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Journal of Structural Biology: X Jun 2024Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is...
Physical properties of biological membranes directly or indirectly govern biological processes. Yet, the interplay between membrane and integral membrane proteins is difficult to assess due to reciprocal effects between membrane proteins, individual lipids, and membrane architecture. Using solid-state NMR (SSNMR) we previously showed that KirBac1.1, a bacterial Inward-Rectifier K channel, nucleates bilayer ordering and microdomain formation through tethering anionic lipids. Conversely, these lipids cooperatively bind cationic residues to activate the channel and initiate K flux. The mechanistic details governing the relationship between cooperative lipid loading and bilayer ordering are, however, unknown. To investigate, we generated KirBac1.1 samples with different concentrations of C-lableded phosphatidyl glycerol (PG) lipids and acquired a full suite of SSNMR 1D temperature series experiments using the ordered all-trans (AT) and disordered -gauche (TG) acyl conformations as markers of bilayer dynamics. We observed increased AT ordered signal, decreased TG disordered signal, and increased bilayer melting temperature with increased PG concentration. Further, we identified cooperativity between ordering and direct binding of PG lipids, indicating KirBac1.1-driven bilayer ordering and microdomain formation is a classically cooperative Hill-type process driven by and predicated upon direct binding of PG lipids. Our results provide unique mechanistic insight into how proteins and lipids in tandem contribute to supramolecular bilayer heterogeneity in the lipid membrane.
PubMed: 38883399
DOI: 10.1016/j.yjsbx.2024.100101 -
ELife Jun 2024The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these...
The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, etention sing elective ooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
Topics: Protein Transport; Endoplasmic Reticulum; Golgi Apparatus; Membrane Microdomains; Secretory Pathway; Humans; Kinetics; Cell Membrane; Membrane Proteins; HeLa Cells
PubMed: 38837189
DOI: 10.7554/eLife.89306 -
International Journal of Molecular... Aug 2023Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization,...
Osteoblast-Derived Matrix Vesicles Exhibit Exosomal Traits and a Unique Subset of microRNA: Their Caveolae-Dependent Endocytosis Results in Reduced Osteogenic Differentiation.
Matrix vesicles (MVs) are nano-sized extracellular vesicles that are anchored in the extracellular matrix (ECM). In addition to playing a role in biomineralization, osteoblast-derived MVs were recently suggested to have regulatory duties. The aims of this study were to establish the characteristics of osteoblast-derived MVs in the context of extracellular vesicles like exosomes, assess their role in modulating osteoblast differentiation, and examine their mechanism of uptake. MVs were isolated from the ECM of MG63 human osteoblast-like cell cultures and characterized via enzyme activity, transmission electron microscopy, nanoparticle tracking analysis, Western blot, and small RNA sequencing. Osteoblasts were treated with MVs from two different culture conditions (growth media [GM]; osteogenic media [OM]) to evaluate their effects on the differentiation and production of inflammatory markers and on macrophage polarization. MV endocytosis was assessed using a lipophilic, fluorescent dye and confocal microscopy with the role of caveolae determined using methyl-β-cyclodextrin. MVs exhibited a four-fold enrichment in alkaline phosphatase specific activity compared to plasma membranes; were 50-150 nm in diameter; possessed exosomal markers CD63, CD81, and CD9 and endosomal markers ALIX, TSG101, and HSP70; and were selectively enriched in microRNA linked to an anti-osteogenic effect and to M2 macrophage polarization. Treatment with GM or OM MVs decreased osteoblast differentiation. Osteoblasts endocytosed MVs using a mechanism that involves caveolae. These results support the hypothesis that osteoblasts produce MVs that participate in the regulation of osteogenesis.
Topics: Humans; MicroRNAs; Caveolae; Osteogenesis; Endocytosis; Cell Differentiation; Culture Media
PubMed: 37628952
DOI: 10.3390/ijms241612770 -
Pharmacological Research Mar 2024The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the...
The uncontrolled bacterial infection-induced cytokine storm and sequential immunosuppression are commonly observed in septic patients, which indicates that the activation of phagocytic cells and the efficient and timely elimination of bacteria are crucial for combating bacterial infections. However, the role of dysregulated immune cells and their disrupted function in sepsis remains unclear. Here, we found that macrophages exhibited the impaired endocytosis capabilities in sepsis by Single-cell RNA sequencing and bulk RNA sequencing. Caveolae protein Caveolin-1 (Cav-1) of macrophages was inactivated by SHP2 rapidly during Escherichia coli (E.coli) infection. Allosteric inhibitor of SHP2 effectively maintains Cav-1 phosphorylation to enhance macrophage to endocytose and eliminate bacteria. Additionally, TLR4 endocytosis of macrophage was also enhanced upon E.coli infection by SHP099, inducing an increased and rapidly resolved inflammatory response. In vivo, pretreatment or posttreatment with inhibitor of SHP2 significantly reduced the bacterial burden in organs and mortality of mice subjected E.coli infection or CLP-induced sepsis. The cotreatment of inhibitor of SHP2 with an antibiotic conferred complete protection against mortality in mice. Our findings suggest that Cav-1-mediated endocytosis and bacterial elimination may play a critical role in the pathogenesis of sepsis, highlighting inhibitor of SHP2 as a potential therapeutic agent for sepsis.
Topics: Animals; Humans; Mice; Bacteria; Caveolae; Endocytosis; Escherichia coli Infections; Macrophages; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Sepsis
PubMed: 38320736
DOI: 10.1016/j.phrs.2024.107096 -
ACS Omega Aug 2023Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in...
Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here, we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium III (Eu) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it is a coordination complex with a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also alters GM1 ganglioside partitioning. GM1 typically partitions into liquid ordered domains, but after labeling phase-separated GUVs with EuTc, cholera toxin B-subunit (CTxB), which binds GM1, labels liquid disordered domains. We also demonstrate that EuTc, but not free Eu or Tc, significantly reduces lipid diffusion coefficients. Finally, we show that EuTc can be used to label cellular membranes similar to a traditional membrane probe. EuTc may find utility as a membrane imaging probe where its large Stokes shift and sharp emission band would enable multicolor imaging.
PubMed: 37599986
DOI: 10.1021/acsomega.3c02721 -
MBio Aug 2023is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect...
is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in , cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in . Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1-TcAC5). We expressed an HA-tagged version of each protein in and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. IMPORTANCE We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in : the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in , while raising new questions on the activation of TcACs and the role of downstream components of this pathway.
Topics: Animals; Trypanosoma cruzi; Adenylyl Cyclases; Chagas Disease; Amino Acid Sequence; Signal Transduction; Mammals
PubMed: 37477489
DOI: 10.1128/mbio.01064-23 -
IScience Oct 2023Signal 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...
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.
PubMed: 37736044
DOI: 10.1016/j.isci.2023.107819 -
Journal of Molecular Medicine (Berlin,... Mar 2024Amyotrophic lateral sclerosis (ALS) is an age-dependent neurodegenerative disease affecting motor neurons in the spinal cord and brainstem whose etiopathogenesis remains...
Amyotrophic lateral sclerosis (ALS) is an age-dependent neurodegenerative disease affecting motor neurons in the spinal cord and brainstem whose etiopathogenesis remains unclear. Recent studies have linked major neurodegenerative diseases with altered function of multimolecular lipid-protein complexes named lipid rafts. In the present study, we have isolated lipid rafts from the anterior horn of the spinal cords of controls and ALS individuals and analysed their lipid composition. We found that ALS affects levels of different fatty acids, lipid classes and related ratios and indexes. The most significant changes affected the contents of n-9/n-7 monounsaturated fatty acids and arachidonic acid, the main n-6 long-chain polyunsaturated fatty acid (LCPUFA), which were higher in ALS lipid rafts. Paralleling these findings, ALS lipid rafts lower saturates-to-unsaturates ratio compared to controls. Further, levels of cholesteryl ester (SE) and anionic-to-zwitterionic phospholipids ratio were augmented in ALS lipid rafts, while sulfatide contents were reduced. Further, regression analyses revealed augmented SE esterification to (mono)unsaturated fatty acids in ALS, but to saturates in controls. Overall, these changes indicate that lipid rafts from ALS spinal cord undergo destabilization of the lipid structure, which might impact their biophysical properties, likely leading to more fluid membranes. Indeed, estimations of membrane microviscosity confirmed less viscous membranes in ALS, as well as more mobile yet smaller lipid rafts compared to surrounding membranes. Overall, these results demonstrate that the changes in ALS lipid rafts are unrelated to oxidative stress, but to anomalies in lipid metabolism and/or lipid raft membrane biogenesis in motor neurons. KEY MESSAGES: The lipid matrix of multimolecular membrane complexes named lipid rafts are altered in human spinal cord in sporadic amyotrophic lateral sclerosis (ALS). Lipid rafts from ALS spinal cord contain higher levels of n-6 LCPUFA (but not n-3 LCPUFA), n-7/n-9 monounsaturates and lower saturates-to-unsaturates ratio. ALS lipid rafts display increased contents of cholesteryl esters, anomalous anionic-to-zwitterionic phospholipids and phospholipid remodelling and reduced sulphated and total sphingolipid levels, compared to control lipid rafts. Destabilization of the lipid structure of lipid raft affects their biophysical properties and leads to more fluid, less viscous membrane microdomains. The changes in ALS lipid rafts are unlikely related to increased oxidative stress, but to anomalies in lipid metabolism and/or raft membrane biogenesis in motor neurons.
Topics: Humans; Amyotrophic Lateral Sclerosis; Neurodegenerative Diseases; Lipids; Membrane Microdomains
PubMed: 38285093
DOI: 10.1007/s00109-024-02419-7 -
Biomedicine & Pharmacotherapy =... Jan 2024Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous...
Blood-brain barrier (BBB) dysfunction plays a pivotal role in the pathology of chronic cerebral hypoperfusion (CCH)-related neurodegenerative diseases. Continuous endothelial cells (EC) that line the blood vessels of the brain are important components of the BBB to strictly control the flow of substances and maintain the homeostatic environment of the brain. However, the molecular mechanisms from the perspective of EC-induced BBB dysfunction after CCH are largely unknown. In this study, the BBB function was assessed using immunostaining and transmission electron microscopy. The EC dysfunction profile was screened by using EC enrichment followed by RNA sequencing. After identified the key EC dysfunction factor, C-kit, we used the C-kit inhibition drug (imatinib) and C-kit down-regulation method (AAV-BR1-C-kit shRNA) to verify the role of C-kit on BBB integrity and EC transcytosis after CCH. Furthermore, we also activated C-kit with stem cell factor (SCF) to observe the effects of C-kit on BBB following CCH. We explored that macromolecular proteins entered the brain mainly through EC transcytosis after CCH and caused neuronal loss. Additionally, we identified receptor tyrosine kinase C-kit as a key EC dysfunction molecule. Furthermore, the pharmacological inhibition of C-kit with imatinib counteracted BBB leakage by reducing caveolae-mediated transcytosis. Moreover, treatment with AAV-BR1-C-kit shRNA, which targets brain EC to inhibit C-kit expression, also ameliorated BBB leakage by reducing caveolae-mediated transcytosis. Furthermore, the SCF increased the permeability of the BBB by actively increasing caveolae-mediated transcytosis. This study provides evidence that C-kit is a key BBB permeability regulator through caveolae-mediated transcytosis in EC after CCH.
Topics: Humans; Blood-Brain Barrier; Caveolae; Endothelial Cells; Imatinib Mesylate; Transcytosis; Brain Ischemia; RNA, Small Interfering; Permeability
PubMed: 38141279
DOI: 10.1016/j.biopha.2023.115778 -
The Journal of Physical Chemistry. B Aug 2023Lipid rafts are condensed regions of cell membranes rich in cholesterol and sphingomyelin, which constitute the target for anticholesterolemic drugs - statins. In this...
Lipid rafts are condensed regions of cell membranes rich in cholesterol and sphingomyelin, which constitute the target for anticholesterolemic drugs - statins. In this work, we use for the first time a combined grazing-incidence X-ray diffraction (GIXD)/polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS)/Brewster angle microscopy (BAM) approach to show the statin effect on model lipid rafts and its components assembled in Langmuir monolayers at the air-water interface. Two representatives of these drugs, fluvastatin (FLU) and cerivastatin (CER), of different hydrophobicity were chosen, while cholesterol (Chol) and sphingomyelin (SM), and their 1:1 mixture were selected to form condensed monolayers of lipid rafts. The effect of statins on the single components of lipid rafts indicated that both the hydrophobicity of the drugs and the organization of the layer determined the drug-lipid interaction. For cholesterol monolayers, only the most hydrophobic CER was effectively changing the film structure, while for the less organized sphingomyelin, the biggest effect was observed for FLU. This drug affected both the polar headgroup region as shown by PM-IRRAS results and the 2D crystalline structure of the SM monolayer as evidenced by GIXD. Measurements performed for Chol/SM 1:1 models proved also that the statin effect depends on the presence of Chol-SM complexes. In this case, the less hydrophobic FLU was not able to penetrate the binary layer at all, while exposure to the hydrophobic CER resulted in the phase separation and formation of ordered assemblies. The changes in the membrane properties were visualized by BAM images and GIXD patterns and confirmed by thermodynamic parameters of hysteresis in the Langmuir monolayer compression-decompression experiments.
Topics: Sphingomyelins; Hydroxymethylglutaryl-CoA Reductase Inhibitors; X-Ray Diffraction; Incidence; Cholesterol; Spectrophotometry, Infrared; Membrane Microdomains; Surface Properties
PubMed: 37551973
DOI: 10.1021/acs.jpcb.3c02574