pH-Dependent physicochemical properties of ornithine lipid in mono- and bilayers.

In certain bacteria, phosphatidylethanolamine lipids (PEL) get largely replaced by phosphate-free ornithine lipids (OL) under conditions of phosphate starvation. It has so far been unknown how much these two lipid types deviate in their physicochemical properties, and how strongly bacteria thus have to adapt in order to compensate for the difference. Here, we use differential scanning calorimetry, X-ray scattering, and X-ray fluorescence to investigate the properties of OL with saturated C14 alkyl chains in mono- and bilayers. OL is found to have a greater tendency than chain-analogous PEL to form ordered structures and, in contrast to PEL, even a molecular superlattice based on a hydrogen bonding network between the headgroups. This superlattice is virtually electrically uncharged and persists over a wide pH range. Our results indicate that OL and PEL behave very differently in ordered single-component membranes but may behave more similarly in fluid multicomponent membranes.

Phospholipid acyl tail affects lipid headgroup orientation and membrane hydration.

Biomembrane hydration is crucial for understanding processes at biological interfaces. While the effect of the lipid headgroup has been studied extensively, the effect (if any) of the acyl chain chemical structure on lipid-bound interfacial water has remained elusive. We study model membranes composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) lipids, the most abundant lipids in biomembranes. We explore the extent to which the lipid headgroup packing and associated water organization are affected by the lipid acyl tail unsaturation and chain length. To this end, we employ a combination of surface-sensitive techniques, including sum-frequency generation spectroscopy, surface pressure measurements, and Brewster angle microscopy imaging. Our results reveal that the acyl tail structure critically affects the headgroup phosphate orientational distribution and lipid-associated water molecules, for both PE and PC lipid monolayers at the air/water interface. These insights reveal the importance of acyl chain chemistry in determining not only membrane fluidity but also membrane hydration.

Driving the catalytic activity of a transmembrane thermosensor kinase.

DesK is a Bacillus thermosensor kinase that is inactive at high temperatures but turns activated when the temperature drops below 25 °C. Surprisingly, the catalytic domain (DesKC) lacking the transmembrane region is more active at higher temperature, showing an inverted regulation regarding DesK. How does the transmembrane region control the catalytic domain, repressing activity at high temperatures, but allowing activation at lower temperatures? By designing a set of temperature minimized sensors that share the same catalytic cytoplasmic domain but differ in number and position of hydrogen-bond (H-bond) forming residues along the transmembrane helix, we are able to tune, invert or disconnect activity from the input signal. By favoring differential H-bond networks, the activation peak could be moved towards lower or higher temperatures. This principle may be involved in regulation of other sensors as environmental physicochemical changes or mutations that modify the transmembrane H-bond pattern can tilt the equilibrium favoring alternative conformations.

Correction to: Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4.

In the published article, the Fig. 2 was published incorrectly. The correct Fig. 2 is given below.

Saturation of acyl chains converts cardiolipin from an antagonist to an activator of Toll-like receptor-4.

Cardiolipins (CLs) are tetra-acylated diphosphatidylglycerols found in bacteria, yeast, plants, and animals. In healthy mammals, CLs are unsaturated, whereas saturated CLs are found in blood cells from Barth syndrome patients and in some Gram-positive bacteria. Here, we show that unsaturated but not saturated CLs block LPS-induced NF-κB activation, TNF-α and IP-10 secretion in human and murine macrophages, as well as LPS-induced TNF-α and IL-1ß release in human blood mononuclear cells. Using HEK293 cells transfected with Toll-like receptor 4 (TLR4) and its co-receptor Myeloid Differentiation 2 (MD2), we demonstrate that unsaturated CLs compete with LPS for binding TLR4/MD2 preventing its activation, whereas saturated CLs are TLR4/MD2 agonists. As a consequence, saturated CLs induce a pro-inflammatory response in macrophages characterized by TNF-α and IP-10 secretion, and activate the alternative NLRP3 inflammasome pathway in human blood-derived monocytes. Thus, we identify that double bonds discriminate between anti- and pro-inflammatory properties of tetra-acylated molecules, providing a rationale for the development of TLR4 activators and inhibitors for use as vaccine adjuvants or in the treatment of TLR4-related diseases.

Characterization by Nano-Infrared Spectroscopy of Individual Aggregated Species of Amyloid Proteins.

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillar structure with a higher ß-sheet content than in their native structure. To characterize them, we used an innovative tool that coupled infrared spectroscopy with atomic force microscopy (AFM-IR). With this method, we show that we can detect different individual aggregated species from oligomers to fibrils and study their morphologies by AFM and their secondary structures based on their IR spectra. AFM-IR overcomes the weak spatial resolution of usual infrared spectroscopy and achieves a resolution of ten nanometers, the size of isolated fibrils. We characterized oligomers, amyloid fibrils of Aß42 and fibrils of α-synuclein. To our surprise, we figured out that the nature of some surfaces (ZnSe) used to study the samples induces destructuring of amyloid samples, leading to amorphous aggregates. We strongly suggest taking this into consideration in future experiments with amyloid fibrils. More importantly, we demonstrate the advantages of AFM-IR, with a high spatial resolution (≤ 10 nm) allowing spectrum recording on individual aggregated supramolecular entities selected thanks to the AFM images or on thin layers of proteins.

DOTAP, a lipidic transfection reagent, triggers Arabidopsis plant defense responses.

MAIN CONCLUSION: DOTAP triggers Arabidopsis thaliana immunity and by priming the defense response is able to reduce bacterial pathogen attack. DOTAP is a cationic lipid widely used as a liposomal transfection reagent and it has recently been identified as a strong activator of the innate immune system in animal cells. Plants are sessile organisms and unlike mammals, that have innate and acquired immunity, plants possess only innate immunity. A key feature of plant immunity is the ability to sense potentially dangerous signals, as it is the case for microbe-associated, pathogen-associated or damage-associated molecular patterns and by doing so, trigger an active defense response to cope with the perturbing stimulus. Here, we evaluated the effect of DOTAP in plant basal innate immunity. An initial plant defense response was induced by the cationic lipid DOTAP in the model plant Arabidopsis thaliana, assessed by callose deposition, reactive oxygen species production, and plant cell death. In addition, a proteomic analysis revealed that these responses are mirrored by changes in the plant proteome, such as up-regulation of proteins related to defense responses, including proteins involved in photorespiration, cysteine and oxylipin synthesis, and oxidative stress response; and down-regulation of enzymes related to photosynthesis. Furthermore, DOTAP was able to prime the defense response for later pathogenic challenges as in the case of the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Disease outcome was diminished in DOTAP-pre-treated leaves and bacterial growth was reduced 100 times compared to mock leaves. Therefore, DOTAP may be considered a good candidate as an elicitor for the study of plant immunity.

Large supramolecular structures of 33-mer gliadin peptide activate toll-like receptors in macrophages.

Gliadin, an immunogenic protein present in wheat, is not fully degraded by humans and after the normal gastric and pancreatic digestion, the immunodominant 33-mer gliadin peptide remains unprocessed. The 33-mer gliadin peptide is found in human faeces and urine, proving not only its proteolytic resistance in vivo but more importantly its transport through the entire human body. Here, we demonstrate that 33-mer supramolecular structures larger than 220 nm induce the overexpression of nuclear factor kappa B (NF-κB) via a specific Toll-like Receptor (TLR) 2 and (TLR) 4 dependent pathway and the secretion of pro-inflammatory cytokines such as IP-10/CXCL10 and TNF-α. Using helium ion microscopy, we elucidated the initial stages of oligomerisation of 33-mer gliadin peptide, showing that rod-like oligomers are nucleation sites for protofilament formation. The relevance of the 33-mer supramolecular structures in the early stages of the disease is paving new perspectives in the understanding of gluten-related disorders.

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells.

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.

Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein.

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel ß-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel ß-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel ß-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.

A lipid-mediated conformational switch modulates the thermosensing activity of DesK.

The thermosensor DesK is a multipass transmembrane histidine-kinase that allows the bacterium Bacillus subtilis to adjust the levels of unsaturated fatty acids required to optimize membrane lipid fluidity. The cytoplasmic catalytic domain of DesK behaves like a kinase at low temperature and like a phosphatase at high temperature. Temperature sensing involves a built-in instability caused by a group of hydrophilic residues located near the N terminus of the first transmembrane (TM) segment. These residues are buried in the lipid phase at low temperature and partially "buoy" to the aqueous phase at higher temperature with the thinning of the membrane, promoting the required conformational change. Nevertheless, the core question remains poorly understood: How is the information sensed by the transmembrane region converted into a rearrangement in the cytoplasmic catalytic domain to control DesK activity? Here, we identify a "linker region" (KSRKERERLEEK) that connects the TM sensor domain with the cytoplasmic catalytic domain involved in signal transmission. The linker adopts two conformational states in response to temperature-dependent membrane thickness changes: (i) random coiled and bound to the phospholipid head groups at the water-membrane interface, promoting the phosphatase state or (ii) unbound and forming a continuous helix spanning a region from the membrane to the cytoplasm, promoting the kinase state. Our results uphold the view that the linker is endowed with a helix/random coil conformational duality that enables it to behave like a transmission switch, with helix disruption decreasing the kinase/phosphatase activity ratio, as required to modulate the DesK output response.

Role of lipid microdomains in TLR-mediated signalling.

Over the last twenty years, evidence has been provided that the plasma membrane is partitioned with microdomains, laterally mobile in the bilayer, providing the necessary microenvironment to specific membrane proteins for signalling pathways to be initiated. We discuss here the importance of such microdomains for Toll-like receptors (TLR) localization and function. First, lipid microdomains favour recruitment and clustering of the TLR machinery partners, i.e. receptors and co-receptors previously identified to be required for ligand recognition and signal transmission. Further, the presence of the so-called Cholesterol Recognition Amino-Acid Consensus (CRAC) sequences in the intracellular juxtamembrane domain of several Toll-like receptors suggests a direct role of cholesterol in the activation process. This article is part of a Special Issue entitled: Lipid-protein interactions.

Structural characterization of novel cationic diC16-amidine bilayers: Evidence for partial interdigitation.

In this work, the bilayer structure of novel cationic lipid diC16-amidine was compared to the one of zwitterionic dipalmitoyl phosphatidylcholine ( DPPC), which shares the same hydrophobic domain. Differential scanning calorimetry shows that DPPC and diC16-am idine bilayers have similar phase transition temperatures, but diC16-a midine membranes display a less cooperative phase transition and an absence of pretransition. Both bilayers were analyzed from surface to core, using 5-, 7-, 10-, 12-, 14-, and 16-PCSL spin labels. As expected, electron spin resonance (ESR) spectra show that the gel phase of DPPC presents a flexibility gradient toward the core. In contrast, this gradient exists in the gel phase of diC16-amidine bilayers but only down to the 12th lipid tail carbon. The 14th and 16th carbons of the cationic lipid are in a very rigid environment, similar to the one observed at the bilayer surface. These data suggest that diC16-amidine molecules are organized in a partially interdigitated gel phase. ESR spectroscopy also shows that the lamellar fluid phase of diC16-amidine is more rigid than the one of DPPC. Fluorescence resonance energy transfer assays reveal that diC16-amidine displays a more efficient fusogenic activity in the gel phase than in the fluid one, suggesting that the partial interdigitation of the gel phase is important for the fusion process to occur. Since the gel- fl uid transition temperature is 42 ·c. diC16-amid ine is fusogenic at the physiological temperature and is therefore a promising lipid for delivery applications without the need of helper lipids.

Protonation drives the conformational switch in the multidrug transporter LmrP.

Multidrug antiporters of the major facilitator superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we used extensive double electron-electron resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from Lactococcus lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations, depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.

Critical residues involved in Toll-like receptor 4 activation by cationic lipid nanocarriers are not located at the lipopolysaccharide-binding interface.

DiC14-amidine is a cationic lipid that was originally designed as a lipid nanocarrier for nucleic acid transport, and turned out to be a Toll-like receptor 4 (TLR4) agonist as well. We found that while E. coli lipopolysaccharide (LPS) is a TLR4 agonist in all species, diC14-amidine nanoliposomes are full agonists for human, mouse and cat receptors but weak horse agonists. Taking advantage of this unusual species specificity, we used chimeric constructs based on the human and horse sequences and identified two regions in the human TLR4 that modulate the agonist activity of diC14-amidine. Interestingly, these regions lie outside the known LPS-binding domain. Competition experiments also support our hypothesis that diC14-amidine interacts primarily with TLR4 hydrophobic crevices located at the edges of the TLR4/TLR4* dimerization interface. We have characterized potential binding modes using molecular docking analysis and suggest that diC14-amidine nanoliposomes activate TLR4 by facilitating its dimerization in a process that is myeloid differentiation 2 (MD-2)-dependent and cluster of differentiation 14 (CD14)-independent. Our data suggest that TLR4 may be activated through binding at different anchoring points, expanding the repertoire of TLR4 ligands to non-MD-2-binding lipids.

Amyloid fibrils are the molecular trigger of inflammation in Parkinson's disease.

Parkinson's disease (PD) is an age-related movement disorder characterized by a progressive degeneration of dopaminergic neurons in the midbrain. Although the presence of amyloid deposits of α-synuclein (α-syn) is the main pathological feature, PD brains also present a severe permanent inflammation, which largely contributes to neuropathology. Although α-syn has recently been implicated in this process, the molecular mechanisms underlying neuroinflammation remain unknown. In the present study, we investigated the ability of different α-syn aggregates to trigger inflammatory responses. We showed that α-syn induced inflammation through activation of Toll-like receptor 2 (TLR2) and the nucleotide oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome only when folded as amyloid fibrils. Oligomeric species, thought to be the primary species responsible for the disease, were surprisingly unable to trigger the same cascades. As neuroinflammation is a key player in PD pathology, these results put fibrils back to the fore and rekindles discussions about the primary toxic species contributing to the disease. Our data also suggest that the inflammatory properties of α-syn fibrils are linked to their intrinsic structure, most probably to their cross-ß structure. Since fibrils of other amyloids induce similar immunological responses, we propose that the canonical fibril-specific cross-ß structure represents a new generic motif recognized by the innate immune system.

Stereospecific requirement of cholesterol in the function of the serotonin1A receptor.

The serotonin1A receptor is an important member of the G protein-coupled receptor (GPCR) family. It is involved in the generation and modulation of a variety of cognitive and behavioral functions and serves as a drug target. Previous work from our laboratory has established the sensitivity of the function of the serotonin1A receptor to membrane cholesterol. Solubilization of the hippocampal serotonin1A receptor utilizing the zwitterionic detergent CHAPS is accompanied by loss of cholesterol and results in reduction in specific ligand binding. Replenishment of cholesterol to solubilized membranes restores specific ligand binding to the receptor. We utilized this strategy of sterol replenishment of solubilized membranes to explore the stereospecific stringency of cholesterol for receptor function. We used two stereoisomers of cholesterol, ent-cholesterol (enantiomer of cholesterol) and epi-cholesterol (a diastereomer of cholesterol), for this purpose. Importantly, we show here that while ent-cholesterol could replace cholesterol in supporting receptor function, epi-cholesterol could not. These results imply that the requirement of membrane cholesterol for the serotonin1A receptor function is diastereospecific, yet not enantiospecific. Our results extend and help define specificity of the interaction of membrane cholesterol with the serotonin1A receptor, and represent the first report utilizing ent-cholesterol to examine stereospecificity of GPCR-cholesterol interaction.

ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins.

Amyloid refers to insoluble protein aggregates that are responsible for amyloid diseases but are also implicated in important physiological functions (functional amyloids). The widespread presence of protein aggregates but also, in most of the cases, their deleterious effects explain worldwide efforts made to understand their formation, structure and biological functions. We emphasized the role of FTIR and especially ATR-FTIR techniques in amyloid protein and/or peptide studies. The multiple advantages provided by ATR-FTIR allow an almost continuous structural view of protein/peptide conversion during the aggregation process. Moreover, it is now well-established that infrared can differentiate oligomers from fibrils simply on their spectral features. ATR-FTIR is certainly the fastest and easiest method to obtain this information. ATR-FTIR occupies a key position in the analysis and comprehension of the complex aggregation mechanism(s) at the oligomer and/or fibril level. These mechanism(s) seem to present strong similarities between different amyloid proteins and might therefore be extremely important to understand for both disease-associated and functional amyloid proteins. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies.

Activation of innate immunity by lysozyme fibrils is critically dependent on cross-ß sheet structure.

Inflammation occurs in many amyloidoses, but its underlying mechanisms remain enigmatic. Here we show that amyloid fibrils of human lysozyme, which are associated with severe systemic amyloidoses, induce the secretion of pro-inflammatory cytokines through activation of the NLRP3 (NLR, pyrin domain containing 3) inflammasome and the Toll-like receptor 2, two innate immune receptors that may be involved in immune responses associated to amyloidoses. More importantly, our data clearly suggest that the induction of inflammatory responses by amyloid fibrils is linked to their intrinsic structure, because the monomeric form and a non-fibrillar type of lysozyme aggregates are both unable to trigger cytokine secretion. These lysozyme species lack the so-called cross-ß structure, a characteristic structural motif common to all amyloid fibrils irrespective of their origin. Since fibrils of other bacterial and endogenous proteins have been shown to trigger immunological responses, our observations suggest that the cross-ß structural signature might be recognized as a generic danger signal by the immune system.