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.

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.

The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. It acts by removing ectopic lipids from the outer leaflet of the outer membrane and returning them to the inner membrane through three proteinaceous assemblies: the MlaA-OmpC complex, situated within the outer membrane; the periplasmic phospholipid shuttle protein, MlaC; and the inner membrane ABC transporter complex, MlaFEDB, proposed to be the founding member of a structurally distinct ABC superfamily. While the function of each component is well established, how phospholipids are exchanged between components remains unknown. This stands as a major roadblock in our understanding of the function of the pathway, and in particular, the role of ATPase activity of MlaFEDB is not clear. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

PtdIns and its phosphorylated derivatives, the phosphoinositides, are the biochemical components of a major pathway of intracellular signaling in all eukaryotic cells. These lipids are few in terms of cohort of unique positional isomers, and are quantitatively minor species of the bulk cellular lipidome. Nevertheless, phosphoinositides regulate an impressively diverse set of biological processes. It is from that perspective that perturbations in phosphoinositide-dependent signaling pathways are increasingly being recognized as causal foundations of many human diseases - including cancer. Although phosphatidylinositol transfer proteins (PITPs) are not enzymes, these proteins are physiologically significant regulators of phosphoinositide signaling. As such, PITPs are conserved throughout the eukaryotic kingdom. Their biological importance notwithstanding, PITPs remain understudied. Herein, we review current information regarding PITP biology primarily focusing on how derangements in PITP function disrupt key signaling/developmental pathways and are associated with a growing list of pathologies in mammals.

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.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Background: PtdIns (3,4,5) P3-dependent Rac exchanger 1 (PREX1), also known as PREX1, a member of the Rac guanine nucleotide exchange factors (Rac-GEF) family. Studies have suggested that PREX1 plays a role in mediating oncogenic pathway activation and controlling various biological mechanisms in different types of cancer, including liver hepatocellular carcinoma (LIHC). However, the function of PREX1 in the pathogenesis of LIHC and its potential role on immunological regulation is not clearly elucidated. Methods: The expression level and the clinical role of PREX1 in LIHC was analyzed based on database from the Cancer Genome Atlas (TCGA), TNM plotter and University of Alabama Cancer Database (UALCAN). We investigated the relationship between PREX1 and immunity in LIHC by TISIDB, CIBERSORT and single cell analysis. Immunotherapy responses were assessed by the immunophenoscores (IPS). Moreover, biological functional assays were performed to further investigate the roles of PREX1 in liver cancer cell lines. Results: Higher expression of PREX1 in LIHC tissues than in normal liver tissues was found based on public datasets. Further analysis revealed that PREX1 was associated with worse clinical characteristics and dismal prognosis. Pathway enrichment analysis indicated that PREX1 participated in immune-related pathways. Through CIBERSORT and single cell analysis, we found a remarkable correlation between the expression of PREX1 and various immune cells, especially macrophages. In addition, high PREX1 expression was found to be associated with a stronger response to immunotherapy. Furthermore, in vitro assays indicated that depletion of PREX1 can suppress invasion and proliferation of LIHC cells. Conclusion: Elevated expression of PREX1 indicates poor prognosis, influences immune modulation and predicts sensitivity of immunosuppression therapy in LIHC. Our results suggested that PREX1 may be a prognostic biomarker and therapeutic target, offering new treatment options for LIHC.

The eukaryotic asparagine (N)-linked glycan is pre-assembled as a fourteen-sugar oligosaccharide on a lipid carrier in the endoplasmic reticulum (ER). Seven sugars are first added to dolichol pyrophosphate (PP-Dol) on the cytoplasmic face of the ER, generating Man5GlcNAc2-PP-Dol (M5GN2-PP-Dol). M5GN2-PP-Dol is then flipped across the bilayer into the lumen by an ER translocator. Genetic studies identified Rft1 as the M5GN2-PP-Dol flippase in vivo but are at odds with biochemical data suggesting Rft1 is dispensable for flipping in vitro. Thus, the question of whether Rft1 plays a direct or an indirect role during M5GN2-PP-Dol translocation has been controversial for over two decades. We describe a completely reconstituted in vitro assay for M5GN2-PP-Dol translocation and demonstrate that purified Rft1 catalyzes the translocation of M5GN2-PP-Dol across the lipid bilayer. These data, combined with in vitro results demonstrating substrate selectivity and rft1∆ phenotypes, confirm the molecular identity of Rft1 as the M5GN2-PP-Dol ER flippase.