To facilitate our understanding of proteome dynamics during signaling events, robust workflows affording fast time resolution without confounding factors are essential. We present Surface-exposed protein Labeling using PeroxidaSe, H2O2, and Tyramide-derivative (SLAPSHOT) to label extracellularly exposed proteins in a rapid, specific, and sensitive manner. Simple and flexible SLAPSHOT utilizes recombinant soluble APEX2 protein applied to cells, thus circumventing the engineering of tools and cells, biological perturbations, and labeling biases. We applied SLAPSHOT and quantitative proteomics to examine the TMEM16F-dependent plasma membrane remodeling in WT and TMEM16F KO cells. Time-course data ranging from 1 to 30 min of calcium stimulation revealed co-regulation of known protein families, including the integrin and ICAM families, and identified proteins known to reside in intracellular organelles as occupants of the freshly deposited extracellularly exposed membrane. Our data provide the first accounts of the immediate consequences of calcium signaling on the extracellularly exposed proteome.

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.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane\'s inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

Amyotrophic lateral sclerosis as a fatal neurodegenerative disease currently lacks effective therapeutic agents. Thus, finding new therapeutic targets to drive disease treatment is necessary. In this study, we utilized brain and plasma proteins as genetic instruments obtained from genome-wide association studies to conduct a Mendelian randomization analysis to identify potential drug targets for amyotrophic lateral sclerosis. Additionally, we validated our results externally using other datasets. We also used Bayesian co-localization analysis and phenotype scanning. Furthermore, we constructed a protein-protein interaction network to elucidate potential correlations between the identified proteins and existing targets. Mendelian randomization analysis indicated that elevated levels of ANO5 (OR = 1.30; 95 % CI, 1.14-1.49; P = 1.52E-04), SCFD1 (OR = 3.82; 95 % CI, 2.39-6.10; P = 2.19E-08), and SIGLEC9 (OR = 1.05; 95% CI, 1.03-1.07; P = 4.71E-05) are associated with an increased risk of amyotrophic lateral sclerosis, with external validation supporting these findings. Co-localization analysis confirmed that ANO5, SCFD1, and SIGLEC9 (coloc.abf-PPH4 = 0.848, 0.984, and 0.945, respectively) shared the same variant with amyotrophic lateral sclerosis, further substantiating potential role of these proteins as a therapeutic target. There are interactive relationships between the potential proteins and existing targets of amyotrophic lateral sclerosis. Our findings suggested that elevated levels of ANO5, SCFD1, and SIGLEC9 are connected with an increased risk of amyotrophic lateral sclerosis and might be promising therapeutic targets. However, further exploration is necessary to fully understand the underlying mechanisms involved.

TMEM16 proteins, which function as Ca2+‑activated Cl‑ channels are involved in regulating a wide variety of cellular pathways and functions. The modulators of Cl‑ channels can be used for the molecule‑based treatment of respiratory diseases, cystic fibrosis, tumors, cancer, osteoporosis and coronavirus disease 2019. The TMEM16 proteins link Ca2+ signaling, cellular electrical activity and lipid transport. Thus, deciphering these complex regulatory mechanisms may enable a more comprehensive understanding of the physiological functions of the TMEM16 proteins and assist in ascertaining the applicability of these proteins as potential pharmacological targets for the treatment of a range of diseases. The present review examined the structures, functions and characteristics of the different types of TMEM16 proteins, their association with the pathogenesis of various diseases and the applicability of TMEM16 modulator‑based treatment methods.

UNASSIGNED: This analysis investigated potential associations between gene variants and clinical end points in the VIEW 1 and 2 randomized clinical trials of intravitreal aflibercept and ranibizumab in neovascular age-related macular degeneration (AMD).
UNASSIGNED: A genome-wide association analysis was conducted in a subgroup of patients from VIEW 1 and 2 consenting to the optional pharmacogenetic analysis.
UNASSIGNED: Data were pooled from 780 samples from patients representative of the overall VIEW 1 and 2 populations. After Bonferroni correction for multiplicity and statistical adjustment for baseline risk factors, no significant associations were found between previously identified prognostic AMD gene variants and treatment response according to key prespecified VIEW 1 and 2 end points. Genome-wide, there were no significant genetic associations in patients experiencing gains of ≥15 Early Treatment of Diabetic Retinopathy Study letters after 1 or 2 years of treatment. A cluster of variants in ANO2 (encoding anoctamin 2, a calcium-activated chloride channel expressed on photoreceptor cells) on chromosome 12 reached the level of significance for loss of ≥5 letters after 1 year of treatment (P < 5 × 10-8), with the ANO2 rs2110166 SNP demonstrating highly significant association (P = 1.99 × 10-8). Carriers of the ANO2 rs2110166 TT genotype showed a robust increase in visual acuity versus baseline compared with a small decrease in those with the TC genotype.
UNASSIGNED: None of the potential prognostic candidate genes were associated with the clinical end points for treated patients. Preliminary analyses suggest an association of ANO2 with retinal function, with a potential impact on vision of approximately one line over at least the first year. Further investigation of the function of ANO2 in retinal pathophysiology is merited.

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.

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.

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.

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.