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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 -
Nutrients Oct 2021Polycystic Ovary Syndrome (PCOS) is the most frequent endocrine disease in females of reproductive age and is characterized by multifactorial unhealthy conditions... (Clinical Trial)
Clinical Trial
Berberine Phospholipid Is an Effective Insulin Sensitizer and Improves Metabolic and Hormonal Disorders in Women with Polycystic Ovary Syndrome: A One-Group Pretest-Post-Test Explanatory Study.
Polycystic Ovary Syndrome (PCOS) is the most frequent endocrine disease in females of reproductive age and is characterized by multifactorial unhealthy conditions related to hormonal unbalance and also to dysmetabolism and inflammation. Recently, increasing evidence has shown that natural plant-based products may play a role in PCOS management. The aim of this one-group pretest-post-test explanatory study was to evaluate, in normal-overweight PCOS women with normal menses, the effectiveness of berberine on: Insulin resistance (IR) by Homeostasis Model Assessment (HOMA); Inflammation by C-Reactive Protein (CRP), Tumor Necrosis Factor α (TNF-α); Lipid metabolism; Sex hormone profile and symptoms correlated to hyperandrogenism, such as acne, by Global Acne Grading System (GAGS) and Cardiff Acne Disability Index (CADI); Body composition by DXA. Finally, adverse effects were assessed by liver and kidney functions and creatine phosphokinase (CPK). All these parameters were collected at baseline and 60 days after supplementation with a new bioavailable and safe berberine formulation. Twelve females (aged 26.6 ± 4.9, BMI 25.3 ± 3.6) were supplied for 60 days with two tablets/day (550 mg/table) of the bioavailable berberine. Results showed a statistically significant decrease in HOMA, CRP, TNF-α, Triglycerides, testosterone, Body Mass Index (BMI), Visceral Adipose Tissue (VAT), fat mass, GAGS and CADI scores, and a statistically significant increase in sex hormone-binding globulin (SHBG). Liver and kidney functions and CPK are not statistically significantly different. Therefore, berberine can represent a safe novel dietary supplement, helpful in treatment strategy for PCOS.
Topics: Adult; Berberine; Body Composition; Endpoint Determination; Female; Hormones; Humans; Inflammation; Insulin; Insulin Resistance; Linear Models; Lipids; Phospholipids; Polycystic Ovary Syndrome
PubMed: 34684666
DOI: 10.3390/nu13103665 -
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 -
Handbook of Experimental Pharmacology 2024The transmembrane protein 16 (TMEM16) family consists of Ca-activated chloride channels and phospholipid scramblases. Ten mammalian TMEM16 proteins, TMEM16A-K (with no... (Review)
Review
The transmembrane protein 16 (TMEM16) family consists of Ca-activated chloride channels and phospholipid scramblases. Ten mammalian TMEM16 proteins, TMEM16A-K (with no TMEM16I), and several non-mammalian TMEM16 proteins, such as afTMEM16 and nhTMEM16, have been discovered. All known TMEM16 proteins are homodimeric proteins containing two subunits. Each subunit consists of ten transmembrane helices with Ca-binding sites and a single ion-permeation/phospholipid transport pathway. The ion-permeation pathway and the phospholipid transport pathway of TMEM16 proteins have a wide intracellular vestibule, a narrow neck, and a smaller extracellular vestibule. Interestingly, the lining wall of the ion-permeation/phospholipid transport pathway may be formed, at least partially, by membrane phospholipids, though the degree of pore-wall forming by phospholipids likely varies among TMEM16 proteins. Thus, the biophysical properties and activation mechanisms of TMEM16 proteins could differ from each other accordingly. Here we review the current understanding of the structure and function of TMEM16 molecules.
Topics: Humans; Animals; Chloride Channels; Phospholipid Transfer Proteins; Anoctamins; Ion Transport; Phospholipids; Calcium; Mammals
PubMed: 35792944
DOI: 10.1007/164_2022_595 -
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 -
International Journal of Biological... Jul 2022Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a...
Autophagy is a process in which parts of the eukaryotic cell are selectively degraded in the lysosome. The materials to be catabolized are first surrounded by a double-membrane structure, the autophagosome. Autophagosome generation is a complex event, in which many proteins are involved. Among the latter, yeast Atg8 or its mammalian orthologues are essential in autophagosome membrane elongation, shaping and closure. A subfamily of the human Atg8 orthologues is formed by the proteins LC3A, LC3B, and LC3C. Previous studies suggest that, at variance with the other two, LC3C does not participate in cardiolipin-mediated mitophagy. The present study was devoted to exploring the binding of LC3C to lipid vesicles, bilayers and monolayers, and the ensuing protein-dependent perturbing effects, in the absence of the mitochondrial lipid cardiolipin. All Atg8 orthologues are covalently bound to a phospholipid prior to their involvement in autophagosome elongation. In our case, a mutant in the C-terminal amino acid, LC3C G126C, together with the use of a maleimide-derivatized phosphatidyl ethanolamine, ensured LC3C lipidation, up to 100% under certain conditions. Ultracentrifugation, surface pressure measurements, spectroscopic and cryo-electron microscopic techniques revealed that lipidated LC3C induced vesicle aggregation (5-fold faster in sonicated than in large unilamellar vesicles) and inter-vesicular lipid mixing (up to 82%), including inner-monolayer lipid mixing (up to 32%), consistent with in vitro partial vesicle fusion. LC3C was also able to cause the release of 80-90% vesicular aqueous contents. The data support the idea that LC3C would be able to help in autophagosome elongation/fusion in autophagy phenomena.
Topics: Autophagy; Cardiolipins; Humans; Microtubule-Associated Proteins; Phospholipids; Protein Binding; Saccharomyces cerevisiae
PubMed: 35618088
DOI: 10.1016/j.ijbiomac.2022.05.129 -
Nature Structural & Molecular Biology Apr 2024Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the...
Class C G-protein-coupled receptors (GPCRs) are activated through binding of agonists to the large extracellular domain (ECD) followed by rearrangement of the transmembrane domains (TMDs). GPR156, a class C orphan GPCR, is unique because it lacks an ECD and exhibits constitutive activity. Impaired GPR156-G signaling contributes to loss of hearing. Here we present the cryo-electron microscopy structures of human GPR156 in the G-free and G-coupled states. We found that an endogenous phospholipid molecule is located within each TMD of the GPR156 dimer. Asymmetric binding of Gα to the phospholipid-bound GPR156 dimer restructures the first and second intracellular loops and the carboxy-terminal part of the elongated transmembrane 7 (TM7) without altering dimer conformation. Our findings reveal that GPR156 is a transducer for phospholipid signaling. Constant binding of abundant phospholipid molecules and the G-protein-induced reshaping of the cytoplasmic face provide a basis for the constitutive activation of GPR156.
Topics: Humans; Cryoelectron Microscopy; Receptors, G-Protein-Coupled; Signal Transduction; GTP-Binding Proteins; Phospholipids
PubMed: 38332368
DOI: 10.1038/s41594-024-01224-7 -
Journal of Lipid Research Jun 2023This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular... (Review)
Review
This review considers the hypothesis that a small portion of plasma membrane cholesterol regulates reverse cholesterol transport in coordination with overall cellular homeostasis. It appears that almost all of the plasma membrane cholesterol is held in stoichiometric complexes with bilayer phospholipids. The minor fraction of cholesterol that exceeds the complexation capacity of the phospholipids is called active cholesterol. It has an elevated chemical activity and circulates among the organelles. It also moves down its chemical activity gradient to plasma HDL, facilitated by the activity of ABCA1, ABCG1, and SR-BI. ABCA1 initiates this process by perturbing the organization of the plasma membrane bilayer, thereby priming its phospholipids for translocation to apoA-I to form nascent HDL. The active excess sterol and that activated by ABCA1 itself follow the phospholipids to the nascent HDL. ABCG1 similarly rearranges the bilayer and sends additional active cholesterol to nascent HDL, while SR-BI simply facilitates the equilibration of the active sterol between plasma membranes and plasma proteins. Active cholesterol also flows downhill to cytoplasmic membranes where it serves both as a feedback signal to homeostatic ER proteins and as the substrate for the synthesis of mitochondrial 27-hydroxycholesterol (27HC). 27HC binds the LXR and promotes the expression of the aforementioned transport proteins. 27HC-LXR also activates ABCA1 by competitively displacing its inhibitor, unliganded LXR. § Considerable indirect evidence suggests that active cholesterol serves as both a substrate and a feedback signal for reverse cholesterol transport. Direct tests of this novel hypothesis are proposed.
Topics: High-Density Lipoproteins, Pre-beta; Cholesterol; Biological Transport; Sterols; Phospholipids; ATP Binding Cassette Transporter 1
PubMed: 37169287
DOI: 10.1016/j.jlr.2023.100385 -
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