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1.
Nature ; 628(8009): 910-918, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38570680

RESUMO

OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.


Assuntos
Canais de Cálcio , Microscopia Crioeletrônica , Ativação do Canal Iônico , Mecanotransdução Celular , Humanos , Anoctaminas/química , Anoctaminas/metabolismo , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Canais de Cálcio/ultraestrutura , Lipídeos/química , Lipossomos/metabolismo , Lipossomos/química , Modelos Moleculares , Nanoestruturas/química
2.
Am J Hum Genet ; 111(6): 1184-1205, 2024 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-38744284

RESUMO

Anoctamins are a family of Ca2+-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca2+ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca2+-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca2+-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease.


Assuntos
Anoctaminas , Mutação de Sentido Incorreto , Humanos , Anoctaminas/genética , Anoctaminas/metabolismo , Mutação de Sentido Incorreto/genética , Masculino , Feminino , Epilepsia/genética , Criança , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Estudos de Associação Genética , Linhagem , Cálcio/metabolismo , Genes Dominantes , Pré-Escolar , Células HEK293 , Adolescente
3.
PLoS Biol ; 22(8): e3002762, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-39173068

RESUMO

During embryonic development, tissues and organs are gradually shaped into their functional morphologies through a series of spatiotemporally tightly orchestrated cell behaviors. A highly conserved organ shape across metazoans is the epithelial tube. Tube morphogenesis is a complex multistep process of carefully choreographed cell behaviors such as convergent extension, cell elongation, and lumen formation. The identity of the signaling molecules that coordinate these intricate morphogenetic steps remains elusive. The notochord is an essential tubular organ present in the embryonic midline region of all members of the chordate phylum. Here, using genome editing, pharmacology and quantitative imaging in the early chordate Ciona intestinalis we show that Ano10/Tmem16k, a member of the evolutionarily ancient family of transmembrane proteins called Anoctamin/TMEM16 is essential for convergent extension, lumen expansion, and connection during notochord morphogenesis. We find that Ano10/Tmem16k works in concert with the plasma membrane (PM) localized Na+/Ca2+ exchanger (NCX) and the endoplasmic reticulum (ER) residing SERCA, RyR, and IP3R proteins to establish developmental stage specific Ca2+ signaling molecular modules that regulate notochord morphogenesis and Ca2+ dynamics. In addition, we find that the highly conserved Ca2+ sensors calmodulin (CaM) and Ca2+/calmodulin-dependent protein kinase (CaMK) show an Ano10/Tmem16k-dependent subcellular localization. Their pharmacological inhibition leads to convergent extension, tubulogenesis defects, and deranged Ca2+ dynamics, suggesting that Ano10/Tmem16k is involved in both the "encoding" and "decoding" of developmental Ca2+ signals. Furthermore, Ano10/Tmem16k mediates cytoskeletal reorganization during notochord morphogenesis, likely by altering the localization of 2 important cytoskeletal regulators, the small GTPase Ras homolog family member A (RhoA) and the actin binding protein Cofilin. Finally, we use electrophysiological recordings and a scramblase assay in tissue culture to demonstrate that Ano10/Tmem16k likely acts as an ion channel but not as a phospholipid scramblase. Our results establish Ano10/Tmem16k as a novel player in the prevertebrate molecular toolkit that controls organ morphogenesis across scales.


Assuntos
Anoctaminas , Ciona intestinalis , Morfogênese , Notocorda , Animais , Notocorda/metabolismo , Notocorda/embriologia , Anoctaminas/metabolismo , Anoctaminas/genética , Ciona intestinalis/metabolismo , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Morfogênese/genética , Sinalização do Cálcio , Regulação da Expressão Gênica no Desenvolvimento , Retículo Endoplasmático/metabolismo , Cálcio/metabolismo
4.
Cell ; 151(1): 111-22, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23021219

RESUMO

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:


Assuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Cálcio/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Ambystoma mexicanum , Animais , Anoctamina-1 , Anoctaminas , Canais de Cloreto/metabolismo , Hemostasia , Metabolismo dos Lipídeos , Megacariócitos/metabolismo , Camundongos , Camundongos Knockout , Oócitos/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Xenopus
5.
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
6.
Proc Natl Acad Sci U S A ; 121(27): e2311831121, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38941274

RESUMO

TMEM16F is a calcium-activated phospholipid scramblase and nonselective ion channel, which allows the movement of lipids bidirectionally across the plasma membrane. While the functions of TMEM16F have been extensively characterized in multiple cell types, the role of TMEM16F in the central nervous system remains largely unknown. Here, we sought to study how TMEM16F in the brain may be involved in neurodegeneration. Using a mouse model that expresses the pathological P301S human tau (PS19 mouse), we found reduced tauopathy and microgliosis in 6- to 7-mo-old PS19 mice lacking TMEM16F. Furthermore, this reduction of pathology can be recapitulated in the PS19 mice with TMEM16F removed from neurons, while removal of TMEM16F from microglia of PS19 mice did not significantly impact tauopathy at this time point. Moreover, TMEM16F mediated aberrant phosphatidylserine exposure in neurons with phospho-tau burden. These studies raise the prospect of targeting TMEM16F in neurons as a potential treatment of neurodegeneration.


Assuntos
Anoctaminas , Neurônios , Fosfatidilserinas , Tauopatias , Proteínas tau , Animais , Anoctaminas/metabolismo , Anoctaminas/genética , Fosfatidilserinas/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Proteínas tau/metabolismo , Proteínas tau/genética , Camundongos , Tauopatias/metabolismo , Tauopatias/patologia , Humanos , Microglia/metabolismo , Microglia/patologia , Fosforilação , Camundongos Transgênicos , Modelos Animais de Doenças , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Encéfalo/metabolismo , Encéfalo/patologia , Camundongos Knockout
7.
J Cell Sci ; 137(14)2024 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-38940198

RESUMO

TMEM16F (also known as ANO6), a Ca2+-activated lipid scramblase (CaPLSase) that dynamically disrupts lipid asymmetry, plays a crucial role in various physiological and pathological processes, such as blood coagulation, neurodegeneration, cell-cell fusion and viral infection. However, the mechanisms through which it regulates these processes remain largely elusive. Using endothelial cell-mediated angiogenesis as a model, here we report a previously unknown intracellular signaling function of TMEM16F. We demonstrate that TMEM16F deficiency impairs developmental retinal angiogenesis in mice and disrupts angiogenic processes in vitro. Biochemical analyses indicate that the absence of TMEM16F enhances the plasma membrane association of activated Src kinase. This in turn increases VE-cadherin phosphorylation and downregulation, accompanied by suppressed angiogenesis. Our findings not only highlight the role of intracellular signaling by TMEM16F in endothelial cells but also open new avenues for exploring the regulatory mechanisms for membrane lipid asymmetry and their implications in disease pathogenesis.


Assuntos
Anoctaminas , Células Endoteliais , Transdução de Sinais , Animais , Anoctaminas/metabolismo , Anoctaminas/genética , Camundongos , Humanos , Células Endoteliais/metabolismo , Quinases da Família src/metabolismo , Quinases da Família src/genética , Neovascularização Fisiológica , Fosforilação , Caderinas/metabolismo , Antígenos CD/metabolismo , Antígenos CD/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Membrana Celular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Angiogênese , Proteínas de Transferência de Fosfolipídeos
8.
J Biol Chem ; 300(7): 107432, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38825009

RESUMO

The Ca2+-activated Cl- channel regulator CLCA1 potentiates the activity of the Ca2+-activated Cl- channel (CaCC) TMEM16A by directly engaging the channel at the cell surface, inhibiting its reinternalization and increasing Ca2+-dependent Cl- current (ICaCC) density. We now present evidence of functional pairing between two other CLCA and TMEM16 protein family members, namely CLCA4 and the CaCC TMEM16B. Similar to CLCA1, (i) CLCA4 is a self-cleaving metalloprotease, and the N-terminal portion (N-CLCA4) is secreted; (ii) the von Willebrand factor type A (VWA) domain in N-CLCA4 is sufficient to potentiate ICaCC in HEK293T cells; and (iii) this is mediated by the metal ion-dependent adhesion site motif within VWA. The results indicate that, despite the conserved regulatory mechanism and homology between CLCA1 and CLCA4, CLCA4-dependent ICaCC are carried by TMEM16B, rather than TMEM16A. Our findings show specificity in CLCA/TMEM16 interactions and suggest broad physiological and pathophysiological links between these two protein families.


Assuntos
Anoctaminas , Canais de Cloreto , Humanos , Anoctamina-1/metabolismo , Anoctamina-1/genética , Anoctaminas/metabolismo , Anoctaminas/genética , Anoctaminas/química , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Canais de Cloreto/genética , Cloretos/metabolismo , Células HEK293 , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Domínios Proteicos
9.
J Physiol ; 602(19): 4889-4905, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39167717

RESUMO

Mammalian olfactory sensory neurons (OSNs) generate an odorant-induced response by sequentially activating two ion channels, which are in their ciliary membranes. First, a cationic, Ca2+-permeable cyclic nucleotide-gated channel is opened following odorant stimulation via a G protein-coupled transduction cascade and an ensuing rise in cAMP. Second, the increase in ciliary Ca2+ opens the excitatory Ca2+-activated Cl- channel TMEM16B, which carries most of the odorant-induced receptor current. While the role of TMEM16B in amplifying the response has been well established, it is less understood how this secondary ion channel contributes to response kinetics and action potential generation during single as well as repeated stimulation and, on the other hand, which response properties the cyclic nucleotide-gated (CNG) channel determines. We first demonstrate that basic membrane properties such as input resistance, resting potential and voltage-gated currents remained unchanged in OSNs that lack TMEM16B. The CNG channel predominantly determines the response delay and adaptation during odorant exposure, while the absence of the Cl- channels shortens both the time the response requires to reach its maximum and the time to terminate after odorant stimulation. This faster response termination in Tmem16b knockout OSNs allows them, somewhat counterintuitively despite the large reduction in receptor current, to fire action potentials more reliably when stimulated repeatedly in rapid succession, a phenomenon that occurs both in isolated OSNs and in OSNs within epithelial slices. Thus, while the two olfactory ion channels act in concert to generate the overall response, each one controls specific aspects of the odorant-induced response. KEY POINTS: Mammalian olfactory sensory neurons (OSNs) generate odorant-induced responses by activating two ion channels sequentially in their ciliary membranes: a Na+, Ca2⁺-permeable cyclic nucleotide-gated (CNG) channel and the Ca2⁺-activated Cl⁻ channel TMEM16B. The CNG channel controls response delay and adaptation during odorant exposure, while TMEM16B amplifies the response and influences the time required for the response to reach its peak and terminate. OSNs lacking TMEM16B display faster response termination, allowing them to fire action potentials more reliably during rapid repeated stimulation. The CNG and TMEM16B channels have distinct and complementary roles in shaping the kinetics and reliability of odorant-induced responses in OSNs.


Assuntos
Anoctaminas , Neurônios Receptores Olfatórios , Animais , Anoctaminas/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Camundongos , Potenciais de Ação/fisiologia , Cálcio/metabolismo , Camundongos Knockout , Canais de Cloreto/metabolismo , Canais de Cloreto/fisiologia , Camundongos Endogâmicos C57BL , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Odorantes
10.
Pflugers Arch ; 476(2): 211-227, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37979051

RESUMO

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.


Assuntos
Asma , COVID-19 , Camundongos , Animais , Anoctamina-1/metabolismo , Ivermectina/farmacologia , Ivermectina/uso terapêutico , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Anoctaminas/metabolismo , Pulmão/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Cálcio/metabolismo , Inflamação/tratamento farmacológico , Anti-Inflamatórios , Canais de Cloreto/metabolismo
11.
Hum Mol Genet ; 31(12): 2063-2077, 2022 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-35043958

RESUMO

Prostate cancer is among the most common cancers in men, with a large fraction of the individual risk attributable to heritable factors. A majority of the diagnosed cases does not lead to a lethal disease, and hence biological markers that can distinguish between indolent and fatal forms of the disease are of great importance for guiding treatment decisions. Although over 300 genetic variants are known to be associated with prostate cancer risk, few have been associated with the risk of an aggressive disease. One such variant is rs77559646 located in ANO7. This variant has a dual function. It constitutes a missense mutation in the short isoform of ANO7 and a splice region mutation in full-length ANO7. In this study, we have analyzed the impact of the variant allele of rs77559646 on ANO7 mRNA splicing using a minigene splicing assay and by performing splicing analysis with the tools IRFinder (intron retention finder), rMATS (replicate multivariate analysis of transcript splicing) and LeafCutter on RNA sequencing data from prostate tissue of six rs77559646 variant allele carriers and 43 non-carriers. The results revealed a severe disruption of ANO7 mRNA splicing in rs77559646 variant allele carriers. Immunohistochemical analysis of prostate samples from patients homozygous for the rs77559646 variant allele demonstrated a loss of apically localized ANO7 protein. Our study is the first to provide a mechanistic explanation for the impact of a prostate cancer risk SNP on ANO7 protein production. Furthermore, the rs77559646 variant is the first known germline loss-of-function mutation described for ANO7. We suggest that loss of ANO7 contributes to prostate cancer progression.


Assuntos
Anoctaminas , Neoplasias da Próstata , Splicing de RNA , Anoctaminas/genética , Sequência de Bases , Humanos , Masculino , Neoplasias da Próstata/genética , RNA Mensageiro/genética
12.
Am J Physiol Heart Circ Physiol ; 326(1): H270-H277, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37999645

RESUMO

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously have shown that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We have also demonstrated that ADAM17 sheddase activity targets IRα; however, the mechanisms driving endothelial ADAM17 activity in T2D are largely unknown. Herein, we report that externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane causes ADAM17-mediated shedding of IRα and blunting of insulin signaling in endothelial cells. Furthermore, we demonstrate that endothelial PS externalization is mediated by the phospholipid scramblase anoctamin-6 (ANO6) and that this process can be stimulated by neuraminidase, a soluble enzyme that cleaves sialic acid residues. Of note, we demonstrate that men and women with T2D display increased levels of neuraminidase activity in plasma, relative to age-matched healthy individuals, and this occurs in conjunction with increased ADAM17 activity and impaired leg blood flow responses to endogenous insulin. Collectively, this work reveals the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.NEW & NOTEWORTHY This work provides the first evidence that neuraminidase, an enzyme increased in the circulation of men and women with type 2 diabetes (T2D), promotes anoctamin-6 (ANO6)-dependent externalization of phosphatidylserine in endothelial cells, which in turn leads to activation of a disintegrin and metalloproteinase-17 (ADAM17) and consequent shedding of the insulin receptor-α from the cell surface. Hence, this work supports that consideration should be given to the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Masculino , Humanos , Feminino , Células Endoteliais/metabolismo , Receptor de Insulina/metabolismo , Fosfatidilserinas/metabolismo , Neuraminidase/metabolismo , Insulina/metabolismo , Desintegrinas , Proteína ADAM17/metabolismo , Anoctaminas/metabolismo
13.
Mov Disord ; 39(5): 887-892, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38469933

RESUMO

BACKGROUND: Biallelic pathogenic variants in the ANO10 gene cause autosomal recessive progressive ataxia (ATX-ANO10). METHODS: Following the MDSGene protocol, we systematically investigated genotype-phenotype relationships in ATX-ANO10 based on the clinical and genetic data from 82 published and 12 newly identified patients. RESULTS: Most patients (>80%) had loss-of-function (LOF) variants. The most common variant was c.1150_1151del, found in all 29 patients of Romani ancestry, who had a 14-year earlier mean age at onset than patients homozygous for other LOF variants. We identified previously undescribed clinical features of ATX-ANO10 (e.g., facial muscle involvement and strabismus) suggesting the involvement of brainstem pathology, and we propose a diagnostic algorithm that may aid clinical ATX-ANO10 diagnosis. CONCLUSIONS: The early disease onset in patients with c.1150_1151del may indicate the existence of genetic/environmental disease-modifying factors in the Romani population. Our findings will inform patient counseling and may improve our understanding of the disease mechanism. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Anoctaminas , Ataxias Espinocerebelares , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Idade de Início , Anoctaminas/genética , Estudos de Associação Genética , Ataxias Espinocerebelares/genética , Idoso
14.
Mov Disord ; 39(6): 983-995, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38581205

RESUMO

BACKGROUND: Based on a limited number of reported families, biallelic CA8 variants have currently been associated with a recessive neurological disorder named, cerebellar ataxia, mental retardation, and dysequilibrium syndrome 3 (CAMRQ-3). OBJECTIVES: We aim to comprehensively investigate CA8-related disorders (CA8-RD) by reviewing existing literature and exploring neurological, neuroradiological, and molecular observations in a cohort of newly identified patients. METHODS: We analyzed the phenotype of 27 affected individuals from 14 families with biallelic CA8 variants (including data from 15 newly identified patients from eight families), ages 4 to 35 years. Clinical, genetic, and radiological assessments were performed, and zebrafish models with ca8 knockout were used for functional analysis. RESULTS: Patients exhibited varying degrees of neurodevelopmental disorders (NDD), along with predominantly progressive cerebellar ataxia and pyramidal signs and variable bradykinesia, dystonia, and sensory impairment. Quadrupedal gait was present in only 10 of 27 patients. Progressive selective cerebellar atrophy, predominantly affecting the superior vermis, was a key diagnostic finding in all patients. Seven novel homozygous CA8 variants were identified. Zebrafish models demonstrated impaired early neurodevelopment and motor behavior on ca8 knockout. CONCLUSION: Our comprehensive analysis of phenotypic features indicates that CA8-RD exhibits a wide range of clinical manifestations, setting it apart from other subtypes within the category of CAMRQ. CA8-RD is characterized by cerebellar atrophy and should be recognized as part of the autosomal-recessive cerebellar ataxias associated with NDD. Notably, the presence of progressive superior vermis atrophy serves as a valuable diagnostic indicator. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Ataxia Cerebelar , Peixe-Zebra , Humanos , Ataxia Cerebelar/genética , Criança , Adolescente , Masculino , Feminino , Pré-Escolar , Animais , Adulto , Adulto Jovem , Anoctaminas/genética , Deficiência Intelectual/genética , Fenótipo , Transtornos do Neurodesenvolvimento/genética
15.
Cell Biol Int ; 48(6): 848-860, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38444077

RESUMO

Oxidized low-density lipoprotein (oxLDL), a key component in atherosclerosis and hyperlipidemia, is a risk factor for atherothrombosis in dyslipidemia, yet its mechanism is poorly understood. In this study, we used oxLDL-induced human aortic endothelial cells (HAECs) and high-fat diet (HFD)-fed mice as a hyperlipidemia model. Phosphatidylserine (PS) exposure, cytosolic Ca2+, reactive oxygen species (ROS), and lipid peroxidation were measured by flow cytometer. TMEM16F expression was detected by immunofluorescence, western blot, and reverse transcription polymerase chain reaction. Procoagulant activity (PCA) was measured by coagulation time, intrinsic/extrinsic factor Xase, and thrombin generation. We found that oxLDL-induced PS exposure and the corresponding PCA of HAECs were increased significantly compared with control, which could be inhibited over 90% by lactadherin. Importantly, TMEM16F expression in oxLDL-induced HAECs was upregulated by enhanced intracellular Ca2+ concentration, ROS, and lipid peroxidation, which led to PS exposure. Meanwhile, the knockdown of TMEM16F by short hairpin RNA significantly inhibited PS exposure in oxLDL-induced HAECs. Moreover, we observed that HFD-fed mice dramatically increased the progress of thrombus formation and accompanied upregulated TMEM16F expression by thromboelastography analysis, FeCl3-induced carotid artery thrombosis model, and western blot. Collectively, these results demonstrate that TMEM16F-mediated PS exposure may contribute to prothrombotic status under hyperlipidemic conditions, which may serve as a novel therapeutic target for the prevention of thrombosis in hyperlipidemia.


Assuntos
Anoctaminas , Células Endoteliais , Lipoproteínas LDL , Fosfatidilserinas , Espécies Reativas de Oxigênio , Animais , Humanos , Camundongos , Anoctaminas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Dieta Hiperlipídica , Células Endoteliais/metabolismo , Hiperlipidemias/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Lipoproteínas LDL/metabolismo , Camundongos Endogâmicos C57BL , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trombose/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo
16.
BMC Neurol ; 24(1): 96, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38491364

RESUMO

BACKGROUND: The Limb Girdle Muscular Dystrophies (LGMDs) are characterized by progressive weakness of the shoulder and hip girdle muscles as a result of over 30 different genetic mutations. This study is designed to develop clinical outcome assessments across the group of disorders. METHODS/DESIGN: The primary goal of this study is to evaluate the utility of a set of outcome measures on a wide range of LGMD phenotypes and ability levels to determine if it would be possible to use similar outcomes between individuals with different phenotypes. We will perform a multi-center, 12-month study of 188 LGMD patients within the established Genetic Resolution and Assessments Solving Phenotypes in LGMD (GRASP-LGMD) Research Consortium, which is comprised of 11 sites in the United States and 2 sites in Europe. Enrolled patients will be clinically affected and have mutations in CAPN3 (LGMDR1), ANO5 (LGMDR12), DYSF (LGMDR2), DNAJB6 (LGMDD1), SGCA (LGMDR3), SGCB (LGMDR4), SGCD (LGMDR6), or SGCG (LGMDR5, or FKRP-related (LGMDR9). DISCUSSION: To the best of our knowledge, this will be the largest consortium organized to prospectively validate clinical outcome assessments (COAs) in LGMD at its completion. These assessments will help clinical trial readiness by identifying reliable, valid, and responsive outcome measures as well as providing data driven clinical trial decision making for future clinical trials on therapeutic agents for LGMD. The results of this study will permit more efficient clinical trial design. All relevant data will be made available for investigators or companies involved in LGMD therapeutic development upon conclusion of this study as applicable. TRIAL REGISTRATION: Clinicaltrials.gov NCT03981289; Date of registration: 6/10/2019.


Assuntos
Distrofia Muscular do Cíngulo dos Membros , Sarcoglicanopatias , Humanos , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Distrofia Muscular do Cíngulo dos Membros/genética , Fenótipo , Músculo Esquelético , Mutação/genética , Proteínas do Tecido Nervoso/genética , Chaperonas Moleculares/genética , Proteínas de Choque Térmico HSP40/genética , Pentosiltransferases/genética , Anoctaminas/genética
17.
Brain ; 146(9): 3800-3815, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36913258

RESUMO

Anoctamin-5 related muscle disease is caused by biallelic pathogenic variants in the anoctamin-5 gene (ANO5) and shows variable clinical phenotypes: limb-girdle muscular dystrophy type 12 (LGMD-R12), distal muscular dystrophy type 3 (MMD3), pseudometabolic myopathy or asymptomatic hyperCKaemia. In this retrospective, observational, multicentre study we gathered a large European cohort of patients with ANO5-related muscle disease to study the clinical and genetic spectrum and genotype-phenotype correlations. We included 234 patients from 212 different families, contributed by 15 centres from 11 European countries. The largest subgroup was LGMD-R12 (52.6%), followed by pseudometabolic myopathy (20.5%), asymptomatic hyperCKaemia (13.7%) and MMD3 (13.2%). In all subgroups, there was a male predominance, except for pseudometabolic myopathy. Median age at symptom onset of all patients was 33 years (range 23-45 years). The most frequent symptoms at onset were myalgia (35.3%) and exercise intolerance (34.1%), while at last clinical evaluation most frequent symptoms and signs were proximal lower limb weakness (56.9%) and atrophy (38.1%), myalgia (45.1%) and atrophy of the medial gastrocnemius muscle (38.4%). Most patients remained ambulatory (79.4%). At last evaluation, 45.9% of patients with LGMD-R12 additionally had distal weakness in the lower limbs and 48.4% of patients with MMD3 also showed proximal lower limb weakness. Age at symptom onset did not differ significantly between males and females. However, males had a higher risk of using walking aids earlier (P = 0.035). No significant association was identified between sportive versus non-sportive lifestyle before symptom onset and age at symptom onset nor any of the motor outcomes. Cardiac and respiratory involvement that would require treatment occurred very rarely. Ninety-nine different pathogenic variants were identified in ANO5 of which 25 were novel. The most frequent variants were c.191dupA (p.Asn64Lysfs*15) (57.7%) and c.2272C>T (p.Arg758Cys) (11.1%). Patients with two loss-of function variants used walking aids at a significantly earlier age (P = 0.037). Patients homozygous for the c.2272C>T variant showed a later use of walking aids compared to patients with other variants (P = 0.043). We conclude that there was no correlation of the clinical phenotype with the specific genetic variants, and that LGMD-R12 and MMD3 predominantly affect males who have a significantly worse motor outcome. Our study provides useful information for clinical follow up of the patients and for the design of clinical trials with novel therapeutic agents.


Assuntos
Doenças Musculares , Distrofia Muscular do Cíngulo dos Membros , Feminino , Masculino , Humanos , Mialgia/genética , Estudos Retrospectivos , Anoctaminas/genética , Mutação/genética , Doenças Musculares/epidemiologia , Doenças Musculares/genética , Doenças Musculares/patologia , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/epidemiologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/diagnóstico , Atrofia/patologia
18.
Prenat Diagn ; 44(9): 1098-1104, 2024 08.
Artigo em Inglês | MEDLINE | ID: mdl-38922934

RESUMO

This article presents two fetal cases of gnathodiaphyseal dysplasia (GDD), a rare autosomal dominant disorder, and reviews the relevant literature. The cases involved two fetuses exhibiting bone bowing, which led to the diagnosis of GDD. Genetic testing revealed two de novo variants of the ANO5 gene, confirming the diagnosis. A literature review was conducted to explore GDD's clinical and paraclinical presentation, diagnosis, and management. GDD is a rare but frequently inherited cause of bone fragility and jaw lesions characterized by a gain-of-function variant within the ANO5 gene. Clinical manifestations range from recurrent dental infections with mild jaw lesions to severe bone fragility with several fractures associated with large jaw lesions requiring disfiguring surgeries. Diagnostic techniques depend on the context and include targeted genetic testing of ANO5, untargeted molecular analysis with whole-exome sequencing, or whole-genome sequencing. This case report highlights the importance of recognizing GDD as a novel cause of bone bowing and fractures during pregnancy. By summarizing the literature, this article contributes to healthcare professionals' knowledge and improves the recognition, diagnosis, and care of patients with GDD.


Assuntos
Anoctaminas , Osteogênese Imperfeita , Humanos , Feminino , Gravidez , Osteogênese Imperfeita/genética , Osteogênese Imperfeita/diagnóstico , Osteogênese Imperfeita/complicações , Osteogênese Imperfeita/diagnóstico por imagem , Anoctaminas/genética , Adulto
19.
Int J Mol Sci ; 25(18)2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39337485

RESUMO

Anoctamin 6 (ANO6, TMEM16F) is a phospholipid (PL) scramblase that moves PLs between both plasma membrane (PM) leaflets and operates as an ion channel. It plays a role in development and is essential for hemostasis, bone mineralization and immune defense. However, ANO6 has also been shown to regulate cellular Ca2+ signaling and PM compartments, thereby controlling the expression of ion channels such as CFTR. Given these pleiotropic effects, we investigated the functional interdependence of the ubiquitous ANO6 with other commonly co-expressed anoctamins. As most expression studies on anoctamins use HEK293 human embryonic kidney cells, we compared ion currents, PL scrambling and Ca2+ signals induced by the overexpression of anoctamins in HEK293 wild-type parental and ANO6-knockout cells. The data suggest that the endogenous expression of ANO6 significantly affects the results obtained from overexpressed anoctamins, particularly after increasing intracellular Ca2+. Thus, a significant interdependence of anoctamins may influence the interpretation of data obtained from the functional analysis of overexpressed anoctamins.


Assuntos
Anoctaminas , Cálcio , Proteínas de Transferência de Fosfolipídeos , Humanos , Anoctaminas/metabolismo , Anoctaminas/genética , Células HEK293 , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo
20.
Int J Mol Sci ; 25(7)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38612670

RESUMO

We aimed to identify serum exosomal microRNAs (miRNAs) associated with the transition from atrial fibrillation (AF) to sinus rhythm (SR) and investigate their potential as biomarkers for the early recurrence of AF within three months post-treatment. We collected blood samples from eight AF patients at Chang Gung Memorial Hospital in Taiwan both immediately before and within 14 days following rhythm control treatment. Exosomes were isolated from these samples, and small RNA sequencing was performed. Using DESeq2 analysis, we identified nine miRNAs (16-2-3p, 22-3p, 23a-3p, 23b-3p, 125a-5p, 328-3p, 423-5p, 504-5p, and 582-3p) associated with restoration to SR. Further analysis using the DIABLO model revealed a correlation between the decreased expression of miR-125a-5p and miR-328-3p and the early recurrence of AF. Furthermore, early recurrence is associated with a longer duration of AF, presumably indicating a more extensive state of underlying cardiac remodeling. In addition, the reads were mapped to mRNA sequences, leading to the identification of 14 mRNAs (AC005041.1, ARHGEF12, AMT, ANO8, BCL11A, DIO3OS, EIF4ENIF1, G2E3-AS1, HERC3, LARS, NT5E, PITX1, SLC16A12, and ZBTB21) associated with restoration to SR. Monitoring these serum exosomal miRNA and mRNA expression patterns may be beneficial for optimizing treatment outcomes in AF patients.


Assuntos
Fibrilação Atrial , Exossomos , MicroRNAs , Humanos , Fibrilação Atrial/genética , MicroRNAs/genética , Coração , Exossomos/genética , RNA Mensageiro , Anoctaminas
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