Explore the vast range of titles available.

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10−36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

A strong association between multiple sclerosis (MS) and Epstein-Barr virus (EBV) has been established but the exact role of EBV in MS remains controversial. Recently, molecular mimicry between EBNA1 and specific GlialCAM, CRYAB and ANO2 peptides has been suggested as a possible pathophysiological mechanism. The aim of this study was to analyse anti-EBV antibodies in MS patients against (I) EBV lifecycle proteins, (II) putative cross-reactive peptides, and (III) during treatment. In this retrospective cross-sectional study, 258 serum samples were included consisting of EBV-negative (n = 25) and EBV-positive (n = 36) controls, 192 MS samples including untreated relapsing-remitting MS (RRMS) with and without relapses, secondary progressive MS (SPMS) and primary progressive MS (PPMS) patients, and 106 patients on 8 different treatment regimens. IgG and IgM antibody titers against EBV docking/fusion proteins (gp350, gh/gp42, gh/gL/gp42), immediate early antigen (BZLF1), early antigens (EA p85, EA P138, EA P54), capsid antigens (VCA P18, VCA P23, VCA gp125) and late antigens (EBNA1) were measured. Specific EBNA1 and GlialCAM, CRYAB and ANO2 peptides were synthesized and also incorporated in our custom magnetic bead based multiplex assay. We observed significantly elevated IgG antibody titers in EBV-positive controls, RRMS with and without relapse, SPMS and PPMS patients for all lifecycle antigens except for several early antigens when compared to EBV-negative controls. Significantly higher IgG antibody titers were observed in RRMS patients for fusion proteins and EBNA1 peptides when compared to EBV-positive controls. An MS specific response was observed for ANO2 but not for GlialCAM or CRYAB. No significant treatment effects or a specific IgM response were detectable. The MS-specific, differential antibody response to EBV antigens confirms an altered immunological response to EBV in MS patients. EBV reactivation does not appear to play an important role in MS pathogenesis and no differential antibody signatures were observed between MS disease phases. The MS-specific anti-ANO2 antibody response suggests a potential role for EBNA1 as an antigenic driver, although the exact role of anti-ANO2 antibodies needs to be determined. The precise pathophysiological role of EBV in MS remains uncertain and requires further investigation.

Anoctamin 1 (ANO1)/TMEM16A is a Cl − channel activated by intracellular Ca 2+ mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca 2+ . Here, we demonstrate that two helices, “reference” and “Ca 2+ sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca 2+ -dependent manner. Positively and negatively charged residues in the two helices are essential for Ca 2+ -dependent activation because neutralization of these charges change the Ca 2+ sensitivity. We now predict that the Ca 2+ sensor helix attaches to the reference helix in the resting state, and as intracellular Ca 2+ rises, Ca 2+ acts on the sensor helix, which repels it from the reference helix. This Ca 2+ -dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis.

COVID-19 is a disease with unique characteristics that include lung thrombosis 1 , frequent diarrhoea 2 , abnormal activation of the inflammatory response 3 and rapid deterioration of lung function consistent with alveolar oedema 4 . 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. Lungs from patients who died from COVID-19 show atypical fused cells, the formation of which is mediated by the SARS-CoV-2 spike protein, and drugs that inhibit TMEM16F can prevent spike-induced syncytia formation.

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. The depletion of ANO9 reduced the binding ability to PD‐1 by downregulating the expression of PD‐L2 in GC cells. This is the first report to clarify the mechanism of ANO9 regulating the immune escape of cancer cells via PD‐L2.

The dual functions of TMEM16F as Ca 2+ -activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca 2+ -activated Cl - channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca 2+ influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling. TMEM16F is a Ca 2+ activated ion channel and lipid scramblase involved in cell fusion. Here authors determine cryo-EM structures of TMEM16F with or without bound blockers, such as the FDA-approved drug niclosamide.

Purinergic P2X 7 receptors (P2X 7 R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X 7 R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X 7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca 2+ dependent phospholipid scramblase and Ca 2+ -activated Cl − channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X 7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6 −/− mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages. Activation of purinergic P2X 7 receptors is important for phagocytosis and bacterial killing. Here the authors show that a phospholipid scramblase, Anoctamin 6, is activated downstream of P2X 7 R and is a critical mediator of bacterial internalization and killing by macrophages.

TMEM16F, a Ca 2+ -activated phospholipid scramblase (CaPLSase), is critical for placental trophoblast syncytialization, HIV infection, and SARS-CoV2-mediated syncytialization, however, how TMEM16F is activated during cell fusion is unclear. Here, using trophoblasts as a model for cell fusion, we demonstrate that Ca 2+ influx through the Ca 2+ permeable transient receptor potential vanilloid channel TRPV4 is critical for TMEM16F activation and plays a role in subsequent human trophoblast fusion. GSK1016790A, a TRPV4 specific agonist, robustly activates TMEM16F in trophoblasts. We also show that TRPV4 and TMEM16F are functionally coupled within Ca 2+ microdomains in a human trophoblast cell line using patch-clamp electrophysiology. Pharmacological inhibition or gene silencing of TRPV4 hinders TMEM16F activation and subsequent trophoblast syncytialization. Our study uncovers the functional expression of TRPV4 and one of the physiological activation mechanisms of TMEM16F in human trophoblasts, thus providing us with novel strategies to regulate CaPLSase activity as a critical checkpoint of physiologically and disease-relevant cell fusion events.

Polycystic kidney diseases are characterized by the development of numerous bilateral renal cysts that continuously enlarge resulting in a decline of kidney function due to compression of intact nephrons. Cyst growth is driven by transepithelial chloride secretion which depends on both intracellular cAMP and calcium. Mechanisms that are involved in the regulation of the underlying secretory pathways remain incompletely understood. Here we show that glucose concentration has a strong impact on cyst growth of renal tubular cells within a collagen matrix as well as in embryonic kidneys deficient or competent for Pkd1 . Glucose-dependent cyst growth correlates with the transcriptional induction of the calcium-activated chloride channel anoctamin 1 (ANO1) and its increased expression in the apical membrane of cyst-forming cells. Inhibition of ANO1 with the specific inhibitor CaCCinh-AO1 significantly decreases glucose-dependent cyst growth in both models. Ussing chamber analyses revealed increased apical chloride secretion of renal tubular cells upon exposure to high glucose medium which can also be inhibited by the use of CaCCinh-AO1. These data suggest that glycemic control may help to reduce renal cyst growth in patients with polycystic kidney disease. Key message Renal cyst growth depends on glucose concentration in two in vitro cyst models. High glucose leads to upregulation of the calcium-activated chloride channel ANO1. High glucose promotes calcium-activated chloride secretion via ANO1. Glucose-dependent secretion can be inhibited by a specific inhibitor of ANO1.

Casein kinase 2 (CK2) is a highly ubiquitous and conserved serine/threonine kinase that forms a tetramer consisting of a catalytic subunit (CK2α) and a regulatory subunit (CK2β). Despite being ubiquitous, CK2 is commonly found at higher expression levels in cancer cells, where it inhibits apoptosis, and supports cell migration and proliferation. The Ca2+-activated chloride channel TMEM16A shows similar effects in cancer cells: TMEM16A increases cell proliferation and migration and is highly expressed in squamous cell carcinoma of the head and neck (HNSCC) as well as other malignant tumors. A microscopy-based high-throughput screening was performed to identify proteins that regulate TMEM16A. Within this screen, CK2 was found to be required for proper membrane expression of TMEM16A. small interfering (si) RNA-knockdown of CK2 reduced plasma membrane expression of TMEM16A and inhibited TMEM16A whole cell currents in (cystic fibrosis bronchial epithelial) CFBE airway epithelial cells and in the head and neck cancer cell lines Cal33 and BHY. Inhibitors of CK2, such as TBB and the preclinical compound CX4549 (silmitasertib), also blocked membrane expression of TMEM16A and Ca2+-activated whole cell currents. siRNA-knockout of CK2 and its pharmacological inhibition, as well as knockdown or inhibition of TMEM16A by either niclosamide or Ani9, attenuated cell proliferation. Simultaneous inhibition of CK2 and TMEM16A strongly potentiated inhibition of cell proliferation. Although membrane expression of TMEM16A is reduced by inhibition of CK2, our data suggest that the antiproliferative effects by inhibition of CK2 are mostly independent of TMEM16A. Simultaneous inhibition of TMEM16A by niclosamide and inhibition of CK2 by silmitasertib was additive with respect to blocking cell proliferation, while cytotoxicity was reduced when compared to solely blockade of CK2. Therefore, parallel blockade TMEM16A by niclosamide may assist with anticancer therapy by silmitasertib.