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.

Lipid-protein interactions regulating the canonical and the non-canonical NLRP3 inflammasome.

The inflammatory response is a complex regulated effector mechanism of the innate immune system that is initiated after tissue injury or infection. The NLRP3 inflammasome is an important initiator of inflammation by regulating the activation of caspase-1, the maturation of pro-inflammatory cytokines and the induction of pyroptotic cell death. Numerous studies demonstrate that the NLRP3 inflammasome could be modulated by lipids, existing a relation between lipids and the activation of different inflammatory processes. In this review we will summarize how the mechanism of NLRP3 inflammasome activation is regulated by different lipids and how these lipids control specific cellular localization of NLRP3 during activation. Although being a cytosolic protein, NLRP3 interacts with lipids accessible in neighbor membranes. Also, the modulation of NLRP3 by endogenous lipids has been found causative of different metabolic diseases and bacterial-pathogenic lipids lead to NLRP3 activation during infection. The understanding of the modulation of the NLRP3 inflammasome by lipids has resulted not only in a better knowledge about the mechanism of NLRP3 activation and its implication in disease, but also opens a new avenue for the development of novel therapeutics and vaccines, as NLRP3 could be modulated by synthetic lipids used as adjuvants.

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.

Interhelical H-Bonds Modulate the Activity of a Polytopic Transmembrane Kinase.

DesK is a Histidine Kinase that allows Bacillus subtilis to maintain lipid homeostasis in response to changes in the environment. It is located in the membrane, and has five transmembrane helices and a cytoplasmic catalytic domain. The transmembrane region triggers the phosphorylation of the catalytic domain as soon as the membrane lipids rigidify. In this research, we study how transmembrane inter-helical interactions contribute to signal transmission; we designed a co-expression system that allows studying in vivo interactions between transmembrane helices. By Alanine-replacements, we identified a group of polar uncharged residues, whose side chains contain hydrogen-bond donors or acceptors, which are required for the interaction with other DesK transmembrane helices; a particular array of H-bond- residues plays a key role in signaling, transmitting information detected at the membrane level into the cell to finally trigger an adaptive response.

A Transmembrane Histidine Kinase Functions as a pH Sensor.

The two-component system DesK-DesR regulates the synthesis of unsaturated fatty acids in the soil bacteria Bacillus subtilis. This system is activated at low temperature and maintains membrane lipid fluidity upon temperature variations. Here, we found that DesK-the transmembrane histidine kinase-also responds to pH and studied the mechanism of pH sensing. We propose that a helix linking the transmembrane region with the cytoplasmic catalytic domain is involved in pH sensing. This helix contains several glutamate, lysine, and arginine residues At neutral pH, the linker forms an alpha helix that is stabilized by hydrogen bonds in the i, i + 4 register and thus favors the kinase state. At low pH, protonation of glutamate residues breaks salt bridges, which results in helix destabilization and interruption of signaling. This mechanism inhibits unsaturated fatty acid synthesis and rigidifies the membrane when Bacillus grows in acidic conditions.

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.

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.

Effect of structure in ionised albumin based nanoparticle: Characterisation, Emodin interaction, and in vitro cytotoxicity.

A γ-irradiated bovine albumin serum-based nanoparticle was characterised structurally, and functionally. The nanoparticle was characterised by A.F.M., D.L.S, zeta potential, T.E.M., gel-electrophoresis, and spectroscopy. We studied the stability of the nanoparticle at different pH values and against time, by fluorescence spectroscopy following the changes in the tryptophan environment in the nanoparticle. The nanoparticle was also functionalized with Folic Acid, its function as a nanovehicle was evaluated through its interaction with the hydrophobic drug Emodin. The binding and kinetic properties of the obtained complex were evaluated by biophysical methods as well as its toxicity in tumor cells. According to its biophysics, the nanoparticle is a spherical nanosized vehicle with a hydrodynamic diameter of 70 nm. Data obtained describe the nanoparticle as nontoxic for cancer cell lines. When combined with Emodin, the nanoparticle proved to be more active on MCF-7 cancer cell lines than the nanoparticle without Emodin. Significantly, the albumin aggregate preserves the main activity-function of albumin and improved characteristics as an excellent carrier of molecules. More than carrier properties, the nanoparticle alone induced an immune response in macrophages which may be advantageous in vaccine and cancer therapy formulation.

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.

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.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum.

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1ß secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants.

ATR-FTIR Analysis of Amyloid Proteins.

Attenuated total reflection FTIR (ATR-FTIR) has been used for decades to study protein secondary structures. More recently, it reveals also to be an exquisite and sensitive tool to study and discriminate amyloid aggregates. Based on the analysis of specific spectral features of ß-sheet structures, we present here a detailed protocol to differentiate oligomers vs. fibrils. This protocol, applicable to all amyloid proteins, demonstrates the power of this inexpensive, rapid, and low protein material-demanding method.

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.

Toll-like receptor 2 promiscuity is responsible for the immunostimulatory activity of nucleic acid nanocarriers.

Lipopolyamines (LPAs) are cationic lipids; they interact spontaneously with nucleic acids to form lipoplexes used for gene delivery. The main hurdle to using lipoplexes in gene therapy lies in their immunostimulatory properties, so far attributed to the nucleic acid cargo, while cationic lipids were considered as inert to the immune system. Here we demonstrate for the first time that di-C18 LPAs trigger pro-inflammatory responses through Toll-like receptor 2 (TLR2) activation, and this whether they are bound to nucleic acids or not. Molecular docking experiments suggest potential TLR2 binding modes reminiscent of bacterial lipopeptide sensing. The di-C18 LPAs share the ability of burying their lipid chains in the hydrophobic cavity of TLR2 and, in some cases, TLR1, at the vicinity of the dimerization interface; the cationic headgroups form multiple hydrogen bonds, thus crosslinking TLRs into functional complexes. Unravelling the molecular basis of TLR1 and TLR6-driven heterodimerization upon LPA binding underlines the highly collaborative and promiscuous ligand binding mechanism. The prevalence of non-specific main chain-mediated interactions demonstrates that potentially any saturated LPA currently used or proposed as transfection agent is likely to activate TLR2 during transfection. Hence our study emphasizes the urgent need to test the inflammatory properties of transfection agents and proposes the use of docking analysis as a preliminary screening tool for the synthesis of new non-immunostimulatory nanocarriers.

Structural Characterization of the Amyloid Precursor Protein Transmembrane Domain and Its γ-Cleavage Site.

Alzheimer's disease is the most common form of dementia that affects about 50 million of sufferers worldwide. A major role for the initiation and progression of Alzheimer's disease has been associated with the amyloid ß-peptide (Aß), which is a protease cleavage product of the amyloid precursor protein. The amyloid precursor protein is an integral membrane protein with a single transmembrane domain. Here, we assessed the structural integrity of the transmembrane domain within oriented phosphatidylcholine lipid bilayers and determined the tilt angle distribution and dynamics of various subdomains using solid-state NMR and attenuated total reflectance Fourier transform infrared spectroscopies. Although the overall secondary structure of the transmembrane domain is α-helical, pronounced conformational and topological heterogeneities were observed for the γ- and, to a lesser extent, the ζ-cleavage site, with pronounced implications for the production of Aß and related peptides, the development of the disease, and pharmaceutical innovation.

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.

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.

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.