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The Journal of Biological Chemistry Sep 2019A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process,...
A hallmark of G-protein-coupled receptors (GPCRs) is the conversion of external stimuli into specific cellular responses. In this tightly-regulated process, extracellular ligand binding by GPCRs promotes specific conformational changes within the seven transmembrane helices, leading to the coupling and activation of intracellular "transducer" proteins, such as heterotrimeric G proteins. Much of our understanding of the molecular mechanisms that govern GPCR activation is derived from experiments with purified receptors reconstituted in detergent micelles. To elucidate the influence of the phospholipid bilayer on GPCR activation, here we interrogated the functional, pharmacological, and biophysical properties of a GPCR, the β-adrenergic receptor (βAR), in high-density lipoprotein (HDL) particles. Compared with detergent-reconstituted βAR, the βAR in HDL particles had greatly enhanced levels of basal (constitutive) activity and displayed increased sensitivity to agonist activation, as assessed by activation of heterotrimeric G protein and allosteric coupling between the ligand-binding and transducer-binding pockets. Using F NMR spectroscopy, we directly linked these functional differences in detergent- and HDL-reconstituted βAR to a change in the equilibrium between inactive and active receptor states. The contrast between the low levels of βAR constitutive activity in cells and the high constitutive activity observed in an isolated phospholipid bilayer indicates that βAR basal activity depends on the reconstitution system and further suggests that various cellular mechanisms suppress βAR basal activity physiologically. Our findings provide critical additional insights into GPCR activation and reveal how dramatically reconstitution systems can impact membrane protein function.
Topics: Detergents; Humans; Phospholipids; Receptors, Adrenergic, beta-2
PubMed: 31362983
DOI: 10.1074/jbc.AC119.009848 -
European Biophysics Journal : EBJ Dec 2015The ability of the membrane skeletal protein spectrin to interact with phospholipids, and aminophospholipids in particular, in both natural and model membranes, is well...
The ability of the membrane skeletal protein spectrin to interact with phospholipids, and aminophospholipids in particular, in both natural and model membranes, is well documented. The present study involves phospholipid-induced quenching of tryptophan fluorescence to probe spectrin-membrane interactions in the presence and absence of cholesterol. We performed the experiments on small unilamellar vesicles of phospholipids made of DMPC and DMPC/DMPE and of DOPC and DOPC/DOPE with and without cholesterol at two different temperatures, one below at 15 °C and another above, at 50 °C, the main phase transition temperature (T m) of the bulk phospholipid. Results indicate that erythroid and brain spectrin binds DMPC/DMPE membranes by tenfold and 40-fold stronger, respectively, in the presence of 20 % cholesterol, up to which both gel (Lβ) and liquid crystalline (Lα) phases coexists, at 15 °C particularly in DMPC-based membranes containing saturated fatty acyl chains and not in DOPC-based membranes with appreciably lower T m. Time-resolved fluorescence and circular dichroism spectroscopic studies indicated no significant change in the mean lifetime of the tryptophan residues in spectrin and in the secondary structures of the proteins upon binding to the phospholipid SUVs.
Topics: Cholesterol; Dimyristoylphosphatidylcholine; Fluorescence; Gels; Liquid Crystals; Phosphatidylcholines; Phosphatidylethanolamines; Protein Binding; Spectrin; Unilamellar Liposomes
PubMed: 26184723
DOI: 10.1007/s00249-015-1057-2 -
Plant Molecular Biology Jul 2023Verticillium wilt which produced by the soil-borne fungus Verticillium dahliae is an important biotic threat that limits cotton (Gossypium hirsutum) growth and...
Verticillium wilt which produced by the soil-borne fungus Verticillium dahliae is an important biotic threat that limits cotton (Gossypium hirsutum) growth and agricultural productivity. It is very essential to explore new genes for the generation of V. dahliae resistance or tolerance cotton varieties. Ca signaling as a secondary messenger is involved in pathogen stress response. Despite Ca-responsive phospholipid-binding BONZAI (BON) genes have intensively been investigated in Arabidopsis, their function has not still been characterized in cotton. Here, we showed that three copies of GhBON1, two copies of GhBON2 and GhBON3 were found from the genome sequences of upland cotton. The expression of GhBON1 was inducible to V. dahliae. Knocking down of GhBON1, GhBON2 and GhBON3 using virus induced gene silencing (VIGS) each increased up-regulation of defense responses in cotton. These GhBON1, GhBON2 and GhBON3-silenced plants enhanced resistance to V. dahliae accompanied by higher burst of hydrogen peroxide and decreased cell death and had more effect on the up-regulation of defense response genes. Further analysis revealed that GhBON1 could interacts with BAK1-interacting receptor-like kinase 1 (GhBIR1) and pathogen-associated molecular pattern (PAMP) receptor regulator BAK1 (GhBAK1) at plasma membrane. Our study further reveals that plant Ca -responsive phospholipid-binding BONZAI genes negatively regulate Verticillium wilt with the conserved function in response to disease resistance or plant immunity.
Topics: Gossypium; Verticillium; Disease Resistance; Signal Transduction; Phospholipids; Plant Diseases; Gene Expression Regulation, Plant; Plant Proteins
PubMed: 37261657
DOI: 10.1007/s11103-023-01359-z -
Current Opinion in Structural Biology Feb 2016The C-terminal hypervariable region (HVR) of the splice variant KRAS4B is disordered. Classically, the role of the post-translationally-modified HVR is to navigate Ras... (Review)
Review
The C-terminal hypervariable region (HVR) of the splice variant KRAS4B is disordered. Classically, the role of the post-translationally-modified HVR is to navigate Ras in the cell and to anchor it in localized plasma membrane regions. Here, we propose additional regulatory roles, including auto-inhibition by shielding the effector binding site in the GDP-bound state and release upon GTP binding and in the presence of certain oncogenic mutations. The released HVR can interact with calmodulin. We show that oncogenic mutations (G12V/G12D) modulate the HVR-phospholipid binding specificity, resulting in preferential interactions with phosphatidic acid. The shifts in the conformational preferences and binding specificity in the disordered state exemplify the critical role of the unstructured tail of K-Ras4B in cancer.
Topics: Animals; Catalytic Domain; Gene Expression Regulation, Neoplastic; Genetic Variation; Humans; Membrane Microdomains; Mutation; Neoplasms; Phospholipids; Protein Binding; Protein Folding; Protein Interaction Domains and Motifs; Protein Isoforms; Protein Transport; Proto-Oncogene Proteins p21(ras); Signal Transduction
PubMed: 26709496
DOI: 10.1016/j.sbi.2015.11.010 -
Chinese Journal of Traumatology =... Apr 2020With the deepening of research, proteomics has developed into a science covering the study of all the structural and functional characteristics of proteins and the... (Review)
Review
With the deepening of research, proteomics has developed into a science covering the study of all the structural and functional characteristics of proteins and the dynamic change rules. The essence of various biological activities is revealed from the perspectives of the biological structure, functional activity and corresponding regulatory mechanism of proteins by proteomics. Among them, phospholipid-binding protein is one of the hotspots of proteomics, especially annexin A1, which is widely present in various tissues and cells of the body. It has the capability of binding to phospholipid membranes reversibly in a calcium ion dependent manner. In order to provide possible research ideas for researchers, who are interested in this protein, the biological effects of annexin A1, such as inflammatory regulation, cell signal transduction, cell proliferation, differentiation and apoptosis are described in this paper.
Topics: Annexin A1; Apoptosis; Calcium; Cell Proliferation; Humans; Inflammation; Phospholipids; Protein Binding; Proteomics; Signal Transduction
PubMed: 32201231
DOI: 10.1016/j.cjtee.2020.02.002 -
Physical Chemistry Chemical Physics :... Mar 2023Amyloid-beta (Aβ) aggregation triggers neurotoxicity and is linked to Alzheimer's disease. Aβ oligomers, rather than extended fibrils, adhere to the cell membrane,...
Amyloid-beta (Aβ) aggregation triggers neurotoxicity and is linked to Alzheimer's disease. Aβ oligomers, rather than extended fibrils, adhere to the cell membrane, causing cell death. Phosphatidylserine (PS), an anionic phospholipid, is prevalent in neuronal membranes (< 20 molar percentage) and, while isolated to the cytoplasmic leaflet of the membrane in healthy cells, its exposure in apoptotic cells and migration to exoplasmic leaflet is triggered by oxidative damage to the membrane. It is widely believed that PS plays a crucial role in the Aβ peptide interaction in the membranes of neuronal cells. However, due to the complexity of the cell membrane, it can be challenging to address molecular level understanding of the PS-Aβ binding and oligomerization processes. Herein, we use microcavity supported lipid bilayers (MSLBs) to analyse PS and Aβ binding, oligomer formation, and membrane damage. MSLBs are a useful model to evaluate protein-membrane interactions because of their cell-like dual aspect fluidity, their addressability and compositional versatility. We used electrochemical impedance spectroscopy (EIS) and confocal fluorescence microscopy to compare the impact of Aβ on simple zwitterioinic membrane, dioleoylphosphatidylcholine (DOPC), with MSLBs comprised of transversally asymmetric binary DOPC and dioleoylphosphatidylserine (DOPS). Monomeric Aβ adsorbs weakly to the pristine zwitterionic DOPC membrane without aggregation. Using a membrane integrity test, with pyranine trapped within the cavities beneath the membrane, Aβ exposure did not result in pyranine leakage, indicating that DOPC membranes were intact. When 10 mol% DOPS was doped asymmetrically into the membrane's outer leaflet, oligomerization of Aβ monomer was evident in EIS and atomic force microscopy (AFM), and confocal imaging revealed that membrane damage, resulted in extensive pyranine leakage from the pores. The effects were time, and DOPS and Aβ concentration-dependent. Membrane pore formation was visible within 30 minutes, and oligomerization, membrane-oligomer multilayer, and Aβ fibril formation evident over 3 to 18 hours. In asymmetric membranes with DOPS localized to the lower leaflet, optothermally (laser induced) damage increased local DOPS concentrations at the distal leaflet, promoting Aβ aggregation.
Topics: Amyloid beta-Peptides; Arylsulfonates; Lipid Bilayers; Phosphatidylserines; Phospholipids
PubMed: 36317678
DOI: 10.1039/d2cp03344e -
Biochimica Et Biophysica Acta.... Sep 2020NMR is a sophisticated method for investigation of structure and dynamics of lipid and protein molecules in membranes. Vibrational spectroscopy is also powerful because... (Review)
Review
NMR is a sophisticated method for investigation of structure and dynamics of lipid and protein molecules in membranes. Vibrational spectroscopy is also powerful because of relatively high resolution and sensitivity, and easier access than NMR. A combined use of these spectroscopies could provide important insights into the membrane biophysics. A structural analysis of phosphatidylethanolamine (PE) bilayers in built-up films by infrared dichroism suggested that polar groups oriented parallel to the membrane surface. A Raman analysis of phosphatidylcholine (PC) revealed that the gauche conformation was preferred for the choline backbone not only in solid, but also in the gel and liquid-crystalline states. The polar group structure of DPPC bilayers in the liquid-crystalline state was determined by analyzing deuterium quadrupole splitting of the choline group and phosphorus chemical shift anisotropy of the phosphate group in combination with restriction of the gauche conformation of the choline group determined by Raman spectroscopy. This was an excellent complementarity of NMR and vibrational spectroscopies. The deuterium quadrupole splitting values mentioned above were found to change on addition of ions such as NaCl, CaCl, and LaCl, suggesting that a structural change takes place on ion binding and the polar group of PC works as an electric charge sensor of membranes. The ion-bound structure was determined by NMR using the restriction from Raman spectroscopy. The PN vector of phosphorylcholine group was inclined by 63° from the membrane surface, while the inclination was 18° in the ion-free form. The deuterium quadrupole splitting values and phosphorus powder patterns revealed that on mixing with phosphatidylglycerol (PG) or cardiolipin (CL), PC did not change its dynamic structure of the glycerol backbone, but PE did. The mixture of PE with PG or CL shared a new dynamic structure, suggesting their adaptive miscibility in the molecular level. PC was molecularly immiscible with any of PE, PG, and CL. The molecular miscibility would regulate not only interactions of proteins with mixed bilayers but also formation of asymmetric lipid membranes. Interactions of crown-ether (CE) modified artificial microbial peptides with phospholipid bilayers were investigated by NMR and FTIR. CE-modified 14-mers with one or two basic amino acid residues revealed position-specific selectivity for the suppression of calcein leakage from PC vesicles but did not for that from PG vesicles, suggesting that structures of the lipid polar groups play crucial roles in different responses of the vesicles to the positively charged peptides. Manipulation of the peptide-polar group interaction can be used for drug design.
Topics: Cardiolipins; Lipid Bilayers; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylethanolamines; Phosphatidylglycerols; Spectrum Analysis, Raman
PubMed: 32407775
DOI: 10.1016/j.bbamem.2020.183352 -
Molecular Biology of the Cell Sep 2018Mitochondrial transport and anchoring mechanisms work in concert to position mitochondria to meet cellular needs. In yeast, Mmr1 functions as a mitochondrial adaptor for...
Mitochondrial transport and anchoring mechanisms work in concert to position mitochondria to meet cellular needs. In yeast, Mmr1 functions as a mitochondrial adaptor for Myo2 to facilitate actin-based transport of mitochondria to the bud. Posttransport, Mmr1 is proposed to anchor mitochondria at the bud tip. Although both functions require an interaction between Mmr1 and mitochondria, the molecular basis of the Mmr1-mitochondria interaction is poorly understood. Our in vitro phospholipid binding assays indicate Mmr1 can directly interact with phospholipid membranes. Through structure-function studies we identified an unpredicted membrane-binding domain composed of amino acids 76-195 that is both necessary and sufficient for Mmr1 to interact with mitochondria in vivo and liposomes in vitro. In addition, our structure-function analyses indicate that the coiled-coil domain of Mmr1 is necessary and sufficient for Mmr1 self-interaction and facilitates the polarized localization of the protein. Disrupting either the Mmr1-membrane interaction or Mmr1 self-interaction leads to defects in mitochondrial inheritance. Therefore, direct membrane binding and self-interaction are necessary for Mmr1 function in mitochondrial inheritance and are utilized as a means to spatially and temporally regulate mitochondrial positioning.
Topics: Inheritance Patterns; Lipid Bilayers; Mitochondria; Mitochondrial Proteins; Phospholipids; Protein Binding; Protein Domains; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30044712
DOI: 10.1091/mbc.E18-02-0122 -
Physiological Reviews Oct 2014All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated... (Review)
Review
All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes.
Topics: Animals; Cell Membrane; Cytoskeleton; Humans; Lipid Metabolism; Phosphatidylinositols; Phospholipids
PubMed: 25287863
DOI: 10.1152/physrev.00040.2013 -
Nature May 2024Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and...
Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized de novo by the Kennedy pathway from choline and ethanolamine, respectively. Despite the essential roles of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here we show that the protein encoded by FLVCR1, whose mutation leads to the neurodegenerative syndrome posterior column ataxia and retinitis pigmentosa, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprising aromatic and polar residues. Despite binding to a common site, FLVCR1 interacts in different ways with the larger quaternary amine of choline in and with the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are crucial for the transport of ethanolamine, but dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLVCR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.
Topics: Humans; Binding Sites; Biological Transport; Choline; Ethanolamine; Membrane Transport Proteins; Models, Molecular; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorylation; Mutagenesis
PubMed: 38693265
DOI: 10.1038/s41586-024-07374-4