Depletion Of Membrane Cholesterol Research Articles

Pathogenic and Apathogenic Strains of Lymphocytic Choriomeningitis Virus Have Distinct Entry and Innate Immune Activation Pathways.

Lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV) share many genetic and biological features including subtle differences between pathogenic and apathogenic strains. Despite remarkable genetic similarity, the viscerotropic WE strain of LCMV causes a fatal LASV fever-like hepatitis in non-human primates (NHPs) while the mouse-adapted Armstrong (ARM) strain of LCMV is deeply attenuated in NHPs and can vaccinate against LCMV-WE challenge. Here, we demonstrate that internalization of WE is more sensitive to the depletion of membrane cholesterol than ARM infection while ARM infection is more reliant on endosomal acidification. LCMV-ARM induces robust NF-κB and interferon response factor (IRF) activation while LCMV-WE seems to avoid early innate sensing and failed to induce strong NF-κB and IRF responses in dual-reporter monocyte and epithelial cells. Toll-like receptor 2 (TLR-2) signaling appears to play a critical role in NF-κB activation and the silencing of TLR-2 shuts down IL-6 production in ARM but not in WE-infected cells. Pathogenic LCMV-WE infection is poorly recognized in early endosomes and failed to induce TLR-2/Mal-dependent pro-inflammatory cytokines. Following infection, Interleukin-1 receptor-associated kinase 1 (IRAK-1) expression is diminished in LCMV-ARM- but not LCMV-WE-infected cells, which indicates it is likely involved in the LCMV-ARM NF-κB activation. By confocal microscopy, ARM and WE strains have similar intracellular trafficking although LCMV-ARM infection appears to coincide with greater co-localization of early endosome marker EEA1 with TLR-2. Both strains co-localize with Rab-7, a late endosome marker, but the interaction with LCMV-WE seems to be more prolonged. These findings suggest that LCMV-ARM's intracellular trafficking pathway may facilitate interaction with innate immune sensors, which promotes the induction of effective innate and adaptive immune responses.

Protopanaxadiol Targeting Membrane Induces HepG2 Cell Apoptosis Via Raft-like Formation and Tubulation Disruption.

Protopanaxadiol (PPD), which has a molecular structure similar to cholesterol, is a potent anticancer agent that has been proposed to target the lipid membrane for the pharmacological effects. However, the underlying mechanism by which PPD modulates the cell membrane leading to cancer cell death is not be fully understood. In this work, we used single cell infrared spectroscopy, scanning electron microscopy and confocal microscopy to investigate the effects of PPD on human hepatocellular carcinoma (HepG2) cells, focusing on the change in membrane structure. We found that PPD significantly reduced the number of membrane tubules over the course of treatment. Interestingly, the addition of PPD could promote the formation of lipid raft-like domains (PPD rafts) and even restore the domain disruption caused by methyl-beta-cyclodextrin depletion of membrane cholesterol. In addition, PPD pre-treatment may increase the induction effect of FasL, which impairs cell viability, although it does not appear to be beneficial for Fas clustering in the PPD rafts. Collectively, these results highlight a non-classical mechanism by which PPD induces HepG2 apoptosis by directly affecting the physical properties of the cell membrane, providing a novel insight into understanding membrane-targeted therapy.

Cyclodextrin derivatives decrease Transient Receptor Potential vanilloid 1 and Ankyrin 1 ion channel activation via altering the surrounding membrane microenvironment by cholesterol depletion.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1) are nonselective cation channels expressed in primary sensory neurons and several other non-neuronal structures such as immune cells, keratinocytes, and vascular smooth muscle cells. They play important roles in nociception, pain processing and their chanellopathies are associated with the development of several pathological conditions. They are located in cholesterol- and sphingolipid-rich membrane lipid raft regions serving as platforms to modulate their activations. We demonstrated earlier that disruption of these lipid rafts leads to decreased TRP channel activation and exerts analgesic effects. Cyclodextrins are macrocyclic molecules able to form host-guest complexes with cholesterol and deplete it from the membrane lipid rafts. The aim of this study was to investigate 8 structurally different (methylated and non-methylated) CD derivatives on cell viability, mitochondrial membrane potential, membrane composition and activation abilities of the TRPV1 and TRPA1 channels. We showed that non-methylated derivatives have preferable safety profiles compared to methylated ones. Furthermore, methylated derivatives reduced mitochondrial membrane potential. However, all investigated derivatives influence the ordered cell membrane structure depleting membrane cholesterol and inhibit the TRPV1 agonist capsaicin- and the TRPA1 agonist allyl isothiocyanate-induced Ca2+-influx. This mechanism of action might provide novel perspectives for the development of peripherally acting analgesics via indirectly decreasing the generation and transmission of nociceptive signals.

Targeting cholesterol impairs cell invasion of all breast cancer types

BackgroundBreast cancer clinical outcome relies on its intrinsic molecular subtype and mortality is almost exclusively due to metastasis, whose mechanism remains unclear. We recently revealed the specific contribution of plasma membrane cholesterol to the invasion of malignant MCF10CAIa but not premalignant MCF10AT and normal MCF10A cell lines in 2D, through invadopodia formation and extracellular matrix (ECM) degradation. In the present study, we address the impact of breast cancer subtypes, mutations and aggressiveness on cholesterol implication in breast cancer cell invasion and 3D spheroid invasion and growth.MethodsWe used nine breast cancer cell lines grouped in four subtypes matching breast tumor classification. Four of these cell lines were also used to generate 3D spheroids. These cell lines were compared for cell invasion in 2D and 3D, spheroid growth in 3D, gelatin degradation, cortactin expression, activation and subcellular distribution as well as cell surface cholesterol distribution and lipid droplets. The effect of plasma membrane cholesterol depletion on all these parameters was determined in parallel and systematically compared with the impact of global matrix metalloproteinase (MMP) inhibition.ResultsThe six invasive cell lines in 2D were sensitive to partial cholesterol depletion, independently of their subtype, aggressiveness or mutation. Nevertheless, the effect was stronger in the three cell lines able to degrade gelatin. 3D spheroid invasion was also reduced after cholesterol depletion in all breast cancer subtypes tested. Notably, targeting cholesterol was more powerful than MMP inhibition in reducing invasion in both 2D and 3D culture models. Moreover, cholesterol depletion in the six invasive cell lines impaired cortactin distribution in the perinuclear region where invadopodia localized. Breast cancer cell line aggressiveness relied on cholesterol-enriched domains at the ECM-free side and intracellular lipid droplets. Furthermore, the three gelatin-degrading cell lines were characterized by increased cholesterol-enriched submicrometric domains at their ECM-contact side.ConclusionTogether, our data suggest cell surface cholesterol combined with lipid droplet labeling as a breast cancer cell aggressiveness marker. They also open the way to test other cholesterol-targeting drugs in more complex models to further evaluate whether cholesterol could represent a strategy in breast cancer therapy.

Cholesterol modulation of interactions between psychostimulants and dopamine transporters.

The dopamine transporter (DAT) is a key site of action for cocaine and amphetamines. Dysfunctional DAT is associated with aberrant synaptic dopamine transmission and enhanced drug-seeking and taking behavior. Studies in cultured cells and ex vivo suggest that DAT function is sensitive to membrane cholesterol content. Although it is largely unknown whether psychostimulants alter cholesterol metabolism in the brain, emerging evidence indicates that peripheral cholesterol metabolism is altered in patients with psychostimulant use disorder and circulating cholesterol levels are associated with vulnerability to relapse. Cholesterol interacts with sphingolipids forming lipid raft microdomains on the membrane. These cholesterol-rich lipid raft microdomains serve to recruit and assemble other lipids and proteins to initiate signal transduction. There are two spatially and functionally distinct populations of the DAT segregated by cholesterol-rich lipid raft microdomains and cholesterol-scarce non-raft microdomains on the plasma membrane. These two DAT populations are differentially regulated by DAT blockers (e.g. cocaine), substrates (e.g. amphetamine), and protein kinase C providing distinct cholesterol-dependent modulation of dopamine uptake and efflux. In this chapter, we summarize the impact of depletion and addition of membrane cholesterol on DAT conformational changes between the outward-facing and the inward-facing states, lipid raft-associated DAT localization, basal and induced DAT internalization, and DAT function. In particular, we focus on how the interactions of the DAT with cocaine and amphetamine are influenced by membrane cholesterol. Lastly, we discuss the therapeutic potential of cholesterol-modifying drugs as a new avenue to normalize DAT function and dopamine transmission in patients with psychostimulant use disorder.

Piezo1 Regulation Involves Lipid Domains and the Cytoskeleton and Is Favored by the Stomatocyte-Discocyte-Echinocyte Transformation.

Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address this question since they display Piezo1 clusters, a strong and dynamic cytoskeleton and three types of submicrometric lipid domains, respectively enriched in cholesterol, GM1 ganglioside/cholesterol and sphingomyelin/cholesterol. We revealed that Piezo1 clusters were present in both the rim and the dimple erythrocyte regions. Upon Piezo1 chemical activation by Yoda1, the Piezo1 cluster proportion mainly increased in the dimple area. This increase was accompanied by Ca2+ influx and a rise in echinocytes, in GM1/cholesterol-enriched domains in the dimple and in cholesterol-enriched domains in the rim. Conversely, the effects of Piezo1 activation were abrogated upon membrane cholesterol depletion. Furthermore, upon Piezo1-independent Ca2+ influx, the above changes were not observed. In healthy donors with a high echinocyte proportion, Ca2+ influx, lipid domains and Piezo1 fluorescence were high even at resting state, whereas the cytoskeleton membrane occupancy was lower. Accordingly, upon decreases in cytoskeleton membrane occupancy and stiffness in erythrocytes from patients with hereditary spherocytosis, Piezo1 fluorescence was increased. Altogether, we showed that Piezo1 was differentially controlled by lipid domains and the cytoskeleton and was favored by the stomatocyte-discocyte-echinocyte transformation.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

Crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels.

The activity of neuronal Kv7.2/Kv7.3 channels is critically dependent on PIP2 and finely modulated by cholesterol. Here, we report the crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels. Our results show that currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells, by acute application of methyl-β-cyclodextrin (MβCD), were less sensitive to PIP2 dephosphorylation strategies than those of control cells, suggesting that cholesterol depletion enhances the Kv7.2/Kv7.3-PIP2 interaction. In contrast, the sensitivity of Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by MβCD was not altered in mutant channels with different apparent affinities for PIP2.

Avasimibe can cooperate with a DC-targeting and integration-deficient lentivector to induce stronger HBV specific T cytotoxic response by regulating cholesterol metabolism

We have reported a lentivector which could effectively induce HBV-specific cytotoxic T lymphocytes (CTLs). Avasimibe is an inhibitor of acetyl-CoA acetyltransferase-1 (ACAT1), and has been shown to enhance T lymphocyte cytotoxicity on tumor cells. However, the role of avasimibe in lentivector-induced HBV-specific T cytotoxic response remains unknown. Based on previous study, we constructed an integration-deficient lentivector LVDC-ID-HBV (harboring HBcAg expression), and the in vitro experiments showed that the combination of avasimibe exhibited better efficacy in inducing HBV-specific CTL responses including cell proliferation, production of cytokines, as well as CTL killing activities. Mechanism experiments showed that increasing cell membrane cholesterol levels by MβCD-coated cholesterol or ACAT1 inhibition efficiently promoted TCR clustering, signaling transduction and immunological synapse formation, thereby mediating augmented CTL responses. Nevertheless, the depletion of plasma membrane cholesterol with MβCD led to obviously decreased CTL responses. The avasimibe-mediated strengthened immune effects were also determined in animal experiments and the results were in agreement with those from the in vitro research. In particular, the in vivo CTL killing activities were identified by the CFSE or BV-labeled splenocyte lysis assay. Moreover, the experiments in HBV transgenic mice showed that the LVDC-ID-HBV plus avasimibe group demonstrated the lowest serum HBsAg and HBV DNA levels, as well as the lowest expression of HBsAg and HBcAg in liver tissues. We concluded that the HBV-specific CTL immune responses could be potentiated by avasimibe through regulating plasma membrane cholesterol levels. Avasimibe may be a potential adjuvant for lentivector vaccine against HBV infection.

Hypo-Osmotic Stress and Pore-Forming Toxins Adjust the Lipid Order in Sheep Red Blood Cell Membranes.

Lipid ordering in cell membranes has been increasingly recognized as an important factor in establishing and regulating a large variety of biological functions. Multiple investigations into lipid organization focused on assessing ordering from temperature-induced phase transitions, which are often well outside the physiological range. However, particular stresses elicited by environmental factors, such as hypo-osmotic stress or protein insertion into membranes, with respect to changes in lipid status and ordering at constant temperature are insufficiently described. To fill these gaps in our knowledge, we exploited the well-established ability of environmentally sensitive membrane probes to detect intramembrane changes at the molecular level. Our steady state fluorescence spectroscopy experiments focused on assessing changes in optical responses of Laurdan and diphenylhexatriene upon exposure of red blood cells to hypo-osmotic stress and pore-forming toxins at room temperature. We verified our utilized experimental systems by a direct comparison of the results with prior reports on artificial membranes and cholesterol-depleted membranes undergoing temperature changes. The significant changes observed in the lipid order after exposure to hypo-osmotic stress or pore-forming toxins resembled phase transitions of lipids in membranes, which we explained by considering the short-range interactions between membrane components and the hydrophobic mismatch between membrane thickness and inserted proteins. Our results suggest that measurements of optical responses from the membrane probes constitute an appropriate method for assessing the status of lipids and phase transitions in target membranes exposed to mechanical stresses or upon the insertion of transmembrane proteins.

Single-particle tracking and machine-learning classification reveals heterogeneous Piezo1 diffusion.

The mechanically-activated ion channel Piezo1 is involved in numerous physiological processes. Piezo1 is activated by diverse mechanical cues and is gated by membrane tension. The channel has been found to be mobile in the plasma membrane. We employed single particle tracking (SPT) of endogenously-expressed, tdTomato-tagged Piezo1 using Total Internal Reflection Fluorescence Microscopy in two cell types, mouse embryonic fibroblasts and liver sinusoidal endothelial cells. Application of SPT unveiled a surprising heterogeneity of Piezo1 mobility in the plasma membrane. Leveraging a machine learning technique, Piezo1 trajectories were sorted into distinct classes (“mobile,” “intermediate,” and “trapped”) by partitioning features that describe the geometric properties of a trajectory. To evaluate the effects of the plasma membrane properties on Piezo1 diffusion, we manipulated membrane composition by depleting or supplementing cholesterol or by adding margaric acid to stiffen the membrane. To address effects of channel activation on Piezo1 mobility, we treated cells with Yoda1, a Piezo1 agonist, and GsMTx-4, a channel inhibitor. We collected thousands of trajectories for each condition, and found that “mobile” Piezo1 in cells supplemented with cholesterol or margaric acid exhibited decreased mobility, whereas Piezo1 in cholesterol-depleted membranes demonstrated increased mobility, compared to their respective controls. Additionally, activation by Yoda1 increased Piezo1 mobility and inhibition by GsMTx-4 decreased Piezo1 mobility compared to their respective controls. The “mobile” trajectories were analyzed further by fitting the time-averaged mean-squared displacement as a function of lag time to a power-law model, revealing Piezo1 consistently exhibits anomalous subdiffusion. This suggests Piezo1 is not freely mobile, but that its mobility may be hindered by subcellular interactions. These studies illuminate the fundamental properties governing Piezo1 diffusion in the plasma membrane and set the stage to determine how specific cellular interactions may influence channel activity and mobility.

Severe COVID-19-A Review of Suggested Mechanisms Based on the Role of Extracellular Matrix Stiffness.

The severity of COVID-19 commonly depends on age-related tissue stiffness. The aim was to review publications that explain the effect of microenvironmental extracellular matrix stiffness on cellular processes. Platelets and endothelial cells are mechanosensitive. Increased tissue stiffness can trigger cytokine storm with the upregulated expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and interleukin IL-6, and tissue integrity disruption, leading to enhanced virus entry and disease severity. Increased tissue stiffness in critically ill COVID-19 patients triggers platelet activation and initiates plague formation and thrombosis development. Cholesterol content in cell membrane increases with aging and further enhances tissue stiffness. Membrane cholesterol depletion decreases virus entry to host cells. Membrane cholesterol lowering drugs, such as statins or novel chitosan derivatives, have to be further developed for application in COVID-19 treatment. Statins are also known to decrease arterial stiffness mitigating cardiovascular diseases. Sulfated chitosan derivatives can be further developed for potential use in future as anticoagulants in prevention of severe COVID-19. Anti-TNF-α therapies as well as destiffening therapies have been suggested to combat severe COVID-19. The inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells pathway must be considered as a therapeutic target in the treatment of severe COVID-19 patients. The activation of mechanosensitive platelets by higher matrix stiffness increases their adhesion and the risk of thrombus formation, thus enhancing the severity of COVID-19.

Atorvastatin differentially regulates the interactions of cocaine and amphetamine with dopamine transporters

The function of the dopamine transporter (DAT) is regulated by membrane cholesterol content. A direct, acute removal of membrane cholesterol by methyl-β-cyclodextrin (MβCD) has been shown to reduce dopamine (DA) uptake and release mediated by the DAT. This is of particular interest because a few widely prescribed statins that lower peripheral cholesterol levels are blood-brain barrier (BBB) penetrants, and therefore could alter DAT function through brain cholesterol modulation. The goal of this study was to investigate the effects of prolonged atorvastatin treatment (24 h) on DAT function in neuroblastoma 2A cells stably expressing DAT. We found that atorvastatin treatment effectively lowered membrane cholesterol content in a concentration-dependent manner. Moreover, atorvastatin treatment markedly reduced DA uptake and abolished cocaine inhibition of DA uptake, independent of surface DAT levels. These deficits induced by atorvastatin treatment were reversed by cholesterol replenishment. However, atorvastatin treatment did not change amphetamine (AMPH)-induced DA efflux. This is in contrast to a small but significant reduction in DA efflux induced by acute depletion of membrane cholesterol using MβCD. This discrepancy may involve differential changes in membrane lipid composition resulting from chronic and acute cholesterol depletion. Our data suggest that the outward-facing conformation of DAT, which favors the binding of DAT blockers such as cocaine, is more sensitive to atorvastatin-induced cholesterol depletion than the inward-facing conformation, which favors the binding of DAT substrates such as AMPH. Our study on statin-DAT interactions may have clinical implications in our understanding of neurological side effects associated with chronic use of BBB penetrant statins.

Protective role of Cav-1 in pneumolysin-induced endothelial barrier dysfunction.

Pneumolysin (PLY) is a bacterial pore forming toxin and primary virulence factor of Streptococcus pneumonia, a major cause of pneumonia. PLY binds cholesterol-rich domains of the endothelial cell (EC) plasma membrane resulting in pore assembly and increased intracellular (IC) Ca2+ levels that compromise endothelial barrier integrity. Caveolae are specialized plasmalemma microdomains of ECs enriched in cholesterol. We hypothesized that the abundance of cholesterol-rich domains in EC plasma membranes confers cellular susceptibility to PLY. Contrary to this hypothesis, we found increased PLY-induced IC Ca2+ following membrane cholesterol depletion. Caveolin-1 (Cav-1) is an essential structural protein of caveolae and its regulation by cholesterol levels suggested a possible role in EC barrier function. Indeed, Cav-1 and its scaffolding domain peptide protected the endothelial barrier from PLY-induced disruption. In loss of function experiments, Cav-1 was knocked-out using CRISPR-Cas9 or silenced in human lung microvascular ECs. Loss of Cav-1 significantly enhanced the ability of PLY to disrupt endothelial barrier integrity. Rescue experiments with re-expression of Cav-1 or its scaffolding domain peptide protected the EC barrier against PLY-induced barrier disruption. Dynamin-2 (DNM2) is known to regulate caveolar membrane endocytosis. Inhibition of endocytosis, with dynamin inhibitors or siDNM2 amplified PLY induced EC barrier dysfunction. These results suggest that Cav-1 protects the endothelial barrier against PLY by promoting endocytosis of damaged membrane, thus reducing calcium entry and PLY-dependent signaling.

Surface cholesterol-enriched domains specifically promote invasion of breast cancer cell lines by controlling invadopodia and extracellular matrix degradation

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer.

Cover Picture: Membrane Cholesterol Depletion Enhances Enzymatic Activity of Cell‐Membrane‐Coated Metal‐Organic‐Framework Nanoparticles (Angew. Chem. Int. Ed. 24/2022)

Enhancement of the enzymatic activity of cell-membrane-coated metal-organic framework nanoparticles by depleting membrane cholesterol is demonstrated by Weiwei Gao, Liangfang Zhang et al. in their Communication (DOI: 10.1002/anie.202203115). Increased membrane permeability, caused by the reduction of cholesterol level, is likely responsible for the increased enzymatic activity. The discovery broadens the use of these biomimetic nanoparticles in drug delivery and biodetoxification.

Clostridioides difficile surface layer protein triggered inflammasome activation is mediated by membrane lipid raft

Clostridioides difficile is the leading cause of nosocomial infectious diarrhea, with clinical symptoms ranging from mild diarrhea to pseudomembranous colitis and toxic megacolon. Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of C. difficileinfection (CDI), clinical relapse rates remain high. Therefore, new alternative therapeutics to treat CDI are urgently required. Surface layer proteins (SLPs) are the most abundant proteins in the C. difficile cell wall, suggesting that they might involve in immune recognition. Here, we found SLPs as well as C. difficile induced inflammasome activation. In addition, the cholesterol‐rich microdomains, lipid rafts, on the cell membrane are thought to be crucial for bacterial adhesion and signal transduction. We demonstrated that lipid rafts participated in C. difficile SLPs binding to the cell membrane. Furthermore, both binding ability and inflammasome activation induced by SLP were abrogated with membrane cholesterol depletion by methyl‐β‐cyclodextrin (MβCD). The coalescence of SLPs in the cholesterol‐rich microdomains was confirmed in C. difficile‐infected cells. Our results demonstrate that SLPs recruit the lipid rafts, which may be a key step for C. difficile colonization and inducing inflammasome activation.

Ultrashort nanosecond electric pulses activate a conductance in bovine adrenal chromaffin cells that involves cation entry through TRPC and NALCN channels

In whole-cell voltage clamped bovine adrenal chromaffin cells maintained at a holding potential of −70 mV, a single 5 ns, 5 MV/m pulse elicited an inward current carried mainly by Na+ that displayed inward rectification and a reversal potential near −3 mV, a voltage consistent with a non-selective cation current. The broad-spectrum inhibitors of transient receptor potential (TRP) channels, La3+ (10 μM), Gd3+ (10 μM), SKF-96365 (50 μM) and 2-aminoethoxydiphenyl borane (2-APB; 100 μM), inhibited the current similarly by ∼72%, ∼83%, ∼68% and ∼76%, respectively. Depleting membrane cholesterol with methyl-β-cyclodextrin (MβCD; 1–6 mg/ml) or inhibiting phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis with wortmannin (20 and 40 μM) produced a similar level of inhibition on the NEP-induced conductance as the broad spectrum TRP channel inhibitors. Moreover, no additive inhibitory effect was detected by combining MβCD (3 mg/ml), wortmannin (20 μM) and La3+ (10 μM), suggesting that each agent targeted different levels of the same pathway to exert a full effect. RT-PCR experiments revealed robust expression at the mRNA level of TRPC4, TRPC5 and TRPM7 channels for which specific blockers were available. Whereas the TRPM7 blocker FTY720 had no effect, the TRPC4/5 channel inhibitor M084 (20 μM) blocked the conductance by ∼50%, indicating that TRPC4 and/or TRPC5 channel(s) may be partially involved in mediating the NEP-induced current. CP-96345 (20 μM), a specific blocker of the sodium leak current channel (NALCN), also reduced the NEP-induced current. The inhibition was ∼30% and additive to that caused by the TRPC4/5 blocker M084. RT-PCR experiments confirmed the expression of this channel at the mRNA level. Taken as a whole, these data provide evidence that a large fraction of the current evoked by a 5 ns pulse in adrenal chromaffin cells may be carried by both TRPC4/5 channels and the NALCN channel. Understanding the biophysical properties of the NEP-elicited conductance in a neural-type cell will be extremely valuable for the future development of NEP stimulation approaches for neuromodulation.

Computational drug repurposing against SARS-CoV-2 reveals plasma membrane cholesterol depletion as key factor of antiviral drug activity.

Comparing SARS-CoV-2 infection-induced gene expression signatures to drug treatment-induced gene expression signatures is a promising bioinformatic tool to repurpose existing drugs against SARS-CoV-2. The general hypothesis of signature-based drug repurposing is that drugs with inverse similarity to a disease signature can reverse disease phenotype and thus be effective against it. However, in the case of viral infection diseases, like SARS-CoV-2, infected cells also activate adaptive, antiviral pathways, so that the relationship between effective drug and disease signature can be more ambiguous. To address this question, we analysed gene expression data from in vitro SARS-CoV-2 infected cell lines, and gene expression signatures of drugs showing anti-SARS-CoV-2 activity. Our extensive functional genomic analysis showed that both infection and treatment with in vitro effective drugs leads to activation of antiviral pathways like NFkB and JAK-STAT. Based on the similarity-and not inverse similarity-between drug and infection-induced gene expression signatures, we were able to predict the in vitro antiviral activity of drugs. We also identified SREBF1/2, key regulators of lipid metabolising enzymes, as the most activated transcription factors by several in vitro effective antiviral drugs. Using a fluorescently labeled cholesterol sensor, we showed that these drugs decrease the cholesterol levels of plasma-membrane. Supplementing drug-treated cells with cholesterol reversed the in vitro antiviral effect, suggesting the depleting plasma-membrane cholesterol plays a key role in virus inhibitory mechanism. Our results can help to more effectively repurpose approved drugs against SARS-CoV-2, and also highlights key mechanisms behind their antiviral effect.

Inactivation of Influenza Viruses with Heteropolyacids

The antiviral activity of Keggin heteropolyacids (HPAs) against the human influenza A/California/07/09 (H1N1) pdm09 virus is studied in MDCK cell culture. Transmission electron microscopy reveals new features of the destruction of viral particles by HPAs. A proton-anion model that considers the main biochemical processes leading to the destruction of enveloped viruses: the depletion of membrane cholesterol by HPA anions, acidification of the M1 matrix protein by protons of the medium, and anion lysis of lipid membranes, is proposed. The Arrhenius dependences of the biological properties of HPAs on temperature are predicted. The general mechanism for the formation of high biological activity HPAs is described, which is underlined by the anion depletion of cholesterol in bilipid membranes, a new effect in the biochemistry of polyoxometalates.