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Cells May 2021Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on... (Review)
Review
Signal transduction, the ability of cells to perceive information from the surroundings and alter behavior in response, is an essential property of life. Studies on tyrosine kinase action fundamentally changed our concept of cellular regulation. The induced assembly of subcellular hubs via the recognition of local protein or lipid modifications by modular protein interactions is now a central paradigm in signaling. Such molecular interactions are mediated by specific protein interaction domains. The first such domain identified was the SH2 domain, which was postulated to be a reader capable of finding and binding protein partners displaying phosphorylated tyrosine side chains. The SH3 domain was found to be involved in the formation of stable protein sub-complexes by constitutively attaching to proline-rich surfaces on its binding partners. The SH2 and SH3 domains have thus served as the prototypes for a diverse collection of interaction domains that recognize not only proteins but also lipids, nucleic acids, and small molecules. It has also been found that particular SH2 and SH3 domains themselves might also bind to and rely on lipids to modulate complex assembly. Some lipid-binding properties of SH2 and SH3 domains are reviewed here.
Topics: Animals; Binding Sites; Humans; Phospholipids; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Signal Transduction; Structure-Activity Relationship; src Homology Domains; src-Family Kinases
PubMed: 34068055
DOI: 10.3390/cells10051191 -
Molecules (Basel, Switzerland) Apr 2020Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance,...
Visceral adipose tissue derived serine protease inhibitor (vaspin) is a member of the serpin family and has been shown to have beneficial effects on glucose tolerance, insulin stability as well as adipose tissue inflammation, parameters seriously affected by obesity. Some of these effects require inhibition of target proteases such as kallikrein 7(KLK7) and many studies have demonstrated vaspin-mediated activation of intracellular signaling cascades in various cells and tissues. So far, little is known about the exact mechanism how vaspin may trigger these intracellular signaling events. In this study, we investigated and characterized the interaction of vaspin with membrane lipids and polyphosphates as well as their potential regulatory effects on serpin activity using recombinant vaspin and KLK7 proteins and functional protein variants thereof. Here, we show for the first time that vaspin binds to phospholipids and polyphosphates with varying effects on KLK7 inhibition. Vaspin binds strongly to monophosphorylated phosphatidylinositol phosphates (PtdInsP) with no effect on vaspin activation. Microscale thermophoresis (MST) measurements revealed high-affinity binding to polyphosphate 45 (K: 466 ± 75 nM) and activation of vaspin in a heparin-like manner. Furthermore, we identified additional residues in the heparin binding site in β-sheet A by mutating five basic residues resulting in complete loss of high-affinity heparin binding. Finally, using lipid overlay assays, we show that these residues are additionally involved in PtdInsP binding. Phospholipids play a major role in membrane trafficking and signaling whereas polyphosphates are procoagulant and proinflammatory agents. The identification of phospholipids and polyphosphates as binding partners of vaspin will contribute to the understanding of vaspins involvement in membrane trafficking, signaling and beneficial effects associated with obesity.
Topics: Binding Sites; Heparin; Humans; Kinetics; Membrane Lipids; Models, Molecular; Multiprotein Complexes; Phospholipids; Polyphosphates; Protein Binding; Serpins; Structure-Activity Relationship
PubMed: 32344508
DOI: 10.3390/molecules25081992 -
Proceedings of the National Academy of... Jan 2022Lipoprotein-associated phospholipase A (Lp-PLA) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized...
Lipoprotein-associated phospholipase A (Lp-PLA) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA's specificity toward oxidized phospholipids.
Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Allosteric Regulation; Binding Sites; Catalysis; Catalytic Domain; Fatty Acids; Humans; Hydrolysis; Lipoproteins, HDL; Membranes; Molecular Dynamics Simulation; Phospholipids; Substrate Specificity
PubMed: 34996868
DOI: 10.1073/pnas.2102953118 -
The Journal of Biological Chemistry May 2022Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including... (Review)
Review
Lipids play critical roles in several major chronic diseases of our times, including those that involve inflammatory sequelae such as metabolic syndrome including obesity, insulin sensitivity, and cardiovascular diseases. However, defining the substrate specificity of enzymes of lipid metabolism is a challenging task. For example, phospholipase A (PLA) enzymes constitute a superfamily of degradative, biosynthetic, and signaling enzymes that all act stereospecifically to hydrolyze and release the fatty acids of membrane phospholipids. This review focuses on how membranes interact allosterically with enzymes to regulate cell signaling and metabolic pathways leading to inflammation and other diseases. Our group has developed "substrate lipidomics" to quantify the substrate phospholipid specificity of each PLA and coupled this with molecular dynamics simulations to reveal that enzyme specificity is linked to specific hydrophobic binding subsites for membrane phospholipid substrates. We have also defined unexpected headgroup and acyl chain specificity for each of the major human PLA enzymes, which explains the observed specificity at a structural level. Finally, we discovered that a unique hydrophobic binding site-and not each enzyme's catalytic residues or polar headgroup binding site-predominantly determines enzyme specificity. We also discuss how PLAs release specific fatty acids after allosteric enzyme association with membranes and extraction of the phospholipid substrate, which can be blocked by stereospecific inhibitors. After decades of work, we can now correlate PLA specificity and inhibition potency with molecular structure and physiological function.
Topics: Allosteric Regulation; Fatty Acids; Humans; Phospholipases A2; Phospholipids; Substrate Specificity
PubMed: 35358512
DOI: 10.1016/j.jbc.2022.101873 -
Neuron Jan 2024Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways...
Astrocytes play crucial roles in regulating neural circuit function by forming a dense network of synapse-associated membrane specializations, but signaling pathways regulating astrocyte morphogenesis remain poorly defined. Here, we show the Drosophila lipid-binding G protein-coupled receptor (GPCR) Tre1 is required for astrocytes to establish their intricate morphology in vivo. The lipid phosphate phosphatases Wunen/Wunen2 also regulate astrocyte morphology and, via Tre1, mediate astrocyte-astrocyte competition for growth-promoting lipids. Loss of s1pr1, the functional analog of Tre1 in zebrafish, disrupts astrocyte process elaboration, and live imaging and pharmacology demonstrate that S1pr1 balances proper astrocyte process extension/retraction dynamics during growth. Loss of Tre1 in flies or S1pr1 in zebrafish results in defects in simple assays of motor behavior. Tre1 and S1pr1 are thus potent evolutionarily conserved regulators of the elaboration of astrocyte morphological complexity and, ultimately, astrocyte control of behavior.
Topics: Animals; Astrocytes; Drosophila; Drosophila Proteins; Phospholipids; Receptors, G-Protein-Coupled; Sphingosine-1-Phosphate Receptors; Zebrafish
PubMed: 38096817
DOI: 10.1016/j.neuron.2023.11.008 -
Chemical & Pharmaceutical Bulletin 2019Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have... (Review)
Review
Biliary lipids consist mainly of bile salts, phospholipids and cholesterol, which form mixed micelles and vesicles. Bile salts play various physiological roles but have damaging effects on cell membranes due to their detergent properties. The cytotoxicity of bile salts on hepatocytes leads to liver injuries and is largely determined by the bile salt species, the concentrations of bile salts, phospholipids and cholesterol, and the lipid composition of cell membranes. In bile, monomers and simple micelles of bile salts coexist with mixed micelles and vesicles in dynamic equilibrium, and contribute to the cytotoxicity on hepatocytes. The ATP-binding cassette (ABC) transporter family members, ABCB11, ABCB4 and ABCG5/ABCG8, mediate the biliary secretion of bile salts, phospholipids and cholesterol, respectively. Mutations in ABCB4 result in severe cholestatic diseases, and the biliary phospholipids are necessary for the attenuation of bile salt cytotoxicity. On the other hand, cholesterol reverses the cytoprotective effects of phospholipids against bile salts. In addition, phosphatidylethanolamine N-methyltransferase increases the cell resistance to bile salts by changing the phospholipid composition and structures of the apical membranes. In this review, we focus on the molecular mechanisms for the protection of hepatocytes against bile salt cytotoxicity. Further understanding of these mechanisms will help to develop new therapeutic strategies for cholestatic liver diseases.
Topics: ATP-Binding Cassette Transporters; Animals; Bile Acids and Salts; Cholesterol; Hepatocytes; Hydrophobic and Hydrophilic Interactions; Micelles; Phospholipids
PubMed: 30930437
DOI: 10.1248/cpb.c18-01029 -
Biophysical Journal Feb 2020The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC),...
The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (K = 46 μM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a K ≈ 0.2 μM for PC/PG 1:1 and 0.6 μM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.
Topics: Calorimetry; Exenatide; Liposomes; Peptides; Phosphatidylglycerols; Phospholipids
PubMed: 31972156
DOI: 10.1016/j.bpj.2019.12.028 -
Nature Communications Aug 2022Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular...
Production of high density lipoprotein (HDL) requires ATP-binding cassette transporter A1 (ABCA1) to drive phospholipid (PL) from the plasma membrane into extracellular apolipoprotein A-I. Here, we use simulations to show that domains of ABCA1 within the plasma membrane remove PL from the membrane's outer leaflet. In our simulations, after the lipid diffuses into the interior of ABCA1's outward-open cavity, PL extracted by the gateway passes through a ring-shaped domain, the annulus orifice, which forms the base of an elongated hydrophobic tunnel in the transporter's extracellular domain. Engineered mutations in the gateway and annulus strongly inhibit lipid export by ABCA1 without affecting cell-surface expression levels. Our finding that ABCA1 extracts lipid from the outer face of the plasma membrane and forces it through its gateway and annulus into an elongated hydrophobic tunnel contrasts with the alternating access model, which proposes that ABCA1 flops PL substrate from the inner leaflet to the outer leaflet of the membrane. Consistent with our model, ABCA1 lacks the charged amino acid residues in the transmembrane domain found in the floppase members of the ABC transporter family.
Topics: ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Apolipoprotein A-I; Cell Membrane; Lipoproteins, HDL; Phospholipids; Protein Domains
PubMed: 35974019
DOI: 10.1038/s41467-022-32437-3 -
Biochimica Et Biophysica Acta.... Feb 2022Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to...
Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.
Topics: Annexin A2; Biophysical Phenomena; Calcium; Carrier Proteins; Cell Communication; Cell Membrane; Cytoskeleton; Humans; Membrane Lipids; Phosphatidylserines; Phospholipids
PubMed: 34699769
DOI: 10.1016/j.bbamem.2021.183810 -
ACS Chemical Biology Apr 2024Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous...
Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca with a ∼ 45 μM. Each subsequent binding affinity ( ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca binding affinity, we explored the extent that Ca binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and dioleoylphosphatidylserine (DOPS) positively modulated Ca binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) was reduced with increasing [Ca]. Overall, we find that specific lipids differentially modulate Ca binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca, and anionic phospholipids that may also control some aspects of vesicular exocytosis.
Topics: Calcium; Exocytosis; Neurotransmitter Agents; Phospholipids; Synaptic Transmission; Synaptic Vesicles; Synaptotagmin I; Animals; Rats
PubMed: 38566504
DOI: 10.1021/acschembio.3c00772