Factors Regulating the Substrate Specificity of A-type Phospholipases : A Mass-Spectrometric study

The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, we developed a mass-spectrometric (MS) assay and implemented it to study in detail the effect of acyl chain length and unsaturation of glycerophospholipids (GPLs) on their hydrolysis by three different secretory PLA2s (sPLA2s) from various sources. The key finding of this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis by these PLAs. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to be more important for substrate specificity. In the next study we used the MS assay developed in study I, to investigate whether substrate efflux propensity regulates the activity of the Ca2+ -independent PLA-Beta (iPLAβ). Our results strongly suggest that the activity of iPLAβ is also determined by the efflux of the GPL substrate from the membrane bilayer. Our last study was on the cytoplasmic PLA-alpha (cPLA2α) that has been implicated in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. The study was carried out to understand to what extent accommodation in the catalytic site determines specificity for arachidonic acid (AA) and if efflux propensity plays a role in the substrate specificity of cPLA2α. Our results indicate that while accommodation of the substrate in the active site greatly contributes to the preferential hydrolysis of AA-containing GPLs by cPLA2α, efflux of the substrate from a membrane bilayer also plays a significant role. In summary, these studies not only provide information on the factors regulating the substrate specificity of various PLAs but also indicate that lateral arrangement of GPLs could be a key regulator of homeostatic PLAs like iPLAβ.The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, a mass-spectrometric high-throughput assay was developed which allows us to monitor the hydrolysis of a multitude of GPL molecular species simultaneously. Specificities of three different sPLA2s were elucidated by this method. The key finding in this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to contribute to substrate specificity. Our second study gives the first experimental evidence that efflux of the substrate from the bilayer is the rate-limiting factor in the hydrolysis of GPL molecules by iPLAβ. We observed that the activity of the enzyme correlates inversely with the hydrophobicity of the substrate. In the final study on cPLA2α, the key finding is that accommodation of the substrate molecule in the active site of the enzyme is crucial for the preferential hydrolysis of AA-containing GPLs along with efflux of the substrate from the bilayer whereas in the case of GPL substrate lacking AA, efflux from the bilayer seemed to be determing the rate of hydrolysis

Osbpl8 Deficiency in Mouse Causes an Elevation of High-Density Lipoproteins and Gender-Specific Alterations of Lipid Metabolism

Peer reviewe

Factors regulating the substrate specificity of cytosolic phospholipase A(2)-alpha in vitro

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha) plays a key role in signaling in mammalian cells by releasing arachidonic acid (AA) from glycerophospholipids (GPLs) but the factors determining the specificity of cPLA(2)alpha for AA- containing GPLs are not well understood. Accordingly, we investigated those factors by determining the activity of human cPLA(2)alpha towards a multitude of GPL species present in micelles or bilayers. Studies on isomeric PC sets containing a saturated acyl chain of 6 to 24 carbons in the sn1 or sn2 position in micelles showed an abrupt decrease in hydrolysis when the length of the snl or sn2 chain exceeded 17 carbons suggesting that the acyl binding cavity on the enzyme is of the corresponding length. Notably, the saturated isomer pairs were hydrolyzed identically in micelles as well as in bilayers suggesting promiscuous binding of acyl chains to the active site of cPLA(2)alpha. Such promiscuous binding would explain the previous finding that cPLA(2)alpha has both PLA(1) and PLA(2) activities. Interestingly, increasing the length of either the sn1 or sn2 acyl chain inhibited the hydrolysis in bilayers far more than that in micelles suggesting that with micelles (loosely packed) substrate accommodation at the active site of cPLA(2)alpha is rate-limiting, while with bilayers (tightly packed) upward movement of the substrate from the bilayer (efflux) is the rate-limiting step. With the AA-containing PCs, the length of the saturated acyl chain also had a much stronger effect on hydrolysis in bilayers vs. micelles in agreement with this model. In contrast to saturated PCs, a marked isomer preference was observed for AA-containing PCs both in micelles and bilayers. In conclusion, these data significantly help to understand the mode of action and specificity of cPLA(2)alpha. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Data Descriptor: Lipid profiling of C. elegans strains administered pro-longevity drugs and drug combinations

10.1038/sdata.2018.231SCIENTIFIC DATA5

Biocompatible Glyconanoparticles by Grafting of Sophorolipid Monolayers on Monodisperse Iron Oxide Nanoparticles

International audienceThis work presents synthesis and characterization of sophorolipid-coated monodisperse iron oxide nanoparticles. Sophorolipids are biological glycosylated amphiphiles produced by the yeast S. bombicola. In their open acidic form, sophorolipids have been used as surface stabilizing agent for metal and metal oxide nanoparticles but with poor control over size and structural properties. In this work, the COOH function of sophorolipids (SL) was modified with nitrodopamine (NDA), a catechol known for its high affinity to iron ions. The resulting new form of sophorolipid-nitrodopamide (SL-NDA) was used as surface ligand for monodisperse iron oxide nanoparticles. We show, by a combination of thermogravimetric analysis and small angle X-ray and neutron scattering, that iron oxide nanoparticles (IONP) are stabilized by a single, high-density SL-NDA layer, which results in excellent colloidal stability under biologically relevant conditions such as high protein and salt concentration. The IONP grafted with SL-NDA showed negligible uptake and no cytotoxicity tested on two representative cell lines. Thus, they reveal the potential of sophorolipids as stable and non-toxic surface coatings for IONP-based biomedical and biotechnological applications

Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry

International audienceAbstract SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed

Drug Synergy Slows Aging and Improves Healthspan through IGF and SREBP Lipid Signaling

10.1016/j.devcel.2018.09.001DEVELOPMENTAL CELL47167-

Western blot analysis of ORP8 in KO mouse tissues and peritoneal macrophage.

<p>Total protein specimens (20 µg protein/lane) of tissues with abundant ORP8 expression: spleen, brain, kidney, and liver (WT and Osbpl8KO animals), and of peritoneal macrophage (WT, Osbpl8+/−, and Osbpl8−/−) were analyzed by Western blotting with ORP8 and β-actin antibodies. ORP8 runs for an unknown reason as a doublet of bands with the apparent molecular masses of 97 and 101 kDa, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058856#pone.0058856-Yan1" target="_blank">[10]</a>.</p