Lipid Rafts Research Articles

Raman spectroscopy and mass spectrometry identifies a unique group of epidermal lipids in active discoid lupus erythematosus

Discoid lupus erythematosus (DLE) is the most common form of cutaneous lupus1. It can cause permanent scarring. The pathophysiology of is not fully understood. Plasmacytoid dendritic cells are found in close association with apoptotic keratinocytes inferring close cellular signalling. Matrix Associated Laser Desorption Ionisation (MALDI) combined with Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) is an exquisitely sensitive combination to examine disease processes at the cellular and molecular level. Active areas of discoid lupus erythematosus were compared with normal perilesional skin using MALDI combined with FT-ICR-MS. A unique set of biomarkers, including epidermal lipids is identified in active discoid lupus. These were assigned as sphingomyelins, phospholipids and ceramides. Additionally, increased levels of proteins from the keratin, and small proline rich family, and aromatic amino acids (tryptophan, phenylalanine, and tyrosine) in the epidermis are observed. These techniques, applied to punch biopsies of the skin, have shown a distinctive lipid profile of active discoid lupus. This profile may indicate specific lipid signalling pathways. Lipid rich microdomains (known as lipid rafts) are involved in cell signalling and lipid abnormalities have been described with systemic lupus erythematosus which correlate with disease activity.

Multifunctional Proximity Labeling Strategy for Lipid Raft-Specific Sialic Acid Tracking and Engineering.

Lipid raft-specific glycosylation has been implicated in many biological processes, including intracellular trafficking, cell adhesion, signal transduction, and host-pathogen interactions. The major predicament in lipid raft-specific glycosylation research is the unavailability of tools for tracking and manipulating glycans on lipid rafts at the microstructural level. To overcome this challenge, we developed a multifunctional proximity labeling (MPL) platform that relies on cholera toxin B subunit to localize horseradish peroxidase on lipid rafts. In addition to the prevailing electron-rich amino acids, modified sialic acid was included in the horseradish peroxidase-mediated proximity labeling substrate via purposefully designed chemical transformation reactions. In combination with sialic acid editing, the self-renewal of lipid raft-specific sialic acid was visualized. The MPL method enabled tracking of lipid raft dynamics under methyl-β-cyclodextrin and mevinolin treatments; in particular, the alteration of lipid rafts markedly affected cell migration. Furthermore, we embedded functional molecules into the method and implemented raft-specific sialic acid gradient engineering. Our novel strategy presents opportunities for tailoring lipid raft-specific sialic acids, thereby regulating interactions associated with lipid raft regions (such as cell-virus and cell-microenvironment interactions), and can aid in the development of lipid raft-based therapeutic regimens for tumors.

The effect of trans-resveratrol on the physicochemical properties of lipid membranes with different cholesterol content

Resveratrol is one of the most popular phytoalexins, which naturally occurs in grapes and red wine. This compound not only has beneficial effects on the human body, especially on the cardiovascular system, but also has antiviral, antibacterial and antifungal properties. In addition, resveratrol may have therapeutic effects against various types of cancer. The mechanism of action of resveratrol is not fully understood, but it is suspected that one of the most important steps is its interaction with the cell membrane and changing its molecular organization. Therefore, in the present study, we investigated the effects of resveratrol at different concentrations (0–75 μM) on model membranes composed of POPC, SM and cholesterol, in systems with different cholesterol contents and a constant POPC/SM molar ratio (1:1). Our tests included systems containing 5, 15 and 33.3 mol% cholesterol. Tests were carried out for monolayers using the Langmuir monolayer technique supported by Brewster angle microscopy and penetration experiments. Bilayer (liposome) experiments included calcein release, steady-state DPH fluorescence anisotropy and partition coefficients. The results showed that resveratrol interacts with model cell membranes (lipid monolayers and lipid bilayers), and its incorporation into membranes is accompanied by changes in their physicochemical parameters, such as lipid packing, fluidity and permeability. Furthermore, we showed that the cholesterol content of the membrane significantly affects the degree of incorporation of resveratrol into the model membrane, which may indicate that the molecular mechanism of action of this compound is closely related to its interactions with lipid rafts, domains responsible for regulating various cellular functions.

GhSMO2-2 is regulated by brassinosteroid signal and involved in cotton fiber elongation via influencing phytosterol and sphingolipid biosynthesis

Cotton fiber cell is an extremely elongated single cell and is regarded as an ideal material to study the growth and development of plant cell. Phytohormone brassinosteroids (BRs) play crucial roles in fiber cell development. However, its action mechanism in fiber growth is largely unknown. In this study, we identified a homologue of Arabidopsis SMO2–2 (Sterol C-4α Methyl Oxidase), GhSMO2–2, in upland cotton (Gossypium hirsutum L.), which predominantly expressed in fiber cell and peaked at the rapid elongation stage, and responded to BR signal. The GhBES1, a transcription factor in BR signaling could directly bind to the promoter of GhSMO2–2 and promoted its expression. Overexpression of GhSMO2–2 significantly promoted fiber cell elongation and phytosterol synthesis in cotton, and elevated some sphingolipid species that was important for fiber elongation. On the contrary, down-regulating GhSMO2–2 suppressed fiber elongation and blocked these chemicals accumulation. These results indicated that GhSMO2–2 is an important gene involved in fiber cell elongation, and is valuable for genetic engineering to improve fiber quality and yield. Taken together, our study revealed that BR regulated fiber cell elongation by influencing phytosterol and sphingolipid biosynthesis, the key components of membrane lipid raft and provided a new clue for further revealing the function and mechanism of phytosterols, sphingolipids, and BRs in plant cell development.

The Tetraspanin Tspan8 Associates with Endothelin Converting Enzyme ECE1 and Regulates Its Activity.

Tspan8 is a member of the tetraspanins family of cell surface molecules. The ability of tetraspanins to organize membrane microdomains with other membrane molecules and interfere with their function suggests that they could act as surface integrators of external or internal signals. Among the first identified tetraspanins, Tspan8 promotes tumor progression and metastasis, presumably by stimulating angiogenesis and cell motility. In patients, its expression on digestive tract tumors seems to be associated with a bad prognosis. We showed previously that Tspan8 associates with E-cadherin and EGFR and modulates their effects on cell motility. Using Mass spectrometry and western blot, we found a new partner, the endothelin converting enzyme ECE1, and showed that Tspan8 amplifies its activity of conversion of the endothelin-1 precursor bigET1 to endothelin. This was observed by transduction of the colon carcinoma cell line Isreco1, which does not express Tspan8, and on ileum tissue fragments of tspan8ko mice versus wild type mice. Given these results, Tspan8 appears to be a modulator of the endothelin axis, which could possibly be targeted in case of over-activity of endothelins in biological processes of tissues expressing Tspan8.

CELL DEATH: A MODERN VIEW OF APOPTOSIS AND AUTOPHAGY

The purpose of the work – to analyze the literature data regarding the current state ofresearch on cell death, the relationship between the signaling pathways of apoptosis andautophagy in various pathologies, as well as the key mediators of both processes.Conclusions. Data from the literature indicate the existence of a clear relationshipbetween signaling pathways of apoptosis and autophagy in the conditions of variouspathological processes, which is carried out through key mediators, in particular, calcium(Ca2+), lipid rafts, ERLIN, PINK1, etc. Cell death is a promising therapeutic target forthe treatment of human diseases.

Understanding the free-energy landscape of phase separation in lipid bilayers using molecular dynamics

Liquid-liquid phase separation inside the cell often results in biological condensates that can critically affect cell homeostasis. Such phase separation events occur in multiple parts of cells, including the cell membranes, where the “lipid raft” hypothesis posits the formation of ordered domains floating in a sea of disordered lipids. The resulting lipid domains often have functional roles. However, the thermodynamics of lipid phase separation and their resulting mechanistic effects on cell function and dysfunction are poorly understood. Understanding such complex phenomena in cell membranes, with their diverse lipid compositions, is exceptionally difficult. For these reasons, simple model systems that can recapitulate similar behavior are widely used to study this phenomenon. Despite these simplifications, the timescale and length scales of domain formation pose a challenge for molecular dynamics (MD) simulations. Thus, most MD studies focus on spontaneous lipid phase separation—essentially measuring the sign (but not the amplitude) of the free-energy change upon separation—rather than directly interrogating the thermodynamics. Here, we propose a proof-of-concept pipeline that can directly measure this free energy by combining coarse-grained MD with enhanced sampling protocols using a novel collective variable. This approach will be a useful tool to help connect the thermodynamics of phase separation with the mechanistic insights already available from MD simulations.

Role of heat-shock proteins in infection of human adenocarcinoma cell line MCF-7 by tumor-adapted rotavirus isolates


 Background:
 Viruses are being used as alternative and complementary tools for treating cancers. Oncolytic viruses exhibit tumor tropism, ability to enhance anti-tumor immunity and ability to be used in combination with conventional chemotherapy and radiotherapy. We have recently selected some rotavirus isolates which are adapted to efficiently infect and kill tumor cell lines.
 
 
 Aim:
 We tested five tumor cell-adapted rotavirus isolates for their ability to infect the human adenocarcinoma cell line MCF-7.
 
 
 Methods:
 Cell surface membrane-associated proteins mediating virus particle attachment were characterized using ELISA, immunoprecipitation, FACS analysis, and antibody blocking.
 
 
 Results:
 It was found that heat shock proteins (HSPs) such as Hsp90, Hsp70, Hsp60, and Hsp40 are expressed on the cell surface forming complexes with protein disulfide isomerase (PDI), integrin β3, and heat shock cognate protein 70 (Hsc70) in lipid raft microdomains. Interaction of rotavirus isolates with these cellular proteins was further confirmed by a competition assay and an inhibition assay involving the HSPs tested.
 
 
 Conclusion:
 Our findings suggest that the tumor cell-adapted rotavirus isolates studied here offer a promising tool for killing tumor cells, thus encouraging further research into this topic, including animal models.

Let-7g Upregulation Attenuated the KRAS-PI3K-Rac1-Akt Axis-Mediated Bioenergetic Functions.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K-Rac1-Akt-BCL-2/BCL-xL-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K-Rac1-Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS.

Engineered CD4 T cells expressing a membrane anchored viral inhibitor restrict HIV-1 through cis and trans mechanisms.

HIV-1 infection of target cells can occur through either cell-free virions or cell-cell transmission in a virological synapse, with the latter mechanism of infection reported to be 100- to 1,000-fold more efficient. Neutralizing antibodies and entry inhibitors effectively block cell-free HIV-1, but with few exceptions, they display much less inhibitory activity against cell-mediated HIV-1 transmission. Previously, we showed that engineering HIV-1 target cells by genetically linking single-chain variable fragments (scFvs) of antibodies to glycosyl phosphatidylinositol (GPI) potently blocks infection by cell-free virions and cell-mediated infection by immature dendritic cell (iDC)-captured HIV-1. Expression of scFvs on CD4+ cell lines by transduction with X5 derived anti-HIV-1 Env antibody linked to a GPI attachment signal directs GPI-anchored scFvs into lipid rafts of the plasma membrane. In this study, we further characterize the effect of GPI-scFv X5 on cell-cell HIV-1 transmission from DCs to target cells. We report that expression of GPI-scFv X5 in transduced CD4+ cell lines and human primary CD4+ T cells potently restricts viral replication in iDC- or mDC-captured HIV-1 in trans. Using live-cell imaging, we observed that when GPI-GFP or GPI-scFv X5 transduced T cells are co-cultured with iDCs, GPI-anchored proteins enrich in contact zones and subsequently migrate from T cells into DCs, suggesting that transferred GPI-scFv X5 interferes with HIV-1 infection of iDCs. We conclude that GPI-scFv X5 on the surface of transduced CD4+ T cells not only potently blocks cell-mediated infection by DCs, but it transfers from transduced cells to the surface of iDCs and neutralizes HIV-1 replication in iDCs. Our findings have important implications for HIV-1 antibody-based immunotherapies as they demonstrate a viral inhibitory effect that extends beyond the transduced CD4+ T cells to iDCs which can enhance HIV-1 replication.

Rafting on the Evidence for Lipid Raft-like Domains as Hubs Triggering Environmental Toxicants' Cellular Effects.

The plasma membrane lipid rafts are cholesterol- and sphingolipid-enriched domains that allow regularly distributed, sub-micro-sized structures englobing proteins to compartmentalize cellular processes. These membrane domains can be highly heterogeneous and dynamic, functioning as signal transduction platforms that amplify the local concentrations and signaling of individual components. Moreover, they participate in cell signaling routes that are known to be important targets of environmental toxicants affecting cell redox status and calcium homeostasis, immune regulation, and hormonal functions. In this work, the evidence that plasma membrane raft-like domains operate as hubs for toxicants' cellular actions is discussed, and suggestions for future research are provided. Several studies address the insertion of pesticides and other organic pollutants into membranes, their accumulation in lipid rafts, or lipid rafts' disruption by polychlorinated biphenyls (PCBs), benzo[a]pyrene (B[a]P), and even metals/metalloids. In hepatocytes, macrophages, or neurons, B[a]P, airborne particulate matter, and other toxicants caused rafts' protein and lipid remodeling, oxidative changes, or amyloidogenesis. Different studies investigated the role of the invaginated lipid rafts present in endothelial cells in mediating the vascular inflammatory effects of PCBs. Furthermore, in vitro and in vivo data strongly implicate raft-localized NADPH oxidases, the aryl hydrocarbon receptor, caveolin-1, and protein kinases in the toxic mechanisms of occupational and environmental chemicals.

Analysis of acid-tolerance mechanism based on membrane microdomains in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae has been used in the biosynthesis of acid products such as organic acids owing to its acid tolerance. Improving the acid tolerance of S. cerevisiae is beneficial for expanding its application range. Our previous study isolated the TAMC strain that was tolerant to a pH 2.3 through adaptive laboratory evolution; however, its mechanism underlying tolerance to low pH environment remains unclear.ResultsIn this study, through visual observation and order analysis of plasma membrane and membrane microdomains, we revealed that the membrane microdomains of TAMC strain play an indispensable role in acid tolerance. Transcriptomic analysis showed an increase in the expression of genes related to key components of membrane microdomains in TAMC strain. Furthermore, an obvious reduction was observed in the acid tolerance of the strain with sterol C-24 methyltransferase encoding gene ERG6 knockout for inhibiting membrane microdomain formation. Finally, colocalization analysis of H+-ATPase PMA1 and plasma membrane protein PMP1 showed that disruption of membrane microdomains could inhibit the formation of the H+-ATPase complex.ConclusionsMembrane microdomains could provide a platform for forming H+-ATPase complexes to facilitate intracellular H+ homeostasis, and thereby improve cell acid resistance. This study proposed a novel acid tolerance mechanism, providing a new direction for the rational engineering of acid-tolerant strains.

Ganglioside GD3 regulates neural stem cell quiescence and controls postnatal neurogenesis.

The postnatal neural stem cell (NSC) pool hosts quiescent and activated radial glia-like NSCs contributing to neurogenesis throughout adulthood. However, the underlying regulatory mechanism during the transition from quiescent NSCs to activated NSCs in the postnatal NSC niche is not fully understood. Lipid metabolism and lipid composition play important roles in regulating NSC fate determination. Biological lipid membranes define the individual cellular shape and help maintain cellular organization and are highly heterogeneous in structure and there exist diverse microdomains (also known as lipid rafts), which are enriched with sugar molecules, such as glycosphingolipids. An often overlooked but key aspect is that the functional activities of proteins and genes are highly dependent on their molecular environments. We previously reported that ganglioside GD3 is the predominant species in NSCs and that the reduced postnatal NSC pools are observed in global GD3-synthase knockout (GD3S-KO) mouse brains. The specific roles of GD3 in determining the stage and cell-lineage determination of NSCs remain unclear, since global GD3S-KO mice cannot distinguish if GD3 regulates postnatal neurogenesis or developmental impacts. Here, we show that inducible GD3 deletion in postnatal radial glia-like NSCs promotes NSC activation, resulting in the loss of the long-term maintenance of the adult NSC pools. The reduced neurogenesis in the subventricular zone (SVZ) and the dentate gyrus (DG) of GD3S-conditional-knockout mice led to the impaired olfactory and memory functions. Thus, our results provide convincing evidence that postnatal GD3 maintains the quiescent state of radial glia-like NSCs in the adult NSC niche.

Exploring the Impact of Lipid Domain Size on the Lifetime: A Dissipative Particle Dynamics Study

In this research, we have used the dissipative particle dynamics (DPD), a mesoscopic simulation technique, in order to investigate the dynamics of lipid domains in near critical temperature. Our specific focus has been on exploring the influence of lipid domain size on its lifetime, which mimics the behavior of lipid rafts within cellular membranes. The lipid membranes used in this study were composed of saturated and unsaturated lipids, which have been immersed in water. Through the simulation of these membranes close to their critical temperature, we have successfully generated fluctuating domains that mimic the lipid rafts observed in cellular systems. We have proposed a method to obtain the lifetime of the fluctuating domains by analyzing the sizes of the lipid domains at specific intervals of time. Our investigations have revealed a linear correlation between the initial size of the lipid domain and its lifetime. Our research finding give an insight into the underlying mechanisms that govern lipid rafts and their vital role in various cellular processes.

Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains

Signal peptide peptidase-like 2a and b (SPPL2a/b) are aspartyl intramembrane proteases and cleave tail-anchored proteins as well as N-terminal fragments (NTFs) derived from type II-oriented transmembrane proteins. How these proteases recruit substrates and cleavage is regulated, is still incompletely understood. We found that SPPL2a/b localize to detergent-resistant membrane (DRM) domains with the characteristics of tetraspanin-enriched microdomains (TEMs). Based on this, association with several tetraspanins was evaluated. We demonstrate that not only SPPL2a/b but also their substrates tumor necrosis factor (TNF) and CD74 associate with tetraspanins like CD9, CD81, and CD82 and/or TEMs and analyze the stability of these complexes in different detergents. CD9 and CD81 deficiency has protease- and substrate-selective effects on SPPL2a/b function. Our findings suggest that reciprocal interactions with tetraspanins may assist protease-substrate encounters of SPPL2a/b within the membrane. Beyond SPP/SPPL proteases, this supports previous concepts that tetraspanins facilitate membrane-embedded proteolytic processes.

Non-Lethal Doses of RSL3 Impair Microvascular Endothelial Barrier through Degradation of Sphingosie-1-Phosphate Receptor 1 and Cytoskeletal Arrangement in A Ferroptosis-Independent Manner.

The excess microvascular endothelial permeability is a hallmark of acute inflammatory diseases. Maintenance of microvascular integrity is critical to preventing leakage of vascular components into the surrounding tissues. Sphingosine-1-phosphate (S1P) is an active lysophospholipid that enhances the endothelial cell (EC) barrier via activation of its receptor S1PR1. Here, we delineate the effect of non-lethal doses of RSL3, an inhibitor of glutathione peroxidase 4 (GPX4), on EC barrier function. Low doses of RSL3 (50-100 nM) attenuated S1P-induced human lung microvascular barrier enhancement and the phosphorylation of AKT. To investigate the molecular mechanisms by which RSL3 attenuates S1P's effect, we examined the S1PR1 levels. RSL3 treatment reduced S1PR1 levels in 1 h, whereas the effect was attenuated by the proteasome and lysosome inhibitors as well as a lipid raft inhibitor. Immunofluorescence staining showed that RSL3 induced S1PR1 internalization from the plasma membrane into the cytoplasm. Furthermore, we found that RSL3 (100 and 200 nM) increased EC barrier permeability and cytoskeletal rearrangement without altering cell viability. Taken together, our data delineates that non-lethal doses of RSL3 impair EC barrier function via two mechanisms. RSL3 attenuates S1P1-induced EC barrier enhancement and disrupts EC barrier integrity through the generation of 4-hydroxynonena (4HNE). All these effects are independent of ferroptosis.

Effect of NaCl on the Lipid Profile of Detergent-Resistant membranes of the Chloroplasts and Mitochondria of Euhalophyte Plants

The effect of 1 M NaCl on the lipid profile of detergent-resistant chloroplast membranes was studied. sts and mitochondria of salt-accumulating halophyte plants – Salicornia perennans Willd. And Suaeda salsa (L.) Pall. (family Amaranthaceae). The composition of lipids and fatty acids in detergent-resistant membranes differs from the membrane lipids of chloroplasts and mitochondria by a large number of cerebrosides and sterols. Under the given salinity conditions, S. perennans experienced many a fold increase in the proportion of cerebrosides in the composition of detergent-resistant chloroplast membranes, and in S. salsa is found in detergent-resistant mitochondrial membranes. The opposite effect was observed in relation to sterols: a decrease in their relative content under the action of salt. Received data indicate the participation of detergent-resistant membranes in the interaction of chloroplast cells and mitochondria in the cellular response of halophytes to salinity.

Extraction and Analysis of Lipid Raft Proteins with Detergent-and Non detergent-based Method

Introduction: Lipid raft is found on the cell membrane and is considered a microstructure rich in cholesterol, phospholipids and target proteins that are insoluble in nonionic detergents at low temperatures. Methods: In this study, detergent and non-detergent methods were used to extract lipid rafts from different cells. With β-cyclodextrin as the negative control group, we analyzed and compared the effects of different extraction methods on the composition of lipid rafts in Caco-2 and U251 cells using three kinds of lysate, namely detergent method 1, detergent method 2 and non-detergent method, which could be extracted and collected via sucrose density gradient centrifugation. Western blotting and immunofluorescence were utilized to determine the location of lipid rafts via the proteins Caveolin-1 and Flotillin-1, which are the characteristic proteins P-gp and TrkA in cells. The total protein in the lipid raft was quantitatively determined through the BCA (detergent compatible) kit method. Results: The results showed that the total amount of lipid raft proteins extracted via the detergent method was more than that of the non-detergent method, while the content of β-cyclodextrin control histone that caused disruption of lipid rafts structure was the lowest. Conclusion: The detergent method extracted more abundant lipid rafts than the non-detergent method. Detergent method 2 did not only extract more fat raft layers, but also the extracted highest total protein content, wherein it demonstrated better extraction effect with more lipid raft layers and higher expression of target protein P-gp.

Abstract P401: Caveolin-1 And Flotillin-1 Facilitates The SNX19-mediated D 1 R Endocytosis

Sorting nexin 19 (SNX19) belongs to the SNX-PXA-RGS-PXC subfamily of sorting nexins. It controls blood pressure by regulating the endocytosis of dopamine D1 receptor (D 1 R) in renal proximal tubule cells (RPTCs), allowing it to reside in the lipid rafts (LRs) of the plasma membrane. The exclusion of the D 1 R from LRs in RPTCs is associated with hypertension. However, it is unknown if SNX19 interacts with caveolin-1 and flotillin-1, two of the most important LR components. Thus, we studied if SNX19 associates with these two proteins and whether this interaction impacts D 1 R endocytosis. In RPTCs, SNX19 co-immunoprecipitated with caveolin-1 and flotillin-1 in basal conditions. Fenoldopam (FEN, 25nM, 30 min, n=3), a selective D 1 R/D 5 R agonist, increased the co-immunoprecipitation of SNX19 with both caveolin-1 and flotillin-1. The treatment with a D 1 R/D 5 R antagonist, SCH 39166 (SCH, 1 μM, 30 min), prevented the FEN-mediated increase in the co-immunoprecipitation of SNX19 with caveolin-1 and flotillin-1. In RPTCs, the transfection of SNX19 plasmid with deletion of either caveolin-1 (ΔCav1-SNX19) or flotilin-1(ΔFlot1-SNX19) binding domain impaired the FEN-mediated D 1 R endocytosis compared with the cells that were only transfected with wild-type (WT) SNX19 (WT-SNX19, 100.0±5.0%; ΔCav1-SNX19, 39.1±2.8%; ΔFlot1-SNX19, 59.6±3.4%, n=5). Moreover, the relative mRNA expression of SNX19 did not change in RPTCs transfected with WT-SNX19, ΔCav1-SNX19, or ΔFlot1-SNX19. Thus, SNX19 expression does not depend on its caveolin-1 or flotillin-1 binding motif. In C57Bl/6 mice fed with normal salt diet (0.9% NaCl), the renal subcapsular infusion of Snx19 siRNA (3 μg/day,7 days) significantly increased systolic blood pressure (SBP, mmHg) (mock siRNA: 99.3±1.5, n=8; Snx19 siRNA: 114.1±5.4, n=8). The SBP also increased in mice fed with high salt and aggravated by Snx19 siRNA (4% NaCl; mock siRNA: 119.8 ± 5.6, n=4; Snx19 siRNA: 125.6 ±3.4, n=5). Low salt diet decreased SBP that was not affected by Snx19 siRNA (0.04% NaCl, mock siRNA: 92. 0 ± 3.5, n=3; Snx19 siRNA: 82.7 ±6.7, n=3). Germline deletion of Snx19 in C57Bl/6 mice fed with normal salt diet increased SBP. In conclusion, caveolin-1 and flotillin-1 are important for SNX19-mediated D 1 R endocytosis and blood pressure.

Host Membranes as Drivers of Virus Evolution.

The molecular mechanisms controlling the adaptation of viruses to host cells are generally poorly documented. An essential issue to resolve is whether host membranes, and especially lipid rafts, which are usually considered passive gateways for many enveloped viruses, also encode informational guidelines that could determine virus evolution. Due to their enrichment in gangliosides which confer an electronegative surface potential, lipid rafts impose a first control level favoring the selection of viruses with enhanced cationic areas, as illustrated by SARS-CoV-2 variants. Ganglioside clusters attract viral particles in a dynamic electrostatic funnel, the more cationic viruses of a viral population winning the race. However, electrostatic forces account for only a small part of the energy of raft-virus interaction, which depends mainly on the ability of viruses to form a network of hydrogen bonds with raft gangliosides. This fine tuning of virus-ganglioside interactions, which is essential to stabilize the virus on the host membrane, generates a second level of selection pressure driven by a typical induced-fit mechanism. Gangliosides play an active role in this process, wrapping around the virus spikes through a dynamic quicksand-like mechanism. Viruses are thus in an endless race for access to lipid rafts, and they are bound to evolve perpetually, combining speed (electrostatic potential) and precision (fine tuning of amino acids) under the selective pressure of the immune system. Deciphering the host membrane guidelines controlling virus evolution mechanisms may open new avenues for the design of innovative antivirals.