Lipid Rafts Research Articles

Omega-3 Fatty Acids and Arrhythmias.

The pro- and antiarrhythmic effects of omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been extensively studied in preclinical and human trials. Despite early evidence of an antiarrhythmic role of n-3 PUFA in the prevention of sudden cardiac death and postoperative and persistent atrial fibrillation (AF), subsequent well-designed randomized trials have largely not shown an antiarrhythmic benefit. Two trials that tested moderate and high-dose n-3 PUFA demonstrated a reduction in sudden cardiac death, but these findings have not been widely replicated, and the potential of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to reduce arrhythmic death in combination, or as monotherapy, remains uncertain. The accumulated clinical evidence does not support supplementation of n-3 PUFA for postoperative AF or secondary prevention of AF. Several large, contemporary, randomized controlled trials of high-dose n-3 PUFA for primary or secondary cardiovascular prevention have demonstrated a small, significant, dose-dependent increased risk of incident AF compared with mineral oil or corn oil comparator. These findings were reproduced with both icosapent ethyl monotherapy and a mixed EPA+DHA formulation. The proarrhythmic mechanism of increased AF in contemporary cohorts exposed to high-dose n-3 PUFA is unknown. EPA and DHA and their metabolites have pleiotropic cardiometabolic and pro- and antiarrhythmic effects, including modification of the lipid raft microenvironment; alteration of cell membrane structure and fluidity; modulation of sodium, potassium, and calcium currents; and regulation of gene transcription, cell proliferation, and inflammation. Further characterization of the complex association between EPA, EPA+DHA, and DHA and AF is needed. Which formulations, dose ranges, and patient subgroups are at highest risk, remain unclear.

Inhibition of RHOA activity preserves the survival and hemostasis function of long-term cold-stored platelets

AbstractPatients with thrombocytopenia require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions, including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis and genetic and pharmacological approaches, we identified that cold-induced activation of RAS homolog family, member A (RHOA) GTPase causes the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage–induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage–induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GPIbα. RHOA inhibition further prevents the metabolic reprogramming of cold storage–induced lesions and allows the maintenance of glycolytic flux and mitochondria-dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room temperature–stored platelets while retaining their hemostatic activity in vivo, as assessed by bleeding time correction in aspirin-treated mice. Our studies provide a mechanism-based translational approach to prevent cold storage–induced damage, which is useful for human platelet transfusion in patients with thrombocytopenia.

Polar Glycerolipids and Membrane Lipid Rafts.

Current understanding of the structure and functioning of biomembranes is impossible without determining the mechanism of formation of membrane lipid rafts. The formation of liquid-ordered and disordered phases (Lo and Ld) and lipid rafts in membranes and their simplified models is discussed. A new consideration of the processes of formation of lipid phases Lo and Ld and lipid rafts is proposed, taking into account the division of each of the glycerophospholipids into several groups. Generally accepted three-component schemes for modeling the membrane structure are critically considered. A four-component scheme is proposed, which is designed to more accurately assume the composition of lipids in the resulting Lo and Ld phases. The role of the polar head groups of phospholipids and, in particular, phosphatidylethanolamine is considered. The structure of membrane rafts and the possible absence of a clear boundary between the Lo and Ld phases are discussed.

How Membrane Phospholipids Containing Long-Chain Polyunsaturated Fatty Acids and Their Oxidation Products Orchestrate Lipid Raft Dynamics to Control Inflammation

BackgroundLong-chain PUFA (LC-PUFA) influence varying aspects of inflammation. One mechanism by which they regulate inflammation is by controlling the size and molecular composition of lipid rafts. Lipid rafts are sphingolipid/cholesterol-enriched plasma membrane microdomains that compartmentalize signaling proteins and thereby control downstream inflammatory gene expression and cytokine production. ObjectivesThis review summarizes developments in our understanding of how LC-PUFA acyl chains of phospholipids, in addition to oxidized derivatives of LC-PUFAs such as oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC), manipulate formation of lipid rafts and thereby inflammation. MethodsWe reviewed the literature, largely from the past 2 decades, on the impact of LC-PUFA acyl chains and oxidized products of LC-PUFAs on lipid raft biophysical organization of myeloid and lymphoid cells. The majority of the studies are based on rodent or cellular experiments with supporting mechanistic studies using biomimetic membranes and molecular dynamic simulations. These studies have focused largely on the LC-PUFA docosahexaenoic acid, with some studies addressing eicosapentaenoic acid. A few studies have investigated the role of oxidized phospholipids on rafts. ResultsThe biophysical literature suggests a model in which n-3 LC-PUFAs, in addition to oxPAPC, localize predominately to nonraft regions and impart a disordering effect in this environment. Rafts become larger because of the ensuing increase in the difference in order between raft and nonrafts. Biochemical studies suggest that some n-3 LC-PUFAs can be found within rafts. This deviation from homeostasis is a potential trigger for controlling aspects of innate and adaptive immunity. ConclusionOverall, select LC-PUFA acyl chains and oxidized acyl chains of phospholipids control lipid raft dynamics and downstream inflammation. Gaps in knowledge remain, particularly on underlying molecular mechanisms by which plasma membrane receptor organization is controlled in response to oxidized LC-PUFA acyl chains of membrane phospholipids. Validation in humans is also an area for future study.

The Role of Alpha Linolenic Acid on Neuroinflammation: A Systemic Review

Neuroinflammation, an inflammatory response within the central nervous system (CNS), plays a critical role in various neurological disorders, including neurodegenerative diseases. Alpha linolenic acid (ALA), an essential omega-3 polyunsaturated fatty acid, has shown promising health benefits due to its antioxidant and anti-inflammatory properties. This article aims to thoroughly examine the potential effects of ALA in reducing neuroinflammation, emphasizing its roles as an anti-inflammatory and antioxidant agent. The review focuses on studies published between 2018 and 2023, sourced from reputable academic databases such as PubMed, Semantic Scholar, Google Scholar, and ScienceDirect, with nine key papers selected. The findings indicate that ALA significantly mitigates neuroinflammation by decreasing reactive oxygen species (ROS) via the Nrf-2/HO-1/JNK signaling pathway. Additionally, ALA reduces inflammatory cytokines by inhibiting p-JNK activation, disrupting lipid rafts, blocking the pro-inflammatory transcription factor NF-κB, and altering cell membrane phospholipid composition. The review concludes that ALA may have potential therapeutic effects in reducing neuroinflammation through its antioxidant and anti-inflammatory actions, offering possible benefits for various neurological conditions, including Alzheimer's and Parkinson's diseases.

Endothelial TRPV4/Cx43 Signaling Complex Regulates Vasomotor Tone in Resistance Arteries.

S-nitrosylation of Cx43 gap junction channels critically regulates communication between smooth muscle cells and endothelial cells. This posttranslational modification also induces the opening of undocked Cx43 hemichannels. However, its specific impact on vasomotor regulation remains unclear. Considering the role of endothelial TRPV4 channel activation in promoting vasodilation through nitric oxide (NO) production, we investigated the direct modulation of endothelial Cx43 hemichannels by TRPV4 channel activation. Using the proximity ligation assay, we identify that Cx43 and TRPV4 are found in close proximity in the endothelium of resistance arteries. In primary endothelial cell cultures from resistance arteries (ECs), GSK-induced TRPV4 activation enhances eNOS activity, increases NO production, and opens Cx43 hemichannels via direct S-nitrosylation. Notably, the elevated intracellular Ca2+ levels caused by TRPV4 activation were reduced by blocking Cx43 hemichannels. In ex vivo mesenteric arteries, inhibiting Cx43 hemichannels reduced endothelial hyperpolarization without affecting NO production in ECs, underscoring a critical role of TRPV4/Cx43 signaling in endothelial electrical behavior. We perturbed the proximity of Cx43/TRPV4 by disrupting lipid rafts in ECs using β-cyclodextrin. Under these conditions, hemichannel activity, Ca2+ influx, and endothelial hyperpolarization were blunted upon GSK stimulation. Intravital microscopy of mesenteric arterioles in vivo further demonstrated that inhibiting Cx43 hemichannels activity, NO production and disrupting endothelial integrity reduce TRPV4-induced relaxation. These findings underscore a new pivotal role of Cx43 hemichannel associated with TRPV4 signaling pathway in modulating endothelial electrical behavior and vasomotor tone regulation.

Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins1–3. The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour3,4. The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors5–9. Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

M6A-Mediated Induction of 7-Dehydrocholesterol Reductase Stimulates Cholesterol Synthesis and cAMP Signaling to Promote Bladder Cancer Metastasis.

Dysregulation of cholesterol homeostasis occurs in multiple types of tumors and promotes cancer progression. Investigating the specific processes that induce abnormal cholesterol metabolism could identify therapeutic targets to improve cancer treatment. In this investigation, we observed upregulation of 7-dehydrocholesterol reductase (DHCR7), a vital enzyme involved in the synthesis of cholesterol, within bladder cancer tissues in comparison to normal tissues, which was correlated with increased bladder cancer metastasis. Increased expression of DHCR7 in bladder cancer was attributed to decreased mRNA degradation mediated by YTHDF2. Loss or inhibition of DHCR7 reduced bladder cancer cell invasion in vitro and metastasis in vivo. Mechanistically, DHCR7 promoted bladder cancer metastasis by activating the cAMP/protein kinase A/FAK pathway. Specifically, DHCR7 increased cAMP levels by elevating cholesterol content in lipid rafts, thereby facilitating the transduction of signaling pathways mediated by cAMP receptors. DHCR7 additionally enhanced the cAMP signaling pathway by reducing the concentration of 7-dehydrocholesterol and promoting the transcription of the G protein-coupled receptor, namely gastric inhibitory polypeptide receptor. Overall, these findings demonstrate that DHCR7 plays an important role in bladder cancer invasion and metastasis by modulating cholesterol synthesis and cAMP signaling. Furthermore, inhibition of DHCR7 shows promise as a viable therapeutic strategy for suppressing bladder cancer invasion and metastasis. Significance: Inhibiting DHCR7 induces cholesterol metabolism reprogramming and lipid raft remodeling to inactivate the cAMP/protein kinase A/FAK axis and suppress bladder cancer metastasis, indicating the therapeutic potential of targeting DHCR7.

Molecular biological role of epithelial splicing regulatory protein1 in intrahepatic cholangiocarcinoma.

Epithelial splicing regulatory protein1 (ESRP1) regulates tumor progression and metastasis through the epithelial‒mesenchymal transition by interacting with zinc finger E-box binding1 (ZEB1) and CD44 in cancers. However, the role of ESRP1 in intrahepatic cholangiocarcinoma (iCCA) remains unclear. Three iCCA cell lines (HuCCT-1, SSP-25, and KKU-100) were analyzed using small interfering RNA to investigate the molecular biological functions of ESRP1 and ZEB1. The association between clinicopathological features and the expression of ESRP1 and ZEB1 in iCCA tissues was analyzed immunohistochemically. Proteomic analysis was performed to identify molecules related to ESRP1 expression. ESRP1 expression was upregulated in HuCCT-1 and SSP-25 cells. Cell migration and invasion were enhanced, and the expression of ZEB1 and CD44s (CD44 standard) isoforms were upregulated in the ESRP1 silencing cells. Moreover, ESRP1 silencing increased the expression of N-cadherin and vimentin, indicating the presence of mesenchymal properties. Conversely, ZEB1 silencing increased the expression of ESRP1 and CD44v (CD44 variant) isoforms. Immunohistochemical analysis revealed that a lower ESRP1-to-ZEB1 expression ratio was associated with poor recurrence-free survival in patients with iCCA. Flotillin 2, a lipid raft marker related to epithelial‒mesenchymal transition, was identified as a protein related to the interactive feedback loop in proteomic analysis. ESRP1 suppresses tumor progression in iCCA by interacting with ZEB1 and CD44 to regulate epithelial‒mesenchymal transition.

Role of palmitoylation on the neuronal glycine transporter GlyT2.

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.

PTGS2 as target of compound Huangbai liquid in the nursing of pressure ulcer.

Pressure ulcer refers to ulceration and necrosis caused by local skin and cell tissues being compressed for a long time, continuous ischemia, hypoxia, and malnutrition. However, role of prostaglandin-endoperoxide synthase 2 (PTGS2) in the management of pressure ulcers in with compound Huangbai liquid is still unclear. Traditional Chinese medicine components and related targets of compound Huangbai liquid were collected through traditional Chinese medicine systems pharmacology (TCMSP) and Batman-traditional Chinese medicine database. Disease-related targets were obtained using the Gene Cards database. The protein-protein interaction (PPI) network was constructed using the Search tool for retrieval of interacting genes (STRING) and analyzed by Cytoscape to obtain the core components. To evaluate the clinical efficacy of the compound Huangbai liquid in the treatment of pressure ulcers, 40 patients with pressure ulcers were selected and divided into an observation group and a control group, with 20 individuals in each group. The observation group received treatment with compound Huangbai liquid. Sixty-five components and 480 targets of compound Huangbai liquid were obtained from TCMSP and Batman - traditional Chinese medicine databases. Two hundred seventy-three pressure ulcer-related targets were obtained. Seventy-two potential targets of compound Huangbai pigment in treatment of pressure ulcer were obtained, and 2 unrelated targets were deleted. There were 70 nodes and 1167 edges in PPI network. Gene ontology (GO) function is involved in biological processes such as reactive oxygen species metabolism and cellular response to chemical stress. Cellular components such as platelet α granules lumen and membrane rafts were involved. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment results showed that compound Huangbai liquid in treatment of pressure ulcer. The clinical results indicate that the compound Huangbai liquid has a good therapeutic effect on pressure ulcers. PTGS2 may be a target for treatment of pressure ulcers with compound Huangbai liquid, providing a new direction for its treatment.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

De novo lipid synthesis and polarized prenylation drive cell invasion through basement membrane.

To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.

Delivery of Lutein by Using Modified Burdock Polysaccharide Aggregates: Preparation, Characterization, and In Vitro Release Properties.

Novel self-assembled aggregates of stearic acid (SA)-modified burdock polysaccharide (BP) for loading lutein were constructed, and the release and absorption properties of lutein in the aggregates in simulated gastrointestinal fluid were investigated. Three different degrees of substitution (DS) of SA-BPs were used to embed lutein, resulting in the encapsulation efficiency exceeding 90%. The aggregates were uniformly spherical, with a particle size range of 227-341 nm. XRD analysis revealed that lutein was present in a non-crystalline state within the aggregates. FT-IR and FS analysis demonstrated that lutein was located in the hydrophobic domains of SA-BP. The highest bioavailability of lutein in these aggregates reached 4.36 times that in the unmodified samples. These aggregates were able to remain stable in gastric juice and enhance the release rate of lutein in intestinal fluid. The transport of lutein-loaded SA-BP aggregates in Caco-2 cells competed with P-glycoprotein inhibitors, mainly promoting the transmembrane absorption of lutein through caveolae (or lipid raft)-related and clathrin-dependent endocytosis pathways. The above results suggest that SA-BP aggregates have the potential to be promising carriers for the efficient delivery of hydrophobic lutein.

The causal relationship between gut microbiota and diabetic neuropathy: a bi-directional two-sample Mendelian randomization study.

Many studies suggest a strong correlation between gut microbiota (GM) and diabetic neuropathy (DN). However, the precise causal relationship between GM and DN has yet to be fully elucidated. Hence, a bi-directional Mendelian randomization (MR) analysis was used to examine the association between GM and DN. Widely known genome-wide association study (GWAS) of GM was collected from the MiBio Gen project. Summary-level datasets for DN were taken from the FinnGen project. Inverse variance weighted approach was used for evaluating the causal relationship between GM and DN. Subsequently, pleiotropy and heterogeneity tests were performed to verify the reliability of the data. Furthermore, a bidirectional two-sample MR analysis was done to investigate the directionality of the causal relationships. Gene Ontology analysis was conducted to identify the associations that could indicate biological functions. We identified potential causal associations between GM and DN (p< 0.05 in all three MR methods). Among them, we found increased levels of Christensenellaceae R-7 (Odds ratio, OR= 1.52; 95% confidence interval, CI = 1.03-2.23; p = 0.03), Ruminococcaceae UCG013 (OR =1.35; 95% CI = 1.00-1.85; p = 0.04), and Eggerthella groups (OR = 1.27; 95% CI = 1.05-1.55; p = 0.01), which may be associated with a higher risk of DN, while increased levels of Peptococcaceae (OR = 0.69; 95% CI = 0.54-0.90; p< 0.01) and Eubacterium coprostanoligenes groups (OR = 0.68; 95% CI = 0.49-0.93; p = 0.01) could be associated with a lower risk. Gene Ontology pathway analysis revealed enrichment of genes regulated by the associated single-nucleotide polymorphisms (SNPs) in the apical plasma membrane, glycosyltransferase activity, hexosyltransferase activity and membrane raft. Reverse MR analyses indicated that DN was associated with five microbial taxa in all three MR methods. The results of our study validate the possible causative relationship between GM and DN. This discovery gives new perspectives into the mechanism on how GM influences DN, and establishes a theoretical foundation for future investigations into targeted preventive measures.

Roles for PMP22 in Schwann cell cholesterol homeostasis in health and disease.

Underexpression, overexpression, and point mutations in peripheral myelin protein 22 (PMP22) cause most cases of Charcot-Marie-Tooth disease (CMTD). While its exact functions remain unclear, PMP22 is clearly essential for formation and maintenance of healthy myelin in the peripheral nervous system. This review explores emerging evidence for roles of PMP22 in cholesterol homeostasis. First, we highlight dysregulation of lipid metabolism in PMP22-based forms of CMTD and recently-discovered interactions between PMP22 and cholesterol biosynthesis machinery. We then examine data that demonstrates PMP22 and cholesterol co-traffic in cells and co-localize in lipid rafts, including how disease-causing PMP22 mutations result in aberrations in cholesterol localization. Finally, we examine roles for interactions between PMP22 and ABCA1 in cholesterol efflux. Together, this emerging body of evidence suggests that PMP22 plays a role in facilitating enhanced cholesterol synthesis and trafficking necessary for production and maintenance of healthy myelin.

The processed Euphorbia lathyris L. alleviates the inflammatory injury via regulating LXRα/ABCA1 expression and TLR4 positioning to lipid rafts

Euphorbia lathyris L. (EL) is a traditional poisonous herbal medicine used to treat dropsy, ascites, amenorrhea, anuria and constipation. Processing to reduce toxicity of EL is essential for its safe and effective application. However, there is little known regarding the molecular mechanism of reducing toxicity after EL processing. This research aimed to screen the differential markers for EL and PEL, explore the differential mechanisms of inflammatory injury induced by EL and processed EL (PEL) to expound the mechanism of alleviating toxicity after EL processing. The results showed that 15 potential biomarkers, mainly belonging to diterpenoids, were screened to distinguish EL from PEL. EL promoted the expressions of TLR4, NLRP3, NF-κB p65, IL-1β and TNF-α, increased lipid rafts abundance and promoted TLR4 positioning to lipid rafts. Meanwhile, EL decreased LXRα and ABCA1 expression, and reduced cholesterol efflux. In contrast to EL, the effects of PEL on these indicators were markedly weakened. In addition, Euphorbia factors L1, L2, and L3 affected LXRα, ABCA1, TLR4, NLRP3, NF-κB p65, TNF-α and IL-1β expression, influenced cholesterol efflux and lipid rafts abundance, and interfered with the colocalization of TLR4 and lipid rafts. The inflammatory injury caused by processed EL was significantly weaker than that caused by crude EL, and reduction of Euphorbia factors L1, L2, and L3 as well as attenuation of inflammatory injury participated in processing-based detoxification of EL. Our results provide valuable insights into the attenuated mechanism of EL processing and will guide future research on the processing mechanism of toxic traditional Chinese medicine.

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

GAL3ST1 Deficiency Reduces Epithelial-Mesenchymal Transition and Tumorigenic Capacity in a Cholangiocarcinoma Cell Line.

Cholangiocarcinoma (CCA), or bile duct cancer, is the second most common liver malignancy, with an increasing incidence in Western countries. The lack of effective treatments associated with the absence of early symptoms highlights the need to search for new therapeutic targets for CCA. Sulfatides (STs), a type of sulfoglycosphingolipids, have been found in the biliary tract, with increased levels in CCA and other types of cancer. STs are involved in protein trafficking and cell adhesion as part of the lipid rafts of the plasma membrane. We aimed to study the role of STs in CCA by the genetic targeting of GAL3ST1, an enzyme involved in ST synthesis. We used the CRISPR-Cas9 system to generate GAL3ST1-deficient TFK1 cells. GAL3ST1 KO cells showed lower proliferation and clonogenic activity and reduced glycolytic activity compared to TFK1 cells. Polarized TFK1 GAL3ST1 KO cells displayed increased transepithelial resistance and reduced permeability compared to TFK1 wt cells. The loss of GAL3ST1 showed a negative effect on growth in 30 out of 34 biliary tract cancer cell lines from the DepMap database. GAL3ST1 deficiency partially restored epithelial identity and barrier function and reduced proliferative activity in CCA cells. Sulfatide synthesis may provide a novel therapeutic target for CCA.