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1.
FASEB J ; 35(9): e21808, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390515

RESUMO

The link between serum uric acid (SUA) and the risk of venous thromboembolism (VTE) is well established. Recent data suggested a causative role of UA in endothelial cells (ECs) dysfunction. However, the molecular mechanism of high UA on thrombogenesis is unknown. We investigate whether high UA induce phosphatidylserine (PS) externalization and microparticle (MP) shedding in cultured EC, and contribute to UA-induced hypercoagulable state. In the present study, we demonstrate that UA induces PS exposure and EMP release of EC in a concentration- and time-dependent manner, which enhances the procoagulant activity (PCA) of EC and inhibited over 90% by lactadherin in vitro. Furthermore, hyperuricemic rat model was used to evaluate the development of thrombi following by flow stasis in the inferior vena cava (IVC). Hyperuricemia group is more likely to form large and hard thrombi compared with control. Importantly, we found that TMEM16F expression is significantly upregulated in UA-treated EC, which is crucial for UA-induced PS exposure and MP formation. Additionally, UA increases the generation of reactive oxygen species (ROS), lipid peroxidation, and cytosolic Ca2+ concentration in EC, which might contribute to increased TMEM16F expression. Using confocal microscopy, we also observed disruption of the actin cytoskeleton, suggesting that depolymerization of actin filaments might be required for TMEM16F activation and followed by PS exposure and membrane blebbing in UA-treated EC. Our results demonstrate a thrombotic role of EC in hyperuricemia through TMEM16F-mediated PS exposure and MPs release.


Assuntos
Anoctaminas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/metabolismo , Hiperuricemia/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperuricemia/sangue , Peroxidação de Lipídeos/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Ácido Úrico/sangue
2.
Nat Commun ; 12(1): 4990, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404808

RESUMO

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Cálcio/metabolismo , Citoplasma , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Membranas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/metabolismo
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445284

RESUMO

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.


Assuntos
Anoctaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Ânions/metabolismo , Anoctaminas/genética , Cátions/metabolismo , Cloretos/metabolismo , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Permeabilidade , Proteínas de Transferência de Fosfolipídeos/genética , Sódio/metabolismo
4.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207150

RESUMO

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.


Assuntos
Anoctaminas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Anoctaminas/química , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Colesterol/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Modelos Biológicos , Oócitos , Receptores Purinérgicos P2X7/química
5.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946319

RESUMO

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca2+ entry (SOCE). Ca2+ release through inositol 1,4,5-trisphosphate receptor (IP3R) and subsequent Ca2+ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca2+, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca2+ entry through TRPC1 channels activated endogenous Ca2+-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca2+ concentration and/or transmembrane potential, conversely lowering of intracellular Ca2+ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca2+ concentration. Experiments with Ca2+ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca2+-activated chloride channels are involved in Ca2+ entry microdomains.


Assuntos
Anoctaminas/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Canais de Cátion TRPC/metabolismo , Cátions Bivalentes/metabolismo , Células HEK293 , Humanos , Técnicas de Patch-Clamp
6.
Nat Commun ; 12(1): 2826, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990555

RESUMO

TMEM16 lipid scramblases transport lipids and also operate as ion channels with highly variable ion selectivities and various physiological functions. However, their molecular mechanisms of ion conduction and selectivity remain largely unknown. Using computational electrophysiology simulations at atomistic resolution, we identified the main ion-conductive state of TMEM16 lipid scramblases, in which an ion permeation pathway is lined by lipid headgroups that directly interact with permeating ions in a voltage polarity-dependent manner. We found that lipid headgroups modulate the ion-permeability state and regulate ion selectivity to varying degrees in different scramblase isoforms, depending on the amino-acid composition of the pores. Our work has defined the structural basis of ion conduction and selectivity in TMEM16 lipid scramblases and uncovered the mechanisms responsible for the direct effects of membrane lipids on the conduction properties of ion channels.


Assuntos
Anoctaminas/metabolismo , Proteínas Fúngicas/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/química , Proteínas Fúngicas/química , Fusarium/metabolismo , Transporte de Íons , Lipídeos de Membrana/química , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/química , Conformação Proteica , Estrutura Quaternária de Proteína , Eletricidade Estática
7.
Nature ; 594(7861): 88-93, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33827113

RESUMO

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.


Assuntos
Anoctaminas/antagonistas & inibidores , COVID-19/patologia , Fusão Celular , Avaliação Pré-Clínica de Medicamentos , Células Gigantes/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/patologia , Células Epiteliais Alveolares/virologia , Animais , Anoctaminas/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Canais de Cloreto/metabolismo , Chlorocebus aethiops , Feminino , Células Gigantes/metabolismo , Células Gigantes/virologia , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Masculino , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Int J Mol Sci ; 22(5)2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800462

RESUMO

Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.


Assuntos
Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Artrite Reumatoide/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Anoctaminas/metabolismo , Artrite Reumatoide/patologia , Membrana Celular/metabolismo , Células HEK293 , Células HT29 , Humanos , Neoplasias/patologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia
9.
Mol Cells ; 44(2): 88-100, 2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33658434

RESUMO

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca2+-activated ion channel and Ca2+-activated phospholipid scramblase activities, requiring a high intracellular Ca2+ concentration (e.g., half-maximal effective Ca2+ concentration [EC50] of [Ca2+]i > 10 µM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca2+- activated chloride channel exhibiting higher Ca2+ sensitivity (EC50 of 1 µM) than ANO6, suggested that a homologous Ca2+-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca2+ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca2+-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6- 1-6 showed higher sensitivity to Ca2+ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca2+ interaction with Nt- CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca2+-interacting acidic amino acids in ANO6 Nt- CaRes resulted in reduced Ca2+ sensitivity, implying direct interactions of Ca2+ with these residues. Based on these results, we cautiously suggest that the net charge of Nt- CaRes is responsible for the difference in Ca2+ sensitivity between ANO1 and ANO6.


Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Cálcio/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sequência de Aminoácidos , Anoctaminas/genética , Motivos EF Hand , Células HEK293 , Humanos , Modelos Biológicos , Simulação de Dinâmica Molecular , Mutação/genética , Proteínas de Transferência de Fosfolipídeos/genética , Domínios Proteicos , Relação Estrutura-Atividade
10.
J Cell Biol ; 220(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33688936

RESUMO

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.


Assuntos
Membrana Celular/fisiologia , Retículo Endoplasmático/fisiologia , Homeostase/fisiologia , Animais , Anoctaminas/metabolismo , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Citosol/metabolismo , Citosol/fisiologia , Retículo Endoplasmático/metabolismo , Feminino , Humanos , Íons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distrofia Muscular do Cíngulo dos Membros/metabolismo
11.
J Mol Biol ; 433(16): 166941, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-33741412

RESUMO

The TMEM16 family of membrane proteins displays a remarkable functional dichotomy - while some family members function as Ca2+-activated anion channels, the majority of characterized TMEM16 homologs are Ca2+-activated lipid scramblases, which catalyze the exchange of phospholipids between the two membrane leaflets. Furthermore, some TMEM16 scramblases can also function as channels. Due to their involvement in important physiological processes, the family has been actively studied ever since their molecular identity was unraveled. In this review, we will summarize the recent advances in the field and how they influenced our view of TMEM16 family function and evolution. Structural, functional and computational studies reveal how relatively small rearrangements in the permeation pathway are responsible for the observed functional duality: while TMEM16 scramblases can adopt both ion- and lipid conductive conformations, TMEM16 channels can only populate the former. Recent data further provides the molecular details of a stepwise activation mechanism, which is initiated by Ca2+ binding and modulated by various cellular factors, including lipids. TMEM16 function and the surrounding membrane properties are inextricably intertwined, with the protein inducing bilayer deformations associated with scrambling, while the surrounding lipids modulate TMEM16 conformation and activity.


Assuntos
Anoctaminas/química , Anoctaminas/metabolismo , Animais , Cálcio/metabolismo , Humanos , Transporte de Íons , Metabolismo dos Lipídeos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
12.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672260

RESUMO

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca2+ in the low µM range. In addition, intracellular Ca2+ or Co2+ at mM concentrations have been shown to further potentiate the saturated Ca2+-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.


Assuntos
Anoctaminas/metabolismo , Cátions Bivalentes/metabolismo , Anoctamina-1/química , Anoctamina-1/genética , Anoctamina-1/metabolismo , Anoctaminas/química , Anoctaminas/genética , Cálcio/metabolismo , Cátions Bivalentes/farmacologia , Cobalto/metabolismo , Eletrofisiologia/métodos , Células HEK293 , Humanos , Magnésio/metabolismo , Manitol/metabolismo , Manitol/farmacologia , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipídeos/metabolismo , Polilisina/farmacologia
13.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33496727

RESUMO

Mutations in ANO5 (TMEM16E) cause limb-girdle muscular dystrophy R12. Defective plasma membrane repair is a likely mechanism. Using myofibers from Ano5 knockout mice, we show that trafficking of several annexin proteins, which together form a cap at the site of injury, is altered upon loss of ANO5. Annexin A2 accumulates at the wound to nearly twice the level observed in WT fibers, while annexin A6 accumulation is substantially inhibited in the absence of ANO5. Appearance of annexins A1 and A5 at the cap is likewise diminished in the Ano5 knockout. These changes are correlated with an alteration in annexin repair cap fine structure and shedding of annexin-positive vesicles. We conclude that loss of annexin coordination during repair is disrupted in Ano5 knockout mice and underlies the defective repair phenotype. Although ANO5 is a phospholipid scramblase, abnormal repair is rescued by overexpression of a scramblase-defective ANO5 mutant, suggesting a novel, scramblase-independent role of ANO5 in repair.


Assuntos
Anexinas/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Anoctaminas/química , Anoctaminas/deficiência , Anoctaminas/genética , Anoctaminas/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Cinética , Camundongos Knockout , Mutação/genética , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Domínios Proteicos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Cancer Sci ; 112(3): 1026-1037, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33404124

RESUMO

The function of ANO9 in gastrointestinal cancer remains unclear. We investigated the biological behaviors and clinical prognostic values of ANO9 in gastric cancer (GC). Knockdown experiments were performed on human GC cell lines using ANO9 siRNA. Eighty-four primary tissue samples from patients with advanced GC were examined immunohistochemically (IHC). Knockdown of ANO9 reduced the progression of cancer cells in MKN7 and MKN74 cells. A microarray analysis revealed that ANO9 regulated PD-L2 via interferon (IFN)-related genes. We confirmed using flow cytometry that the depletion of ANO9 reduced the binding ability to PD-1 by downregulating the expression of PD-L2 in MKN7 and MKN74 cells. IHC revealed a correlation between the expression of ANO9 and PD-L2 and also that the strong expression of ANO9 was an independent poor prognostic factor in patients with advanced GC. The present results indicate that ANO9 regulates PD-L2 and binding ability to PD-1 via IFN-related genes in GC. Therefore, ANO9 has potential as a biomarker and target of immune checkpoint blockage (ICB) for GC.


Assuntos
Anoctaminas/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/genética , Neoplasias Gástricas/genética , Idoso , Anoctaminas/antagonistas & inibidores , Anoctaminas/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/imunologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Seguimentos , Gastrectomia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Interferons/metabolismo , Masculino , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genética , Prognóstico , Receptor de Morte Celular Programada 1/metabolismo , Estômago/patologia , Estômago/cirurgia , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/terapia , Taxa de Sobrevida
15.
J Gen Physiol ; 153(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33346788

RESUMO

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pHi). We found that low pHi attenuates, whereas high pHi potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca2+ ([Ca2+]i). We further demonstrate that TMEM16F pHi sensitivity depends on [Ca2+]i and exhibits a bell-shaped relationship with [Ca2+]i: TMEM16F channel activation becomes increasingly pHi sensitive from resting [Ca2+]i to micromolar [Ca2+]i, but when [Ca2+]i increases beyond 15 µM, pHi sensitivity gradually diminishes. The mutation of a Ca2+-binding residue that markedly reduces TMEM16F Ca2+ sensitivity (E667Q) maintains the bell-shaped relationship between pHi sensitivity and Ca2+ but causes a dramatic shift of the peak [Ca2+]i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pHi regulatory effects on TMEM16F stem from the competition between Ca2+ and protons for the primary Ca2+-binding residues in the pore. Within the physiological [Ca2+]i range, the protonation state of the primary Ca2+-binding sites influences Ca2+ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pHi and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pHi, including cancer.


Assuntos
Anoctaminas , Infecções por HIV , Anoctaminas/genética , Anoctaminas/metabolismo , Cálcio/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Transporte de Íons , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo
16.
Biochem Biophys Res Commun ; 529(3): 720-725, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32736698

RESUMO

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.


Assuntos
Anoctaminas/metabolismo , Canais de Cálcio Tipo L/metabolismo , Disferlina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Anoctaminas/análise , Canais de Cálcio Tipo L/análise , Caveolina 3/análise , Caveolina 3/metabolismo , Disferlina/análise , Disferlina/genética , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia
17.
J Biol Chem ; 295(35): 12537-12544, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32709749

RESUMO

TMEM16 Ca2+-activated phospholipid scramblases (CaPLSases) mediate rapid transmembrane phospholipid flip-flop and as such play essential roles in various physiological and pathological processes such as blood coagulation, skeletal development, viral infection, cell-cell fusion, and ataxia. Pharmacological tools specifically targeting TMEM16 CaPLSases are urgently needed to understand these novel membrane transporters and their contributions to health and disease. Tannic acid (TA) and epigallocatechin gallate (EGCG) were recently reported as promising TMEM16F CaPLSase inhibitors. However, our present study shows that TA and EGCG do not inhibit the phospholipid-scrambling or ion conduction activities of the dual-functional TMEM16F. Instead, we found that TA and EGCG mainly acted as fluorescence quenchers that rapidly suppress the fluorophores conjugated to annexin V, a phosphatidylserine-binding probe commonly used to report on TMEM16 CaPLSase activity. These data demonstrate the false positive effects of TA and EGCG on inhibiting TMEM16F phospholipid scrambling and discourage the use of these polyphenols as CaPLSase inhibitors. Appropriate controls as well as a combination of both fluorescence imaging and electrophysiological validation are necessary in future endeavors to develop TMEM16 CaPLSase inhibitors.


Assuntos
Anoctaminas/química , Proteínas de Transferência de Fosfolipídeos/química , Fosfolipídeos/química , Animais , Anoctaminas/antagonistas & inibidores , Anoctaminas/metabolismo , Catequina/análogos & derivados , Catequina/química , Catequina/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Polifenóis/química , Polifenóis/farmacologia , Taninos/química , Taninos/farmacologia
18.
Nat Commun ; 11(1): 3298, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620747

RESUMO

Communication between organelles is essential for their cellular homeostasis. Neurodegeneration reflects the declining ability of neurons to maintain cellular homeostasis over a lifetime, where the endolysosomal pathway plays a prominent role by regulating protein and lipid sorting and degradation. Here we report that TMEM16K, an endoplasmic reticulum lipid scramblase causative for spinocerebellar ataxia (SCAR10), is an interorganelle regulator of the endolysosomal pathway. We identify endosomal transport as a major functional cluster of TMEM16K in proximity biotinylation proteomics analyses. TMEM16K forms contact sites with endosomes, reconstituting split-GFP with the small GTPase RAB7. Our study further implicates TMEM16K lipid scrambling activity in endosomal sorting at these sites. Loss of TMEM16K function led to impaired endosomal retrograde transport and neuromuscular function, one of the symptoms of SCAR10. Thus, TMEM16K-containing ER-endosome contact sites represent clinically relevant platforms for regulating endosomal sorting.


Assuntos
Anoctaminas/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Lisossomos/metabolismo , Animais , Anoctaminas/genética , Transporte Biológico , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , Retículo Endoplasmático/ultraestrutura , Endossomos/ultraestrutura , Células HEK293 , Humanos , Metabolismo dos Lipídeos , Lisossomos/ultraestrutura , Camundongos Knockout , Microscopia Eletrônica , Mutação , Transporte Proteico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo
19.
Int J Mol Sci ; 21(11)2020 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-32517157

RESUMO

Anoctamins such as TMEM16A and TMEM16B are Ca2+-dependent Cl- channels activated through purinergic receptor signaling. TMEM16A (ANO1), TMEM16B (ANO2) and TMEM16F (ANO6) are predominantly expressed at the plasma membrane and are therefore well accessible for functional studies. While TMEM16A and TMEM16B form halide-selective ion channels, TMEM16F and probably TMEM16E operate as phospholipid scramblases and nonselective ion channels. Other TMEM16 paralogs are expressed mainly in intracellular compartments and are therefore difficult to study at the functional level. Here, we report that TMEM16E (ANO5), -H (ANO8), -J (ANO9) and K (ANO10) are targeted to the plasma membrane when fused to a C-terminal CAAX (cysteine, two aliphatic amino acids plus methionin, serine, alanin, cystein or glutamin) motif. These paralogs produce Ca2+-dependent ion channels. Surprisingly, expression of the TMEM16 paralogs in the plasma membrane did not produce additional scramblase activity. In contrast, endogenous scrambling induced by stimulation of purinergic P2X7 receptors was attenuated, in parallel with reduced plasma membrane blebbing. This could suggest that intracellular TMEM16 paralogs operate differently when compared to plasma membrane-localized TMEM16F, and may even stabilize intracellular membranes. Alternatively, CAAX tagging, which leads to expression in non-raft compartments of the plasma membrane, may antagonize phosphatidylserine exposure by endogenous raft-located TMEM16F. CAAX-containing constructs may be useful to further investigate the molecular properties of intracellular TMEM16 proteins.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Imunofluorescência , Expressão Gênica , Espaço Intracelular/metabolismo , Ionomicina/farmacologia , Família Multigênica , Fosfolipídeos/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
Ann Surg Oncol ; 27(9): 3218-3230, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32227267

RESUMO

BACKGROUND: Few studies have reported the function and activation mechanism of ANO9 in esophageal squamous cell carcinoma (ESCC). The current study aimed to investigate the role of ANO9 in the regulation of tumor progression. METHODS: Knockdown experiments with human ESCC cell lines were performed using ANO9 siRNA, and the effects on cell proliferation, the cell cycle, apoptosis, and cellular movement were analyzed. Immunohistochemistry (IHC) analysis was performed on 57 primary tumor samples obtained from ESCC patients. RESULTS: In an in vitro study, depletion of ANO9 reduced cell proliferation, invasion, and migration in KYSE150 and KYSE 790 cells. In the cell cycle analysis, depletion of ANO9 increased the number of cells in G0/G1 arrest. In addition, the knockdown of ANO9 increased apoptosis. The results of the microarray analysis indicated that various centrosome-related genes such as CEP120, CNTRL, and SPAST were up- or downregulated in ANO9-depleted KYSE150 cells. The IHC results showed that high expression of ANO9 was associated with poor prognosis. CONCLUSIONS: The results of the current study suggest that ANO9 regulates the cell cycle via centrosome-related genes in ESCC.


Assuntos
Anoctaminas/metabolismo , Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Proteínas de Transferência de Fosfolipídeos/metabolismo , Anoctaminas/genética , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica , Proteínas de Transferência de Fosfolipídeos/genética , Espastina/genética , Espastina/metabolismo
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