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Journal of Colloid and Interface Science Dec 2023Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the...
Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of 'raft'-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation.
PubMed: 37690301
DOI: 10.1016/j.jcis.2023.08.117 -
Acta Histochemica Et Cytochemica Aug 2023Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types...
Adrenal medullary chromaffin (AMC) and sympathetic ganglion cells are derived from the neural crest and show a similar developmental path. Thus, these two cell types have many common properties in membrane excitability and signaling. However, AMC cells function as endocrine cells while sympathetic ganglion cells are neurons. In rat sympathetic ganglion cells, muscarinic M and M receptors mediate excitation and inhibition via suppression of M-type K channels and suppression of voltage-dependent Ca channels, respectively. On the other hand, M receptor stimulation in rat AMC cells also produces excitation by suppressing TWIK-related acid sensitive K (TASK) channels. However, whether M receptors are coupled with voltage-dependent Ca channel suppression is unclear. We explore this issue electrophysiologically and biochemically. Electrical stimulation of nerve fibers in rat adrenal glands trans-synaptically increased the Ca signal in AMC cells. This electrically evoked increased Ca signal was not altered during muscarine-induced increase in Ca signal, whereas it decreased significantly during a GABA-induced increase, due to a shunt effect of increased Cl conductance. The whole-cell current recordings revealed that voltage-dependent Ca currents in AMC cells were suppressed by adenosine triphosphate, but not by muscarinic agonists. The fractionation analysis and immunocytochemistry indicated that Ca1.2 Ca channels and M receptors are located in the raft and non-raft membrane domains, respectively. We concluded that muscarinic stimulation in rat AMC cells does not produce voltage-dependent Ca channel inhibition. This lack of muscarinic inhibition is at least partly due to physical separation of voltage-dependent Ca channels and M receptors in the plasma membrane.
PubMed: 37680574
DOI: 10.1267/ahc.23-00042 -
Frontiers in Bioscience (Landmark... Aug 2023Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as...
BACKGROUND
Plasmolipin (PLLP) is a membrane protein located in lipid rafts that participates in the formation of myelin. It is also implicated in many pathologies, such as neurological disorders, type 2 diabetes, and cancer metastasis. To better understand how PLLP interacts with raft components (gangliosides and cholesterol), we undertook a global study combining simulations and physicochemical measurements of molecular interactions in various PLLP-ganglioside systems.
METHODS
studies consisted of molecular dynamics simulations in reconstructed membrane environments. PLLP-ganglioside interaction measurements were performed by microtensiometry at the water-air interface on ganglioside monolayers.
RESULTS
We have elucidated the mode of interaction of PLLP with ganglioside GM1 and characterized this interaction at the molecular level. We showed that GM1 induces the structuring of the extracellular loops of PLLP and that this interaction propagates a conformational signal through the plasma membrane, involving a cholesterol molecule located between transmembrane domains. This conformational wave is finally transmitted to the intracellular domain of the protein, consistent with the role of PLLP in signal transduction.
CONCLUSIONS
This study is a typical example of the epigenetic dimension of protein structure, a concept developed by our team to describe the chaperone effect of gangliosides on disordered protein motifs which associate with lipid rafts. From a physiological point of view, these data shed light on the role of gangliosides in myelin formation. From a pathological point of view, this study will help to design innovative therapeutic strategies focused on ganglioside-PLLP interactions in various PLLP-associated diseases.
Topics: Humans; G(M1) Ganglioside; Gangliosides; Membrane Microdomains; Myelin Sheath; Proteolipids; Myelin and Lymphocyte-Associated Proteolipid Proteins
PubMed: 37664934
DOI: 10.31083/j.fbl2808157 -
Scientific Reports Aug 2023Oncogenic cell-surface membrane proteins contribute to the phenotypic and functional characteristics of cancer stem cells (CSCs). We employed a proximity-labeling...
Oncogenic cell-surface membrane proteins contribute to the phenotypic and functional characteristics of cancer stem cells (CSCs). We employed a proximity-labeling proteomic approach to quantitatively analyze the cell-surface membrane proteins in close proximity to CD147 in CSCs. Furthermore, we compared CSCs to non-CSCs to identify CSC-specific cell-surface membrane proteins that are closely interact with CD147 and revealed that lateral interaction between CD147 and CD276 concealed within the lipid raft microdomain in CSCs, confers resistance to docetaxel, a commonly used chemotherapy agent for various cancer types, including metastatic breast cancer. Moreover, we investigated the clinical relevance of CD147 and CD276 co-expression in HER2+ breast cancer (BC) and triple-negative breast cancer patients who underwent chemotherapy. We observed poor disease-free survival and Overall survival rates in patients of CD147 and CD276 (p = 0.04 and 0.08, respectively). Subsequent immunohistochemical analysis in independent cohorts of HER2+ BC support for the association between co-expression of CD147 and CD276 and a poor response to chemotherapy. Collectively, our study suggests that the lateral interaction between CD147 and its proximal partners, such as CD276, may serve as a poor prognostic factor in BC and a predictive marker for the critical phenotypic determinant of BC stemness.
Topics: Humans; Proteome; Proteomics; Triple Negative Breast Neoplasms; Docetaxel; Membrane Proteins; Transcription Factors; B7 Antigens
PubMed: 37648771
DOI: 10.1038/s41598-023-41416-7 -
Antibiotics (Basel, Switzerland) Aug 2023Ammonium group containing polymers possess inherent antimicrobial properties, effectively eliminating or preventing infections caused by harmful microorganisms. Here,...
Ammonium group containing polymers possess inherent antimicrobial properties, effectively eliminating or preventing infections caused by harmful microorganisms. Here, homopolymers based on monomers containing ammonium groups were synthesized via Reversible Addition Fragmentation Chain Transfer Polymerization (RAFT) and evaluated as potential antibacterial agents. The antimicrobial activity was evaluated against Gram-positive ( and ) and Gram-negative bacteria ( and ). Three polymers, poly(diallyl dimethyl ammonium chloride), poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), and poly(vinyl benzyl trimethylammonium chloride), were examined to explore the effect of molecular weight (10 kDa, 20 kDa, and 40 kDa) on their antimicrobial activity and toxicity to mammalian cells. The mechanisms of action of the polymers were investigated with dye-based assays, while Scanning Electron Microscopy (SEM) showed collapsed and fused bacterial morphologies due to the interactions between the polymers and components of the bacterial cell envelope, with some polymers proving to be bactericidal and others bacteriostatic, while being non-hemolytic. Among all the homopolymers, the most active, non-Gram-specific polymer was poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), with a molecular weight of 40 kDa, with minimum inhibitory concentrations between 16 and 64 µg/mL, showing a bactericidal mode of action mediated by disruption of the cytoplasmic membrane. This homopolymer could be useful in biomedical applications such as surface dressings and in areas such as eye infections.
PubMed: 37627740
DOI: 10.3390/antibiotics12081320 -
Membranes Jul 2023One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aβ) peptides in the brain. The processing of amyloid precursor protein (APP) into...
One of the hallmarks of Alzheimer's disease (AD) is the accumulation of amyloid beta (Aβ) peptides in the brain. The processing of amyloid precursor protein (APP) into Aβ is dependent on the location of APP in the membrane, membrane lipid composition and, possibly, presence of lipid rafts. In this study, we used atomic force microscopy (AFM) to investigate the interaction between transmembrane fragment APP (corresponding to Aβ) and its amyloidogenic mutant L723P with membranes combining liquid-ordered and liquid-disordered lipid phases. Our results demonstrated that most of the APP is located either in the liquid-disordered phase or at the boundary between ordered and disordered phases, and hardly ever in rafts. We did not notice any major changes in the domain structure induced by APP. In membranes without cholesterol APP, and especially its amyloidogenic mutant L723P formed annular structures and clusters rising above the membrane. Presence of cholesterol led to the appearance of concave membrane regions up to 2 nm in depth that were deeper for wild type APP. Thus, membrane cholesterol regulates changes in membrane structure and permeability induced by APP that might be connected with further formation of membrane pores.
PubMed: 37623767
DOI: 10.3390/membranes13080706 -
Heliyon Aug 2023Vascular endothelial cell dysfunction involving syndecan (SDC) proteoglycans contributes to acute sepsis-associated lung injury (ALI), but the exact SDC isoform involved...
Vascular endothelial cell dysfunction involving syndecan (SDC) proteoglycans contributes to acute sepsis-associated lung injury (ALI), but the exact SDC isoform involved is unclear. We aimed to clarify which SDCs are involved in ALI. A relevant gene expression dataset (GSE5883) was analysed for differentially expressed genes (DEGs) between lipopolysaccharide (LPS)-treated and control lung endothelial cells and for SDC isoform expression. Bioinformatic analyses to predict DEG function were conducted using R language, Gene Ontology, and the Kyoto Encyclopedia of Genes and Genomes. SDC2 and SDC4 expression profiles were examined under inflammatory conditions in human lung vascular endothelial cell and mouse sepsis-associated ALI models. Transcription factors regulating SDC2/4 were predicted to indirectly assess SDC involvement in septic inflammation. Of the DEGs, 224 and 102 genes were up- and downregulated, respectively. Functional analysis indicated that DEGs were involved in modulating receptor ligand and signalling receptor activator activities, cytokine receptor binding, responses to LPS and molecules of bacterial origin, regulation of cell adhesion, tumour necrosis factor signalling, and other functions. DEGs were also enriched for cytoplasmic ribonucleoprotein granules, transcription regulator complexes, and membrane raft cellular components. gene expression was 4.5-fold higher in the LPS group than in the control group, while levels were similar in both groups. SDC4 mRNA and protein expression was markedly upregulated in response to inflammatory injury, and SDC4 downregulation severely exacerbated inflammatory responses in both and models. Overall, our data demonstrate that SDC4, rather than SDC2, is involved in LPS-induced sepsis-associated ALI.
PubMed: 37576224
DOI: 10.1016/j.heliyon.2023.e18600 -
International Journal of Molecular... Aug 2023The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific...
Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.
The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.
Topics: Humans; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily B; Cell Membrane; Cyclodextrins; Cholesterol; Membrane Microdomains
PubMed: 37569709
DOI: 10.3390/ijms241512335 -
ACS Central Science Jul 2023Intracellular delivery of therapeutic biomacromolecules is often challenged by the poor transmembrane and limited endosomal escape. Here, we establish a combinatorial...
Intracellular delivery of therapeutic biomacromolecules is often challenged by the poor transmembrane and limited endosomal escape. Here, we establish a combinatorial library composed of 150 molecular weight-defined gemini amphiphiles (GAs) to identify the vehicles that facilitate robust cytosolic delivery of proteins in vitro and in vivo. These GAs display similar skeletal structures but differential amphiphilicity by adjusting the length of alkyl tails, type of ionizable cationic heads, and hydrophobicity or hydrophilicity of a spacer. The top candidate is highly efficient in translocating a broad spectrum of proteins with various molecular weights and isoelectric points into the cytosol. Particularly, we notice that the entry mechanism is predominantly mediated via the lipid raft-dependent membrane fusion, bypassing the classical endocytic pathway that limits the cytosolic delivery efficiency of many presently available carriers. Remarkably, the top GA candidate is capable of delivering hard-to-deliver Cas9 ribonucleoprotein in vivo, disrupting mutation in the tumor-bearing mice to inhibit tumor growth and extend their survival. Our study reveals a GA-based small-molecule carrier platform for the direct cytosolic delivery of various types of proteins for therapeutic purposes.
PubMed: 37521791
DOI: 10.1021/acscentsci.3c00207 -
Redox Biology Sep 2023Stress-induced release of glucocorticoid is an important amyloidogenic factor that upregulates amyloid precursor protein (APP) and β secretase 1 (BACE1) levels....
Stress-induced release of glucocorticoid is an important amyloidogenic factor that upregulates amyloid precursor protein (APP) and β secretase 1 (BACE1) levels. Glucocorticoid also contributes to the pathogenesis of Alzheimer's disease (AD) by increasing ER-mitochondria connectivity, in which amyloid β (Aβ) processing occurs rigorously because of its lipid raft-rich characteristics. However, the mechanism by which glucocorticoid enhances γ-secretase activity in the mitochondrial-associated membrane of ER (MAM) and subsequent accumulation of mitochondrial Aβ is unclear. In this study, we determined how glucocorticoid enhances Aβ production in MAM using SH-SY5Y cells and ICR mice. First, we observed that cortisol-induced Aβ accumulation in mitochondria preceded its extracellular apposition by enhancing γ-secretase activity, which was the result of increased presenilin 1 (PSEN1) localization in MAM. Screening data revealed that cortisol selectively downregulated the ER retrieval protein Rer1, which triggered its maturation and subsequent entry into the endocytic secretory pathway of PSEN1. Accordingly, overexpression of RER1 reversed the deleterious effects of mitochondrial Aβ on mitochondrial respiratory function and neuronal cell viability. Notably, we found that cortisol guided the glucocorticoid receptor (GR) to bind directly to the RER1 promoter, thus trans-repressing its expression. Inhibiting GR function reduced Aβ accumulation at mitochondria and improved the outcome of a spatial memory task in mice exposed to corticosterone. Taken together, glucocorticoid enhances PSEN1-mediated Aβ generation at MAM by downregulating Rer1, which is a potential target at early stages of AD pathogenesis.
Topics: Humans; Mice; Animals; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Glucocorticoids; Hydrocortisone; Aspartic Acid Endopeptidases; Mice, Inbred ICR; Neuroblastoma; Alzheimer Disease; Adaptor Proteins, Vesicular Transport
PubMed: 37494768
DOI: 10.1016/j.redox.2023.102821