Inducin Triggers LC3-Lipidation and ESCRT-Mediated Lysosomal Membrane Repair.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

High pressure treatment promotes the deteriorating effect of cationic antimicrobial peptides on bacterial membranes.

The helical structure that cationic antimicrobial peptides (cAMPs) adopt upon interaction with membranes is key to their activity. We show that a high hydrostatic pressure not only increases the propensity of cAMPs to adopt a helical conformation in the presence of bacterial lipid bilayer membranes, but also in bulk solution, and the effect on bacterial membranes persists even up to 10 kbar. Therefore, high-pressure treatment could boost cAMP activity in high-pressure food processing to extend the shelf-life of food.

Linear ubiquitination induces NEMO phase separation to activate NF-κB signaling.

The NF-κB essential modulator NEMO is the core regulatory component of the inhibitor of κB kinase complex, which is a critical checkpoint in canonical NF-κB signaling downstream of innate and adaptive immune receptors. In response to various stimuli, such as TNF or IL-1ß, NEMO binds to linear or M1-linked ubiquitin chains generated by LUBAC, promoting its oligomerization and subsequent activation of the associated kinases. Here we show that M1-ubiquitin chains induce phase separation of NEMO and the formation of NEMO assemblies in cells after exposure to IL-1ß. Phase separation is promoted by both binding of NEMO to linear ubiquitin chains and covalent linkage of M1-ubiquitin to NEMO and is essential but not sufficient for its phase separation. Supporting the functional relevance of NEMO phase separation in signaling, a pathogenic NEMO mutant, which is impaired in both binding and linkage to linear ubiquitin chains, does not undergo phase separation and is defective in mediating IL-1ß-induced NF-κB activation.

Life in Multi-Extreme Environments: Brines, Osmotic and Hydrostatic Pressure─A Physicochemical View.

Elucidating the details of the formation, stability, interactions, and reactivity of biomolecular systems under extreme environmental conditions, including high salt concentrations in brines and high osmotic and high hydrostatic pressures, is of fundamental biological, astrobiological, and biotechnological importance. Bacteria and archaea are able to survive in the deep ocean or subsurface of Earth, where pressures of up to 1 kbar are reached. The deep subsurface of Mars may host high concentrations of ions in brines, such as perchlorates, but we know little about how these conditions and the resulting osmotic stress conditions would affect the habitability of such environments for cellular life. We discuss the combined effects of osmotic (salts, organic cosolvents) and hydrostatic pressures on the structure, stability, and reactivity of biomolecular systems, including membranes, proteins, and nucleic acids. To this end, a variety of biophysical techniques have been applied, including calorimetry, UV/vis, FTIR and fluorescence spectroscopy, and neutron and X-ray scattering, in conjunction with high pressure techniques. Knowledge of these effects is essential to our understanding of life exposed to such harsh conditions, and of the physical limits of life in general. Finally, we discuss strategies that not only help us understand the adaptive mechanisms of organisms that thrive in such harsh geological settings but could also have important ramifications in biotechnological and pharmaceutical applications.

Bipolar Imidazolium-Based Lipid Analogues for Artificial Archaeosomes.

Archaeal lipids have harvested biomedical and biotechnological interest because of their ability to form membranes with low permeability and enhanced temperature and pressure stability. Because of problems in isolating archaeal lipids, chemical synthesis appears to be a suitable means of producing model lipids that mimic the biological counterparts. Here, we introduce a new concept: we synthesized bipolar alkylated imidazolium salts of different chain lengths (BIm10-32) and studied their structure and lyotropic phase behavior. Furthermore, mixtures of the bolalipid analogues with phospholipid model biomembranes of diverse complexity were studied. DSC, fluorescence and FTIR spectroscopy, confocal fluorescence microscopy, DLS, SAXS, and TEM were used to reveal changes in lipid phase behavior, fluidity, the lipid's conformational order, and membrane morphology over a wide range of temperatures and for selected pressures. It could be shown that the long-chain BImN32 can form monolayer sheets. Integrated in phospholipid membranes, it reveals a fluidizing effect. Here, the two polar head groups, connected by a long alkyl chain, enable the integration into the bilayer. Interestingly, addition of BImN32 to fluid DPPC liposomes increased the lipid packing markedly, rendering the membrane system more stable at higher temperatures. The membrane system is also stable against compression as indicated by the high-pressure stability of the system, mimicking an archaeal lipid-like behavior. BImN32 incorporation into raft-like anionic model biomembranes led to marked changes in lateral membrane organization, topology, and fusogenicity of the membrane. Overall, it was found that long-chain imidazolium-based bolalipid analogues can help adjust membrane's biophysical properties, while the imidazolium headgroup provides the ability for crucial electrostatic interaction for vesicle fusion or selective interaction with membrane-related signaling molecules and polypeptides in a synthetically tractable manner. The results obtained may help to develop new approaches for rational design of extremophilic bolalipid-based liposomes for various applications, including delivery of drugs and vaccines.

Structural responses of model biomembranes to Mars-relevant salts.

Lipid membranes are a key component of contemporary living systems and are thought to have been essential to the origin of life. Most research on membranes has focused on situations restricted to ambient physiological or benchtop conditions. However, the influence of more extreme conditions, such as the deep subsurface on Earth or extraterrestrial environments are less well understood. The deep subsurface environments of Mars, for instance, may harbor high concentrations of chaotropic salts in brines, yet we know little about how these conditions would influence the habitability of such environments for cellular life. Here, we investigated the combined effects of high concentrations of salts, including sodium and magnesium perchlorate and sulfate, and high hydrostatic pressure on the stability and structure of model biomembranes of varying complexity. To this end, a variety of biophysical techniques have been applied, which include calorimetry, fluorescence spectroscopies, small-angle X-ray scattering, dynamic light scattering, and microscopy techniques. We show that the structure and phase behavior of lipid membranes is sensitively dictated by the nature of the salt, in particular its anion and its concentration. We demonstrate that, with the exception of magnesium perchlorate, which can also induce cubic lipid arrangements, long-chain saturated lipid bilayer structures can still persist at high salt concentrations across a range of pressures. The lateral organization of complex heterogeneous raft-like membranes is affected by all salts. For simple, in particular bacterial membrane-type bilayer systems with unsaturated chains, vesicular structures are still stable at Martian brine conditions, also up to the kbar pressure range, demonstrating the potential compatibility of environments containing such ionic and pressure extremes to lipid-encapsulated life.

Remodeling of the Fibrillation Pathway of α-Synuclein by Interaction with Antimicrobial Peptide LL-III.

Liquid-liquid phase separation (LLPS) has emerged as a key mechanism for intracellular organization, and many recent studies have provided important insights into the role of LLPS in cell biology. There is also evidence that LLPS is associated with a variety of medical conditions, including neurodegenerative disorders. Pathological aggregation of α-synuclein, which is causally linked to Parkinson's disease, can proceed via droplet condensation, which then gradually transitions to the amyloid state. We show that the antimicrobial peptide LL-III is able to interact with both monomers and condensates of α-synuclein, leading to stabilization of the droplet and preventing conversion to the fibrillar state. The anti-aggregation activity of LL-III was also confirmed in a cellular model. We anticipate that studying the interaction of antimicrobial-type peptides with liquid condensates such as α-synuclein will contribute to the understanding of disease mechanisms (that arise in such condensates) and may also open up exciting new avenues for intervention.

Interaction of imidazolium-based lipids with phospholipid bilayer membranes of different complexity.

In recent years, alkylated imidazolium salts have been shown to affect lipid membranes and exhibit general cytotoxicity as well as significant anti-tumor activity. Here, we examined the interactions of a sterically demanding, biophysically unexplored imidazolium salt, 1,3-bis(2,6-diisopropylphenyl)-4,5-diundecylimidazolium bromide (C11IPr), on the physico-chemical properties of various model biomembrane systems. The results are compared with those for the smaller headgroup variant 1,3-dimethyl-4,5-diundecylimidazolium iodide (C11IMe). We studied the influence of these two lipid-based imidazolium salts at concentrations from 1 to about 10 mol% on model biomembrane systems of different complexity, including anionic heterogeneous raft membranes which are closer to natural membranes. Fluorescence spectroscopic, DSC, surface potential and FTIR measurements were carried out to reveal changes in membrane thermotropic phase behavior, lipid conformational order, fluidity and headgroup charge. Complementary AFM and confocal fluorescence microscopy measurements allowed us to detect changes in the lateral organization and membrane morphology. Both lipidated imidazolium salts increase the membrane fluidity and lead to a deterioration of the lateral domain structure of the membrane, in particular for C11IPr owing to its bulkier headgroup. Moreover, partitioning of the lipidated imidazolium salts into the lipid vesicles leads to marked changes in lateral organization, curvature and morphology of the lipid vesicles at high concentrations, with C11IPr having a more pronounced effect than C11IMe. Hence, these compounds seem to be vastly suitable for biochemical and biotechnological engineering, with high potentials for antimicrobial activity, drug delivery and gene transfer.