Apolipoprotein A–I binding to anionic vesicles and lipopolysaccharides: Role for lysine residues in antimicrobial properties

AbstractHuman apolipoprotein A–I (apoA–I) is a 28kDa protein and a major component of high-density lipoproteins, mediating several essential metabolic functions related to heart disease. In the present study the potential protective role against bacterial pathogens was explored. ApoA–I suppressed bacterial growth of Escherichia coli and Klebsiella pneumoniae. The protein was able to bind lipopolysaccharides and showed a strong preference for bilayer vesicles made of phosphatidylglycerol over phosphatidylcholine. Lysine side chains of apoA–I were acetylated to evaluate the importance of electrostatic forces in the binding interaction with both membrane components. Electrophoresis properties, dot blot analysis, circular dichroism, and fluorescence spectroscopy to probe for changes in protein structure indicated that the acetylated protein displayed a strongly reduced lipopolysaccharide and phosphatidylglycerol binding. A mutant containing only the N-terminal domain of apoA–I also showed a reduced ability to interact with the membrane components, although to a lesser extent. These results indicate the potential for apoA–I to function as an antimicrobial protein and exerts this function through lysine residues

Isolation, Characterization, and Stability of Discretely-Sized Nanolipoprotein Particles Assembled with Apolipophorin-III

Background: Nanolipoprotein particles (NLPs) are discoidal, nanometer-sized particles comprised of self-assembled phospholipid membranes and apolipoproteins. NLPs assembled with human apolipoproteins have been used for myriad biotechnology applications, including membrane protein solubilization, drug delivery, and diagnostic imaging. To expand the repertoire of lipoproteins for these applications, insect apolipophorin-III (apoLp-III) was evaluated for the ability to form discretely-sized, homogeneous, and stable NLPs. Methodology: Four NLP populations distinct with regards to particle diameters (ranging in size from 10 nm to.25 nm) and lipid-to-apoLp-III ratios were readily isolated to high purity by size exclusion chromatography. Remodeling of the purified NLP species over time at 4uC was monitored by native gel electrophoresis, size exclusion chromatography, and atomic force microscopy. Purified 20 nm NLPs displayed no remodeling and remained stable for over 1 year. Purified NLPs with 10 nm and 15 nm diameters ultimately remodeled into 20 nm NLPs over a period of months. Intra-particle chemical cross-linking of apoLp-III stabilized NLPs of all sizes. Conclusions: ApoLp-III-based NLPs can be readily prepared, purified, characterized, and stabilized, suggesting their utilit

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory. Athena is a versatile observatory designed to address the Hot and Energetic Universe science theme, as selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), X-IFU aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over a hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR (i.e. in the course of its preliminary definition phase, so-called B1), browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters, such as the instrument efficiency, spectral resolution, energy scale knowledge, count rate capability, non X-ray background and target of opportunity efficiency. Finally, we briefly discuss the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, touch on communication and outreach activities, the consortium organisation and the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. The X-IFU will be provided by an international consortium led by France, The Netherlands and Italy, with ESA member state contributions from Belgium, Czech Republic, Finland, Germany, Poland, Spain, Switzerland, with additional contributions from the United States and Japan.The French contribution to X-IFU is funded by CNES, CNRS and CEA. This work has been also supported by ASI (Italian Space Agency) through the Contract 2019-27-HH.0, and by the ESA (European Space Agency) Core Technology Program (CTP) Contract No. 4000114932/15/NL/BW and the AREMBES - ESA CTP No.4000116655/16/NL/BW. This publication is part of grant RTI2018-096686-B-C21 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”. This publication is part of grant RTI2018-096686-B-C21 and PID2020-115325GB-C31 funded by MCIN/AEI/10.13039/501100011033

The Athena X-ray Integral Field Unit: a consolidated design for the system requirement review of the preliminary definition phase

The Athena X-ray Integral Unit (X-IFU) is the high resolution X-ray spectrometer, studied since 2015 for flying in the mid-30s on the Athena space X-ray Observatory, a versatile observatory designed to address the Hot and Energetic Universe science theme, selected in November 2013 by the Survey Science Committee. Based on a large format array of Transition Edge Sensors (TES), it aims to provide spatially resolved X-ray spectroscopy, with a spectral resolution of 2.5 eV (up to 7 keV) over an hexagonal field of view of 5 arc minutes (equivalent diameter). The X-IFU entered its System Requirement Review (SRR) in June 2022, at about the same time when ESA called for an overall X-IFU redesign (including the X-IFU cryostat and the cooling chain), due to an unanticipated cost overrun of Athena. In this paper, after illustrating the breakthrough capabilities of the X-IFU, we describe the instrument as presented at its SRR, browsing through all the subsystems and associated requirements. We then show the instrument budgets, with a particular emphasis on the anticipated budgets of some of its key performance parameters. Finally we briefly discuss on the ongoing key technology demonstration activities, the calibration and the activities foreseen in the X-IFU Instrument Science Center, and touch on communication and outreach activities, the consortium organisation, and finally on the life cycle assessment of X-IFU aiming at minimising the environmental footprint, associated with the development of the instrument. Thanks to the studies conducted so far on X-IFU, it is expected that along the design-to-cost exercise requested by ESA, the X-IFU will maintain flagship capabilities in spatially resolved high resolution X-ray spectroscopy, enabling most of the original X-IFU related scientific objectives of the Athena mission to be retained. (abridged).Comment: 48 pages, 29 figures, Accepted for publication in Experimental Astronomy with minor editin

Characterization of the ApoLp-III/LPS Complex: Insight into the Mode of Binding Interaction

Apolipoproteins are able to associate with lipopolysaccharides (LPS), potentially providing protection against septic shock. To gain insight into the molecular details of this binding interaction, apolipophorin III (apoLp-III) from <i>Galleria mellonella</i> was used as a model. The binding of apoLp-III to LPS was optimal around 37–40 °C, close to the LPS phase transition temperature. ApoLp-III formed complexes with LPS from <i>E. coli</i> (serotype O55:B5) with a diameter of ∼20 nm and a molecular weight of ∼390 kDa, containing four molecules of apoLp-III and 24 molecules of LPS. The LPS-bound form of the protein was substantially more resistant to guanidine-induced denaturation compared to unbound protein. The denaturation profile displayed a multiphase character with a steep drop in secondary structure between 0 and 1 M guanidine-HCl and a slower decrease above 1 M guanidine-HCl. In contrast, apoLp-III bound to detoxified LPS was only slightly more resistant to guanidine-HCl induced denaturation compared to unbound protein. Analysis of size-exclusion FPLC elution profiles of mixtures of apoLp-III with LPS or detoxified LPS indicated a much weaker binding interaction with detoxified LPS compared to intact LPS. These results indicate that apoLp-III initially interacts with exposed carbohydrate regions, but that the lipid A region is required for a more stable LPS binding interaction

Amphotericin B induces interdigitation of apolipoprotein stabilized nanodisk bilayers.

Amphotericin B nanodisks (AMB-ND) are ternary complexes of AMB, phospholipid and apolipoprotein organized as discrete nanometer scale disk-shaped bilayers. In gel filtration chromatography experiments, empty ND lacking AMB elute as a single population of particles with a molecular weight in the range of 200 kDa. AMB-ND formulated at a 4:1 phospholipid:AMB weight ratio separated into two peaks. One peak eluted at the position of control ND lacking AMB while the second peak, containing all of the AMB present in the original sample, eluted in the void volume. When ND prepared with increased AMB were subjected to gel filtration chromatography an increased proportion of phospholipid and apolipoprotein was recovered in the void volume with AMB. Native gradient gel electrophoresis corroborated the gel filtration chromatography data and electron microscopy studies revealed an AMB concentration-dependent heterogeneity in ND particle size. Stability studies revealed that introduction of AMB into ND decreases the ability of apoA-I to resist denaturation. Atomic force microscopy experiments showed that AMB induces compression of ND bilayer thickness while infrared spectroscopy analysis revealed that the presence of AMB does not induce extreme lipid disorder or alter the mean angle of the molecular axis along fatty acyl chains of ND phospholipids. Taken together the results are consistent with AMB-induced bilayer interdigitation, a phenomenon that likely contributes to AMB-dependent pore formation in susceptible membranes.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.SCOPUS: ar.jinfo:eu-repo/semantics/publishe