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Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China 1 – 3 . However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited 4 – 8 , with current analyses of HCC mainly from non-HBV-enriched populations 9 , 10 . Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain ( FGA ), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications. The Chinese Liver Cancer Atlas project depicts a panoramic genomic landscape of hepatocellular carcinoma, covering candidate coding and non-coding drivers, mutational signatures, extrachromosomal circular DNA, subclonal catastrophic events and detailed evolutionary history.

Anoctamin1 (ANO1)/transmembrane protein 16A (TMEM16A), a calcium-activated chloride channel (CaCC), is involved in many physiological functions such as fluid secretion, smooth muscle contraction, nociception and cancer progression. To date, only a few ANO1 inhibitors have been described, and these have low potency and selectivity for ANO1. Here, we performed a high-throughput screening to identify highly potent and selective small molecule inhibitors of ANO1. Three novel ANO1 inhibitors were discovered from screening of 54,400 synthetic small molecules, and they were found to fully block ANO1 channel activity with an IC50 < 3 μM. Electrophysiological analysis revealed that the most potent inhibitor, 2-(4-chloro-2-methylphenoxy)-N-[(2-methoxyphenyl)methylideneamino]-acetamide (Ani9), completely inhibited ANO1 chloride current with submicromolar potency. Notably, unlike previous small-molecule ANO1 inhibitors identified to date, Ani9 displayed high selectivity for ANO1 as compared to ANO2, which shares a high amino acid homology to ANO1. In addition, Ani9 did not affect the intracellular calcium signaling and CFTR chloride channel activity. Our results suggest that Ani9 may be a useful pharmacological tool for studying ANO1 and a potential development candidate for drug therapy of cancer, hypertension, pain, diarrhea and asthma.

The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca 2+ , prolonged Ca 2+ -dependent activation results in channel desensitization or rundown, the mechanism of which is unclear. Here we show that phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3–5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca 2+ -binding module formed by TMs 6–8 and a PIP 2 -binding regulatory module formed by TMs 3–5, which mediate channel activation and desensitization, respectively. PIP 2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation. The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability and muscle contraction. Here authors show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site.

ObjectiveUC is a chronic inflammatory disease of the colonic mucosa. Growing evidence supports a role for epithelial cell defects in driving pathology. Moreover, long-lasting changes in the epithelial barrier have been reported in quiescent UC. Our aim was to investigate whether epithelial cell defects could originate from changes in the epithelial compartment imprinted by the disease.DesignEpithelial organoid cultures (EpOCs) were expanded ex vivo from the intestinal crypts of non-IBD controls and patients with UC. EpOCs were induced to differentiate (d-EpOCs), and the total RNA was extracted for microarray and quantitative real-time PCR (qPCR) analyses. Whole intestinal samples were used to determine mRNA expression by qPCR, or protein localisation by immunostaining.ResultsEpOCs from patients with UC maintained self-renewal potential and the capability to give rise to differentiated epithelial cell lineages comparable with control EpOCs. Nonetheless, a group of genes was differentially regulated in the EpOCs and d-EpOCs of patients with UC, including genes associated with antimicrobial defence (ie, LYZ, PLA2G2A), with secretory (ie, ZG16, CLCA1) and absorptive (ie, AQP8, MUC12) functions, and with a gastric phenotype (ie, ANXA10, CLDN18 and LYZ). A high rate of concordance was found in the expression profiles of the organoid cultures and whole colonic tissues from patients with UC.ConclusionsPermanent changes in the colonic epithelium of patients with UC could be promoted by alterations imprinted in the stem cell compartment. These changes may contribute to perpetuation of the disease.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family. Cell membranes are made up of two layers of oily molecules, called lipids, embedded with a variety of proteins. Each type of membrane protein carries out a particular activity for the cell, and many are involved in transporting other molecules from one side of the membrane to the other. The TMEM16 proteins are a large family of membrane proteins. Most are known as lipid scramblases and move lipids between the two layers of the membrane. However, some TMEM16 proteins transport ions in or out of the cell, and are instead called ion channels. TMEM16 proteins are found in animals, plants and fungi but not bacteria, and play key roles in many biological activities that keep these organisms alive. For example, in humans, ion channels belonging to the TMEM16 family help keep the lining of the lung moist, and allow muscles in the gut to contract. The structure of a scramblase shows that two protein units interact, with each unit containing a furrow that spans the membrane, through which lipids can move from one layer to the other. However, to date, the shape of a TMEM16 ion channel has not been determined. It was therefore not clear how a protein with features that let it transport large, oily molecules like lipids had evolved to transport small, charged particles instead. TMEM16A is a member of the TMEM16 family that transports negatively charged chloride ions. Using a technique called cryo-electron microscopy, Paulino et al. have determined the three-dimensional shape of the version of TMEM16A from a mouse. Overall, TMEM16A is organized similarly to the lipid scramblase. However, some parts of the TMEM16A protein have undergone rearrangements such that the membrane-exposed furrow that provides a path for lipids in scramblases is now partially sealed in TMEM16A. This results in an enclosed pore that is largely shielded from the oily membrane and through which ions can pass. Additionally, biochemical analysis suggests that TMEM16A forms a narrow pore that may widen towards the side facing the inside of the cell, though further work is needed to understand if this is relevant to the protein’s activity. The three-dimensional structure of TMEM16A reveals how the protein’s architecture differs from other family members working as lipid scramblases. It also gives insight into how TMEM16 proteins might work as ion channels. These findings can now form a strong basis for future studies into the activity of TMEM16 proteins.

Osteoclast over-activation leads to bone loss and chloride homeostasis is fundamental importance for osteoclast function. The calcium-activated chloride channel Anoctamin 1 (also known as TMEM16A) is an important chloride channel involved in many physiological processes. However, its role in osteoclast remains unresolved. Here, we identified the existence of Anoctamin 1 in osteoclast and show that its expression positively correlates with osteoclast activity. Osteoclast-specific Anoctamin 1 knockout mice exhibit increased bone mass and decreased bone resorption. Mechanistically, Anoctamin 1 deletion increases intracellular Cl − concentration, decreases H + secretion and reduces bone resorption. Notably, Anoctamin 1 physically interacts with RANK and this interaction is dependent upon Anoctamin 1 channel activity, jointly promoting RANKL-induced downstream signaling pathways. Anoctamin 1 protein levels are substantially increased in osteoporosis patients and this closely correlates with osteoclast activity. Finally, Anoctamin 1 deletion significantly alleviates ovariectomy induced osteoporosis. These results collectively establish Anoctamin 1 as an essential regulator in osteoclast function and suggest a potential therapeutic target for osteoporosis. Osteoclast over-activation leads to bone loss and chloride homeostasis is important for osteoclast function. Here, the authors show that Anoctamin 1 controls bone resorption by coupling Cl − channel activation with RANKL-RANK signaling transduction.

The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca 2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca 2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca 2+ . The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck. Here authors use cryo-EM and electrophysiology to identify three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate.

IntroductionClostridioides difficile infection (CDI), the leading cause of hospital-acquired diarrhoea, is primarily driven by two potent toxins, TcdA and TcdB. However, the molecular mechanisms underpinning toxin-mediated disruption of intestinal barrier function and the role of fluid and ion exchange regulators in intestinal permeability during CDI, and their modulation by faecal microbiota transplantation (FMT) remain incompletely understood.MethodsCaecal tissues from mice infected with C. difficile and treated with or without FMT were analysed via RT-qPCR for expression of aquaporins (AQP1, AQP4, AQP8, AQP11), carbonic anhydrases (CAI, CAII, CAIV), and ion transporters (ANO1, CLCA1, SGLT1, SLC9A3, SLC26A3, SCNN1A, SCNN1B, SCNN1G). Their expression was also validated in colonic biopsies from CDI patients and healthy individuals and primary human colonoids treated with TcdA and TcdB. AQP1, AQP11, CAI, and CAII were knocked down in Caco-2/TC-7 cells by means of shRNAs, and epithelial barrier formation was assessed by transepithelial electrical resistance (TEER) and tight junction immufluorescence in response to TcdA and B.ResultsCDI significantly downregulated AQP1, AQP11, CAI, CAII, and CAIV expression in murine models, an effect reversed following FMT. Knockdown of AQP1 and CAII in Caco-2/TC-7 cells impaired barrier function, with further deterioration upon TcdB exposure. Validation in human CDI colonic tissues confirmed reduced AQP1 and CAII expression. AQP1 expression was directly suppressed by TcdB in primary human colonoids.ConclusionsWe demonstrate that AQP1 and CAII are key regulators of gut epithelial barrier integrity during CDI. Their downregulation by C. difficile contributes to increased intestinal permeability, a hallmark of disease pathogenesis and their restoration by FMT suggests a potential mechanism for its therapeutic efficacy. These findings identify AQP1 and CAII as promising therapeutic targets for the restoration of barrier function in CDI.

Bitter taste receptors (taste 2 receptors, TAS2Rs) serve as warning sensors in the lingual system against the ingestion of potentially poisonous food. Here, we investigated the functional role of TAS2Rs in the human gut and focused on their potential to trigger an additional host defense pathway in the intestine. Human jejunal crypts, especially those from individuals with obesity, responded to bitter agonists by inducing the release of antimicrobial peptides (α-defensin 5 and regenerating islet-derived protein 3 α [REG3A]) but also regulated the expression of other innate immune factors (mucins, chemokines) that affected E. coli growth. We found that the effect of aloin on E. coli growth and on the release of the mucus glycoprotein CLCA1, identified via proteomics, was affected by TAS2R43 deletion polymorphisms and thus confirmed a role for TAS2R43. RNA-Seq revealed that denatonium benzoate induced an NRF2-mediated nutrient stress response and an unfolded protein response that increased the expression of the mitokine GDF15 but also ADM2 and LDLR, genes that are involved in anorectic signaling and lipid homeostasis. In conclusion, TAS2Rs in the intestine constitute a promising target for treating diseases that involve disturbances in the innate immune system and body weight control. TAS2R polymorphisms may be valuable genetic markers to predict therapeutic responses.

Specific functions of the immune system are essential to protect us from infections caused by pathogens such as viruses and bacteria. However, as we age, the immune system shows a functional decline that can be attributed in large part to age-associated defects in hematopoietic stem cells (HSCs)—the cells at the apex of the immune cell hierarchy. Here, we find that the Hippo pathway coactivator TAZ is potently induced in old HSCs and protects these cells from functional decline. We identify Clca3a1 as a TAZ-induced gene that allows us to trace TAZ activity in vivo. Using CLCA3A1 as a marker, we can isolate “young-like” HSCs from old mice. Mechanistically, Taz acts as coactivator of PU.1 and to some extent counteracts the gradual loss of PU.1 expression during HSC aging. Our work thus uncovers an essential role for Taz in a previously undescribed fail-safe mechanism in aging HSCs. Immune system function declines with age, a consequence of defects in hematopoietic stem cells (HSCs). Here the authors show that TAZ buffers age-related loss of PU.1 activity to maintain HSC functionality and identify the surface protein Clca3a1 as a marker of “young-like” HSCs, even in old mice.