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

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

Abstract3’-Phosphoinositide-dependent-Kinase-1 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, FCS 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 establish an anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple homodimer states. The dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 homodimer conformations. Our findings indicate that the alteration of specific basic residues of PDK1-PH domain leads to its constitutive activation, a potential significance in different types of carcinomas.

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 Spatio-temporal Structure of Filler in Rubber Studied with X-ray Scattering

2014 ◽  
Vol 70 (a1) ◽  
pp. C1327-C1327
Author(s):  
Yuya Shinohara ◽  
Hiroyuki Kishimoto ◽  
Yoshiyuki Amemiya
Keyword(s):  
Temporal Structure ◽  
Hierarchical Structures ◽  
Vital Role ◽  
Correlation Spectroscopy ◽  
Reinforcement Effect ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Scattering ◽  
Spatio Temporal ◽  
Ray Scattering

Filler nanoparticles such as carbon black and silica play a vital role in the reinforcement effect of rubber, whereby its viscoelastic and mechanical properties are dramatically improved. The reinforcement effect is of great importance for developing rubber products such as vehicle tires. Its mechanism, however, has not been clarified despite a large number of studies. We have aimed at elucidating the mechanism by clarifying the spatio-temporal hierarchical structure of filler nanoparticles in rubber with various X-ray scattering techniques: ultra-small-angle X-ray scattering (USAXS) for elucidating the hierarchical structures of filler aggregates and their deformation under stretching, and X-ray photon correlation spectroscopy (XPCS) for observing the translational and rotational dynamics of nanoparticles in rubber. For that purpose, we have developed time-resolved two-dimensional USAXS at BL20XU [1] and XPCS at BL40XU, SPirng-8 [2, 3]. Based on the results of these novel scattering experiments, we have characterized spatio-temporal structure of filled rubber system, thereby developing tire products. In this presentation, experimental details and their results will be presented.

E3 ubiquitin ligases

2005 ◽  
Vol 41 ◽  
pp. 15-30 ◽  
Author(s):  
Helen C. Ardley ◽  
Philip A. Robinson
Keyword(s):  
Protein Complexes ◽  
Loss Of Function ◽  
Direct Role ◽  
Cellular Processes ◽  
Substrate Protein ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
Full Complement ◽  
Spatio Temporal ◽  
Eukaryotic Organisms

The selectivity of the ubiquitin–26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin–protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

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 A data reduction and compression description for high throughput time-resolved electron microscopy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Abhik Datta ◽  
Kian Fong Ng ◽  
Deepan Balakrishnan ◽  
Melissa Ding ◽  
See Wee Chee ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Temporal Resolution ◽  
Data Reduction ◽  
Accurate Estimation ◽  
Data Streaming ◽  
Compression Scheme ◽  
Raw Data ◽  
Time Resolved ◽  
Detection And Counting ◽  
Spatio Temporal

AbstractFast, direct electron detectors have significantly improved the spatio-temporal resolution of electron microscopy movies. Preserving both spatial and temporal resolution in extended observations, however, requires storing prohibitively large amounts of data. Here, we describe an efficient and flexible data reduction and compression scheme (ReCoDe) that retains both spatial and temporal resolution by preserving individual electron events. Running ReCoDe on a workstation we demonstrate on-the-fly reduction and compression of raw data streaming off a detector at 3 GB/s, for hours of uninterrupted data collection. The output was 100-fold smaller than the raw data and saved directly onto network-attached storage drives over a 10 GbE connection. We discuss calibration techniques that support electron detection and counting (e.g., estimate electron backscattering rates, false positive rates, and data compressibility), and novel data analysis methods enabled by ReCoDe (e.g., recalibration of data post acquisition, and accurate estimation of coincidence loss).

scholarly journals Super-resolution time-resolved imaging using computational sensor fusion

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
C. Callenberg ◽  
A. Lyons ◽  
D. den Brok ◽  
A. Fatima ◽  
A. Turpin ◽  
...  
Keyword(s):  
Sensor Fusion ◽  
Spatial Resolution ◽  
Temporal Resolution ◽  
Single Photon ◽  
Super Resolution ◽  
Numerical Data ◽  
Data Cube ◽  
Fluorescence Lifetime Imaging ◽  
Time Resolved ◽  
Temporal Precision

AbstractImaging across both the full transverse spatial and temporal dimensions of a scene with high precision in all three coordinates is key to applications ranging from LIDAR to fluorescence lifetime imaging. However, compromises that sacrifice, for example, spatial resolution at the expense of temporal resolution are often required, in particular when the full 3-dimensional data cube is required in short acquisition times. We introduce a sensor fusion approach that combines data having low-spatial resolution but high temporal precision gathered with a single-photon-avalanche-diode (SPAD) array with data that has high spatial but no temporal resolution, such as that acquired with a standard CMOS camera. Our method, based on blurring the image on the SPAD array and computational sensor fusion, reconstructs time-resolved images at significantly higher spatial resolution than the SPAD input, upsampling numerical data by a factor $$12 \times 12$$ 12 × 12 , and demonstrating up to $$4 \times 4$$ 4 × 4 upsampling of experimental data. We demonstrate the technique for both LIDAR applications and FLIM of fluorescent cancer cells. This technique paves the way to high spatial resolution SPAD imaging or, equivalently, FLIM imaging with conventional microscopes at frame rates accelerated by more than an order of magnitude.

Electron Correlation Microscopy: A New Technique for Studying Local Atom Dynamics Applied to a Supercooled Liquid

2015 ◽  
Vol 21 (4) ◽  
pp. 1026-1033 ◽  
Author(s):  
Li He ◽  
Pei Zhang ◽  
Matthew F. Besser ◽  
Matthew Joseph Kramer ◽  
Paul M. Voyles
Keyword(s):  
Electron Correlation ◽  
Supercooled Liquid ◽  
Scanning Transmission Electron Microscope ◽  
Correlation Spectroscopy ◽  
New Technique ◽  
Time Resolved ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
A New Technique ◽  
Correlation Microscopy

AbstractElectron correlation microscopy (ECM) is a new technique that utilizes time-resolved coherent electron nanodiffraction to study dynamic atomic rearrangements in materials. It is the electron scattering equivalent of photon correlation spectroscopy with the added advantage of nanometer-scale spatial resolution. We have applied ECM to a Pd40Ni40P20 metallic glass, heated inside a scanning transmission electron microscope into a supercooled liquid to measure the structural relaxation time τ between the glass transition temperature Tg and the crystallization temperature, Tx. τ determined from the mean diffraction intensity autocorrelation function g2(t) decreases with temperature following an Arrhenius relationship between Tg and Tg+25 K, and then increases as temperature approaches Tx. The distribution of τ determined from the g2(t) of single speckles is broad and changes significantly with temperature.

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