compartmentalized calcium (Ca2+) levels and transducing this cellular signal to Orai1 channel proteins. Our understanding of the molecular mechanisms underlying STIM signaling has been dramatically improved through available X-ray crystal and solution NMR structures. This high-resolution structural data has revealed that intricate intramolecular and intermolecular protein-protein interactions are involved in converting STIMs from the quiescent to activation-competent states. This review article summarizes the current high resolution structural data on specific EF-hand, sterile α motif and coiled-coil interactions which drive STIM function in the activation of Orai1 channels. Further, the work discusses the effects of post-translational modifications on the structure and function of STIMs. Future structural studies on larger STIM:Orai complexes will be critical to fully defining the molecular bases for STIM function and how post-translational modifications influence these mechanisms.


Assuntos
Proteínas de Neoplasias/química , Proteínas de Neoplasias/fisiologia , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/fisiologia , Molécula 2 de Interação Estromal/química , Molécula 2 de Interação Estromal/fisiologia , Animais , Citosol/fisiologia , Humanos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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