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Molecular and Cellular Biology Sep 1997The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several...
The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.
Topics: Animals; Binding Sites; COS Cells; Models, Molecular; Phospholipids; Phosphotyrosine; Protein Conformation; Signal Transduction; src Homology Domains
PubMed: 9271429
DOI: 10.1128/MCB.17.9.5540 -
Journal of Medicinal Chemistry May 2022Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic...
Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic dysregulation in the liver. LRH-1 contains a large ligand-binding pocket, but generating synthetic modulators has been challenging. We have had recent success generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding and residues at the mouth of the pocket to mimic interactions made by phospholipids. Here, we unite these two designs into one molecule to synthesize the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and structural studies, we show that selective modulation can be driven through contacting deep versus surface polar regions in the pocket. While deep pocket contacts convey high affinity, contacts with the pocket mouth dominate allostery and provide a phospholipid-like transcriptional response in cultured cells.
Topics: Cell Line; Phospholipids; Receptors, Cytoplasmic and Nuclear
PubMed: 35503419
DOI: 10.1021/acs.jmedchem.2c00235 -
The Journal of Physical Chemistry. B Sep 2016The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and...
The interactions of two highly positively charged short peptide sequences with negatively charged lipid bilayers were explored by fluorescence binding assays and all-atom molecular dynamics simulations. The bilayers consisted of mixtures of phosphatidylglycerol (PG) and phosphatidylcholine (PC) lipids as well as a fluorescence probe that was sensitive to the interfacial potential. The first peptide contained nine arginine repeats (Arg9), and the second one had nine lysine repeats (Lys9). The experimentally determined apparent dissociation constants and Hill cooperativity coefficients demonstrated that the Arg9 peptides exhibited weakly anticooperative binding behavior at the bilayer interface at lower PG concentrations, but this anticooperative effect vanished once the bilayers contained at least 20 mol % PG. By contrast, Lys9 peptides showed strongly anticooperative binding behavior at all PG concentrations, and the dissociation constants with Lys9 were approximately 2 orders of magnitude higher than with Arg9. Moreover, only arginine-rich peptides could bind to the phospholipid bilayers containing just PC lipids. These results along with the corresponding molecular dynamics simulations suggested two important distinctions between the behavior of Arg9 and Lys9 that led to these striking differences in binding and cooperativity. First, the interactions of the guanidinium moieties on the Arg side chains with the phospholipid head groups were stronger than for the amino group. This helped facilitate stronger Arg9 binding at all PG concentrations that were tested. However, at PG concentrations of 20 mol % or greater, the Arg9 peptides came into sufficiently close proximity with each other so that favorable like-charge pairing between the guanidinium moieties could just offset the long-range electrostatic repulsions. This led to Arg9 aggregation at the bilayer surface. By contrast, Lys9 molecules experienced electrostatic repulsion from each other at all PG concentrations. These insights may help explain the propensity for cell penetrating peptides containing arginine to more effectively cross cell membranes in comparison with lysine-rich peptides.
Topics: Lipid Bilayers; Peptides; Phospholipids; Polylysine
PubMed: 27571288
DOI: 10.1021/acs.jpcb.6b05604 -
Journal of Bacteriology Jul 1978Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate...
Mutants of Escherichia coli containing a defective sn-glycerol 3-phosphate acyltransferase are conditionally defective in the synthesis of acylglycerol phosphate (acylglycerol-P). Incubation of a deep rough derivative of one of these plsB strains with 1-[3H]oleoylglycerol-32P resulted in the binding of up to 70 nmol of oleoylglycerol-P per 100 nmol of cellular phospholipid. The binding was dependent on time, oleoylglycerol-P concentration, and the quantity of cells employed. The rate and extent of oleoylglycerol-P binding was affected by the deep rough mutation. The altered phospholipid composition due to oleoylglycerol-P binding was without consequence on cell growth and viability, but caused the appearance of intracellular multilamellar structures. Use of the double-labeled oleoylglycerol P demonstrated that the entire molecule was bound to the cell. Intact [3H]-oleoylglycerol-32P was converted to phosphatidylethanolamine and phosphotidyl-glycerol at a rate about 40% of that of de novo phospholipid synthesis. These data demonstrate the transmembrane movement of oleoylglycerol-P to the inner surface of the cytoplasmic membrane and suggest that it may become possible to supplement plsB strains of E. coli with acylglycerol-P's.
Topics: Cell Membrane; Escherichia coli; Glycerol-3-Phosphate O-Acyltransferase; Glycerophosphates; Mutation; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids
PubMed: 353031
DOI: 10.1128/jb.135.1.215-226.1978 -
IUBMB Life Aug 2006Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, phosphatidylcholine producing phosphatidic acid and free choline. This... (Review)
Review
Phospholipase D (PLD) hydrolyzes the phosphodiester bond of the predominant membrane phospholipid, phosphatidylcholine producing phosphatidic acid and free choline. This activity can participate in signal transduction pathways and impact on vesicle trafficking for secretion and endocytosis, as well as receptor signalling. Phospholipids can regulate PLD activity directly, through specific intermolecular interactions, or indirectly, through their effect on the localization or activity of PLD's protein effectors. This short review highlights these various phospholipid inputs into the regulation of PLD activity and also reviews potential roles for PLD-generated phosphatidic acid, particularly a mechanism by which the phospholipid may participate in the process of vesicular trafficking.
Topics: ADP-Ribosylation Factors; Amino Acid Motifs; Amino Acid Sequence; Animals; Biological Transport; Cell Membrane; Humans; Lipid Metabolism; Models, Biological; Phosphatidic Acids; Phospholipase D; Phospholipids; Protein Kinase C; Protein Structure, Tertiary; Signal Transduction; rho GTP-Binding Proteins
PubMed: 16916782
DOI: 10.1080/15216540600871142 -
European Journal of Biochemistry Apr 1988A novel inhibitor of blood coagulation has been isolated from the intima of bovine aorta. The inhibitor, vascular anticoagulant (VAC), has been purified to an active...
Purification and characterization of a novel protein from bovine aorta that inhibits coagulation. Inhibition of the phospholipid-dependent factor-Xa-catalyzed prothrombin activation, through a high-affinity binding of the anticoagulant to the phospholipids.
A novel inhibitor of blood coagulation has been isolated from the intima of bovine aorta. The inhibitor, vascular anticoagulant (VAC), has been purified to an active fraction that contains two Coomassie-blue-staining bands (Mr = 34,000 and Mr = 32,000, as judged by sodium dodecyl sulfate/polyacrylamide electrophoresis). Both bands are single-chain proteins, having no glycoprotein features. Furthermore, they do not contain any detectable 4-carboxyglutamic acid residues. Both proteins have an identical isoelectric pH of approximately 4.5. VAC binds in the presence of calcium ions to a bilayer consisting of 20% dioleoylglycerophosphoserine and 80% dioleoylglycerophosphocholine with a Kd = 6 nM. The binding is dependent on the calcium concentration: half-saturation of binding occurs at a calcium concentration of 0.8 mM. The binding is completely reversible with EDTA. Furthermore the phospholipid/VAC ratio at saturation was n = 112 and n = 32 mol/mol for 0.5 mM Ca2+ and 2 mM Ca2+, respectively. Binding does not occur between VAC and pure dioleoylglycerophosphocholine. In a system with purified coagulation factors VAC inhibits the activation of prothrombin by factor Xa and calcium only in the presence of negatively charged phospholipids. VAC decreases the Vmax and increases the Km of the factor-Xa-catalyzed prothrombin activation. Based on these results, we conclude that we have purified from bovine aortic intima an anticoagulant protein, which exerts its activity through a calcium-dependent binding to negatively charged phospholipids, and thus interferes with the assembly of prothrombinase on the phospholipid surface.
Topics: Animals; Annexins; Aorta; Blood Coagulation; Catalysis; Cattle; Factor V; Factor X; Factor Xa; Peptides; Phospholipids; Protein Binding; Prothrombin; Serine Endopeptidases
PubMed: 2965640
DOI: 10.1111/j.1432-1033.1988.tb13981.x -
Fly 2011Obesity has reached pandemic proportions globally and is often associated with lipotoxic heart diseases. In the obese state, caloric surplus is accommodated in the...
Obesity has reached pandemic proportions globally and is often associated with lipotoxic heart diseases. In the obese state, caloric surplus is accommodated in the adipocytes as triglycerides. As the storage capacity of adipocytes is exceeded or malfunctioning, lipids begin to infiltrate and accumulate in non-adipose tissues, including the myocardium of the heart, leading to organ dysfunction. While the disruption of caloric homeostasis has been widely viewed as a principal mechanism in contributing to peripheral tissue steatosis and lipotoxicity, our recent studies in Drosophila have led to the novel finding that deregulation of phospholipid homeostasis may also significantly contribute to the pathogenesis of lipotoxic cardiomyopathy. Fly mutants that bear perturbations in phosphatidylethanolamine (PE) biosynthesis, such as the easily-shocked (eas) mutants defective in ethanolamine kinase, incurred aberrant activation of the sterol regulatory element binding protein (SREBP) pathway, thereby causing chronic lipogenesis and cardiac steatosis that culminates in the development of lipotoxic cardiomyopathy. Here, we describe the potential relationship between SREBP and other eas-associated phenotypes, such as neuronal excitability defects. We will further discuss the additional implications presented by our work toward the effects of altered lipid metabolism on cellular growth and/or proliferation in response to defective phospholipid homeostasis.
Topics: Animals; Cardiomyopathies; Drosophila; Homeostasis; Lipogenesis; Obesity; Phenotype; Phospholipids; Sterol Regulatory Element Binding Proteins
PubMed: 21494094
DOI: 10.4161/fly.5.3.15708 -
The Journal of Biological Chemistry Feb 2020PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that...
PlsX plays a central role in the coordination of fatty acid and phospholipid biosynthesis in Gram-positive bacteria. PlsX is a peripheral membrane acyltransferase that catalyzes the conversion of acyl-ACP to acyl-phosphate, which is in turn utilized by the polytopic membrane acyltransferase PlsY on the pathway of bacterial phospholipid biosynthesis. We have recently studied the interaction between PlsX and membrane phospholipids and , and observed that membrane association is necessary for the efficient transfer of acyl-phosphate to PlsY. However, understanding the molecular basis of such a channeling mechanism remains a major challenge. Here, we disentangle the binding and insertion events of the enzyme to the membrane, and the subsequent catalysis. We show that PlsX membrane binding is a process mostly mediated by phospholipid charge, whereas fatty acid saturation and membrane fluidity remarkably influence the membrane insertion step. Strikingly, the PlsX mutant, whose biological functionality was severely compromised but remains catalytically active , was able to superficially bind to phospholipid vesicles, nevertheless, it loses the insertion capacity, strongly supporting the importance of membrane insertion in acyl-phosphate delivery. We propose a mechanism in which membrane fluidity governs the insertion of PlsX and thus regulates the biosynthesis of phospholipids in Gram-positive bacteria. This model may be operational in other peripheral membrane proteins with an unprecedented impact in drug discovery/development strategies.
Topics: Bacillus subtilis; Bacterial Proteins; Enterococcus faecalis; Escherichia coli; Gram-Positive Bacteria; Membrane Fluidity; Phosphates; Phospholipids
PubMed: 31796629
DOI: 10.1074/jbc.RA119.011122 -
The Journal of Biological Chemistry Jan 2013The phospholipase A(2) (PLA(2)) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due... (Review)
Review
Using hydrogen/deuterium exchange mass spectrometry to define the specific interactions of the phospholipase A2 superfamily with lipid substrates, inhibitors, and membranes.
The phospholipase A(2) (PLA(2)) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA(2) types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA(2) enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA(2)/membrane interactions are addressed.
Topics: Allosteric Site; Binding Sites; Catalytic Domain; Cell Membrane; Deuterium; Deuterium Exchange Measurement; Humans; Hydrogen; Mass Spectrometry; Models, Molecular; Phosphodiesterase Inhibitors; Phospholipase A2 Inhibitors; Phospholipases A2; Phospholipids; Protein Binding
PubMed: 23209293
DOI: 10.1074/jbc.R112.421909 -
The Journal of Biological Chemistry May 1990We have used fluorescence and circular dichroism spectroscopy to investigate the effect of phospholipid on the structure and molecular stability of human apolipoprotein...
We have used fluorescence and circular dichroism spectroscopy to investigate the effect of phospholipid on the structure and molecular stability of human apolipoprotein A-IV (apo-A-IV). Binding of apo-A-IV to egg phosphatidylcholine vesicles was rapid and did not cause release of encapsulated 6-carboxyfluorescein. Fluorometric titration established that apo-A-IV bound to the vesicles with an association constant of 1.36 x 10(6) liters/mol and a binding maximum of 2 molecules per vesicle. Binding of apo-A-IV to the vesicle surface caused a progressive increase in alpha helicity from 43% at baseline to 83% at saturation; denaturation studies showed that the free energy of stabilization of binding was 6.31 kcal/mol. Fluorescence quenching studies revealed that binding of apo-A-IV to the vesicles was associated with a dramatic decrease in the fractional exposure of tyrosine to iodide, and a decrease in the efficiency of intramolecular tyrosine-tryptophan energy transfer. These findings suggest that the binding of apo-A-IV to the vesicle surface may involve a relaxation of the globular protein conformation in which the tyrosine containing alpha-helical domains surrounding the tryptophan "unfold" and reorient their hydrophobic faces toward the phospholipid monolayer, with a consequent induction of additional alpha-helical structure. However, our data also suggest that apo-A-IV does not penetrate deeply into the region of the phospholipid fatty acyl chains, but rather sits higher in the monolayer, intercalated between the charged phospholipid head groups. This characteristic may determine the labile interaction of apo-A-IV with high density lipoproteins.
Topics: Apolipoproteins A; Circular Dichroism; Energy Transfer; Fluoresceins; Humans; Liposomes; Molecular Structure; Phosphatidylcholines; Phospholipids; Protein Conformation; Protein Denaturation; Spectrometry, Fluorescence; Thermodynamics; Tryptophan; Tyrosine
PubMed: 2335517
DOI: No ID Found