scholarly journals Correlation of Vesicle Binding and Phospholipid Dynamics with Phospholipase C Activity

2009 ◽  
Vol 284 (24) ◽  
pp. 16099-16107 ◽  
Author(s):  
Mingming Pu ◽  
Xiaomin Fang ◽  
Alfred G. Redfield ◽  
Anne Gershenson ◽  
Mary F. Roberts
Keyword(s):  
Enzyme Activity ◽  
Phospholipase C ◽  
Lateral Diffusion ◽  
Lipid Vesicles ◽  
Membrane Lipid ◽  
Correlation Spectroscopy ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Substrate Analogue ◽  
Nmr Methods

The enzymatic activity of the peripheral membrane protein, phosphatidylinositol-specific phospholipase C (PI-PLC), is increased by nonsubstrate phospholipids with the extent of enhancement tuned by the membrane lipid composition. For Bacillus thuringiensis PI-PLC, a small amount of phosphatidylcholine (PC) activates the enzyme toward its substrate PI; above 0.5 mol fraction PC (XPC), enzyme activity decreases substantially. To provide a molecular basis for this PC-dependent behavior, we used fluorescence correlation spectroscopy to explore enzyme binding to multicomponent lipid vesicles composed of PC and anionic phospholipids (that bind to the active site as substrate analogues) and high resolution field cycling 31P NMR methods to estimate internal correlation times (τc) of phospholipid headgroup motions. PI-PLC binds poorly to pure anionic phospholipid vesicles, but 0.1 XPC significantly enhances binding, increases PI-PLC activity, and slows nanosecond rotational/wobbling motions of both phospholipid headgroups, as indicated by increased τc. PI-PLC activity and phospholipid τc are constant between 0.1 and 0.5 XPC. Above this PC content, PI-PLC has little additional effect on the substrate analogue but further slows the PC τc, a motional change that correlates with the onset of reduced enzyme activity. For PC-rich bilayers, these changes, together with the reduced order parameter and enhanced lateral diffusion of the substrate analogue in the presence of PI-PLC, imply that at high XPC, kinetic inhibition of PI-PLC results from intravesicle sequestration of the enzyme from the bulk of the substrate. Both methodologies provide a detailed view of protein-lipid interactions and can be readily adapted for other peripheral membrane proteins.

scholarly journals A Complex Interplay of Anionic Phospholipid Binding Regulates 3′-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gloria de las Heras-Martínez ◽  
Véronique Calleja ◽  
Remy Bailly ◽  
Jean Dessolin ◽  
Banafshé Larijani ◽  
...  
Keyword(s):  
Conformational Dynamics ◽  
Pi3k Pathway ◽  
Correlation Spectroscopy ◽  
Fluorescence Lifetime Imaging ◽  
Anionic Phospholipid ◽  
Direct Role ◽  
Time Resolved ◽  
Anionic Phospholipids ◽  
Spatio Temporal ◽  
Phosphoinositide Dependent Kinase

Abstract 3′-Phosphoinositide-dependent-Kinase-1 (PDK1) is a master regulator whereby its PI3-kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging (Fluorescence Lifetime Imaging and Fluorescence Correlation Spectroscopy) and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We have established a new anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple homodimer states. We show that the dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 homodimer conformations. Our findings provide a role for the PtdSer binding site and its previously unrewarding role in PDK1 downregulation, suggesting a possible therapeutic strategy where the constitutively active dimer conformer of PDK1 may be rendered inactive by small molecules that drive it to its PtdSer-bound conformer.

scholarly journals Size-Tunable Paclitaxel Nanoparticles Stabilized by Anionic Phospholipids for Transdermal Delivery Applications

2020 ◽  
Vol 15 (3) ◽  
pp. 1934578X1990068
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Hiroki Hamada
Keyword(s):  
Stratum Corneum ◽  
Transdermal Delivery ◽  
Skin Tissue ◽  
Composite Nanoparticles ◽  
Rat Skin ◽  
Cooling Process ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Subsequent Heating

Composite nanoparticles composed of an anionic phospholipid of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG) and paclitaxel (PTX) were successfully prepared by mixing them in water followed by a subsequent heating/cooling process. The size of DPPG-PTX nanoparticle could be easily tuned by ultrasonic fragmentation. Upon addition of small-sized fluorescently labeled paclitaxel (FLPTX) nanoparticles with DPPG (DPPG-FLPTX) to rat skin tissue, part of the FLPTX molecules permeated to the stratum corneum.

scholarly journals Extension of the Scope of Anionic Phospholipid-Based Nanoformulation to Kaempferol and Indometacin

2021 ◽  
Vol 16 (3) ◽  
pp. 1934578X2110026
Author(s):  
Noriyuki Uchida ◽  
Masayoshi Yanagi ◽  
Kei Shimoda ◽  
Hiroki Hamada
Keyword(s):  
Phosphatidic Acid ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Dispersion Agent

In this work, resveratrol was dispersed with anionic phospholipids of 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, and 1,2-distearoyl-sn-glycero-3-phosphoglycerol. Moreover, small-sized nanoparticles of kaempferol and indometacin were successfully prepared by using DPPG as a dispersion agent.

Solid state NMR of membrane proteins: methods and applications

2021 ◽  
Author(s):  
Vivien Yeh ◽  
Boyan B. Bonev
Keyword(s):  
Solid State ◽  
High Resolution ◽  
Membrane Proteins ◽  
Solid State Nmr ◽  
Lipid Membrane ◽  
Cell Function ◽  
Membrane Lipid ◽  
Pharmacological Intervention ◽  
Nmr Methods ◽  
Informative Approach

Membranes of cells are active barriers, in which membrane proteins perform essential remodelling, transport and recognition functions that are vital to cells. Membrane proteins are key regulatory components of cells and represent essential targets for the modulation of cell function and pharmacological intervention. However, novel folds, low molarity and the need for lipid membrane support present serious challenges to the characterisation of their structure and interactions. We describe the use of solid state NMR as a versatile and informative approach for membrane and membrane protein studies, which uniquely provides information on structure, interactions and dynamics of membrane proteins. High resolution approaches are discussed in conjunction with applications of NMR methods to studies of membrane lipid and protein structure and interactions. Signal enhancement in high resolution NMR spectra through DNP is discussed as a tool for whole cell and interaction studies.

scholarly journals Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

2016 ◽  
Vol 148 (3) ◽  
pp. 227-237 ◽  
Author(s):  
Sun-Joo Lee ◽  
Feifei Ren ◽  
Eva-Maria Zangerl-Plessl ◽  
Sarah Heyman ◽  
Anna Stary-Weinzinger ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Inward Rectifier ◽  
Primary Site ◽  
Structural Basis ◽  
Channel Activity ◽  
Anionic Phospholipid ◽  
X Ray ◽  
Anionic Phospholipids ◽  
Kir Channel

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

scholarly journals Molecular determinants of KA1 domain-mediated autoinhibition and phospholipid activation of MARK1 kinase

2017 ◽  
Vol 474 (3) ◽  
pp. 385-398 ◽  
Author(s):  
Ryan P. Emptage ◽  
Mark A. Lemmon ◽  
Kathryn M. Ferguson
Keyword(s):  
Membrane Surface ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Anionic Phospholipid ◽  
Anionic Phospholipids ◽  
Disease States ◽  
C Terminus ◽  
Regulatory Module ◽  
Correct Location ◽  
Mechanistic Basis

Protein kinases are frequently regulated by intramolecular autoinhibitory interactions between protein modules that are reversed when these modules bind other ‘activating’ protein or membrane-bound targets. One group of kinases, the MAP/microtubule affinity-regulating kinases (MARKs) contain a poorly understood regulatory module, the KA1 (kinase associated-1) domain, at their C-terminus. KA1 domains from MARK1 and several related kinases from yeast to humans have been shown to bind membranes containing anionic phospholipids, and peptide ligands have also been reported. Deleting or mutating the C-terminal KA1 domain has been reported to activate the kinase in which it is found — also suggesting an intramolecular autoinhibitory role. Here, we show that the KA1 domain of human MARK1 interacts with, and inhibits, the MARK1 kinase domain. Using site-directed mutagenesis, we identify residues in the KA1 domain required for this autoinhibitory activity, and find that residues involved in autoinhibition and in anionic phospholipid binding are the same. We also demonstrate that a ‘mini’ MARK1 becomes activated upon association with vesicles containing anionic phospholipids, but only if the protein is targeted to these vesicles by a second signal. These studies provide a mechanistic basis for understanding how MARK1 and its relatives may require more than one signal at the membrane surface to control their activation at the correct location and time. MARK family kinases have been implicated in a plethora of disease states including Alzheimer's, cancer, and autism, so advancing our understanding of their regulatory mechanisms may ultimately have therapeutic value.

NMR Studies of the Order and Dynamics of Dipalmitoylphosphatidylchoiine Bilayers as a Function of Pressure

1996 ◽  
Author(s):  
Ana Jonas ◽  
Xiangdong Peng
Keyword(s):  
Liquid Crystalline ◽  
Relaxation Times ◽  
Lateral Diffusion ◽  
Lattice Relaxation ◽  
Pressure Effects ◽  
Spin Lattice Relaxation ◽  
2H Nmr ◽  
Spin Lattice ◽  
Spin Locking ◽  
Nmr Methods

We have used 2H NMR methods to examine the order and dynamics of dipalmitoylphosphatidylcholine (DPPC) in multilamellar and small unilamellar vesicles in water as a function of pressure. Multipulse 2H NMR techniques were used with selectively deuterated DPPC on both chains at positions C-2, C-9, or C-13, to obtain lineshapes, spin-lattice relaxation times (T1), and spin-spin relaxation times (T2) at 50 °C from 1 bar to 5.2 kbar pressure. This pressure range allowed us to explore the phase behavior of DPPC from the liquid crystalline (LC) phase through various gel phases (Gl, Gll, Glll, GX), including the interdigited Gi phase. Pressure has an ordering effect: on all chain segments in all the phases. In the LC phase, the order parameter (SCD) decreases from C-2 > C-9 > C-13, while in the gel phases SCD decreases from C-9 > C-13 > C-2, indicating that in the gel phases the middle segments of the chains are more restricted in their motions than the ends. In the LC phase, T1 and T2 values for all segments decrease with pressure and have an order from C-13 > C-9 > C-2. These results suggest that similar conformational motions and molecular rotational motions occur in the LC state in all segments, but have increased amplitudes and frequencies toward the methyl ends. At the phase transitions, discontinuities and abrupt reversal of the slopes for the T1 or T2 dependences on pressure indicate major changes in motional modes and rates for DPPC molecules in the different structures. In the second part of this study, we have measured the lateral diffusion of DPPC in sonicated vesicles in D2O as a function of pressure. The spin-lattice relaxation rate in the rotating frame T−11p was plotted as a function of the square root of the spin-locking field angular frequency (ω1)1/2, and the lateral diffusion coefficient (D) was calculated from the slope. Pressure effects are observed on lateral diffusion in the LC phase (D = 5.4 − 2 × 10−9 cm2 seconds, from 1 to 300 bar) but are negligible in the GI phase (D ≈ 1.0 × 10−9 cm2 seconds, from 400 to 800 bar).

Ligand-regulated secretion of recombinant annexin V from cultured thyroid epithelial cells

2002 ◽  
Vol 282 (6) ◽  
pp. C1313-C1321 ◽  
Author(s):  
Xiuqiong Wang ◽  
Marcia A. Kaetzel ◽  
Sung E. Yoo ◽  
Paul S. Kim ◽  
John R. Dedman
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
External Surface ◽  
Annexin V ◽  
Therapeutic Agents ◽  
Anionic Phospholipid ◽  
Thyroid Cells ◽  
Tissue Ischemia ◽  
Anionic Phospholipids ◽  
Activated Platelets

The exposure of anionic phospholipids on the external surface of injured endothelial cells and activated platelets is a primary biological signal to initiate blood coagulation. Disease conditions that promote the formation of ectopic thrombi result in tissue ischemia. Annexins, Ca2+-dependent anionic phospholipid binding proteins, are potential therapeutic agents for the inhibition of coagulation. We have designed a transgene that targets secretion of annexin V from cultured thyroid cells under the control of doxycycline. Our results indicate that annexin V in the endoplasmic reticulum (ER)/Golgi lumen does not affect the synthesis, processing, and secretion of thyroglobulin. ER luminal Ca2+was moderately increased and can be released by inositol 1,4,5-trisphosphate. Our study demonstrates that targeting and secretion of annexin V through the secretory pathway of mammalian cells does not adversely affect cellular function. Regulated synthesis and release of annexin V may exert anticoagulatory and anti-inflammatory effects systemically and may prove useful in further developing therapeutic strategies for conditions including antiphospholipid syndrome.

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