Membranes constitute a meeting point for lipids and proteins. Not only do they define the entity of cells and cytosolic organelles but they also display a wide variety of important functions previously ascribed to the activity of proteins alone. Indeed, lipids have commonly been considered a mere support for the transient or permanent association of membrane proteins, while acting as a selective cell/organelle barrier. However, mounting evidence demonstrates that lipids themselves regulate the location and activity of many membrane proteins, as well as defining membrane microdomains that serve as spatio-temporal platforms for interacting signalling proteins. Membrane lipids are crucial in the fission and fusion of lipid bilayers and they also act as sensors to control environmental or physiological conditions. Lipids and lipid structures participate directly as messengers or regulators of signal transduction. Moreover, their alteration has been associated with the development of numerous diseases. Proteins can interact with membranes through lipid co-/post-translational modifications, and electrostatic and hydrophobic interactions, van der Waals forces and hydrogen bonding are all involved in the associations among membrane proteins and lipids. The present study reviews these interactions from the molecular and biomedical point of view, and the effects of their modulation on the physiological activity of cells, the aetiology of human diseases and the design of clinical drugs. In fact, the influence of lipids on protein function is reflected in the possibility to use these molecular species as targets for therapies against cancer, obesity, neurodegenerative disorders, cardiovascular pathologies and other diseases, using a new approach called membrane-lipid therapy.

a b s t r a c tThis paper reviews the current knowledge on the various mechanisms for transbilayer, or flip-flop, lipid motion in model and cell membranes, enzyme-assisted lipid transfer by flippases, floppases and scramblases is briefly discussed, while non-catalyzed lipid flip-flop is reviewed in more detail. Transbilayer lipid motion may occur as a result of the insertion of foreign molecules (detergents, lipids, or even proteins) in one of the membrane leaflets. It may also be the result of the enzymatic generation of lipids, e.g. diacylglycerol or ceramide, at one side of the membrane. Transbilayer motion rates decrease in the order diacylglycerol ) ceramide ) phospholipids. Ceramide, but not diacylglycerol, can induce transbilayer motion of other lipids, and bilayer scrambling. Transbilayer lipid diffusion and bilayer scrambling are defined as two conceptually and mechanistically different processes. The mechanism of scrambling appears to be related to local instabilities caused by the nonlamellar ceramide molecule, or by other molecules that exhibit a relatively slow flip-flop rate, when asymmetrically inserted or generated in one of the monolayers in a cell or model membrane.

Many cancers are termed immunoevasive due to expression of immunomodulatory ligands. Programmed death ligand-1 (PD-L1) and cluster of differentiation 80/86 (CD80/86) interact with their receptors, programmed death receptor-1 (PD-1) and cytotoxic Tlymphocyte associated protein-4 (CTLA-4) respectively, on tumorinfiltrating leukocytes eliciting immunosuppression. Immunotherapies aimed at blocking these interactions are revolutionizing cancer treatments, albeit in an inadequately described patient subset. To address the issue of patient stratification for immune checkpoint intervention, we quantitatively imaged PD-1/PD-L1 interactions in tumor samples from patients, employing an assay that readily detects these intercellular proteinprotein interactions in the less than or equal to 10nm range. These analyses across multiple patient cohorts demonstrated the intercancer, inter-patient, and intra-tumoral heterogeneity of interacting immune checkpoints. The PD-1/PD-L1 interaction was not correlated with clinical PD-L1 expression scores in malignant melanoma. Crucially, amongst anti-PD-1 treated metastatic NSCLC patients, those with lower PD-1/PD-L1 interaction had significantly worsened survival. It is surmised that within tumors selecting for an elevated level of PD-1/PD-L1 interaction, there is a greater dependence on this pathway for immune evasion and hence they exhibit more impressive patient response to intervention. Research.

However, the C-terminal domain by itself is devoid of membrane lytic properties. The present results can be interpreted in the light of our previous studies that involved in receptor binding a peptide in the C-terminal region of HlyA. We had also shown experimentally the distinction between reversible membrane adsorption and irreversible lytic insertion of the toxin. In this context, the present data allow us to propose that both major domains of HlyA are directly involved in membrane-toxin interaction, the nonapeptide repeat, calcium-binding RTX domain being responsible for the early stages of HlyA docking to the target membrane.

Escherichia coli ␣-hemolysin (HlyA) is a protein exotoxin that binds and lyses eukaryotic cell and model membranes in the presence of calcium. Previous studies have been able to distinguish between reversible toxin binding to the membrane and irreversible insertion into the lipid matrix. Membrane lysis occurs as the combined effect of protein insertion plus a transient perturbation of the membrane bilayer structure. In the past, insertion and bilayer perturbation have not been experimentally dissected. This has now been achieved by studying HlyA penetration into lipid monolayers at the air-water interface, in which three-dimensional effects (of the kind required to break down the bilayer permeability barrier) cannot occur. The study of native HlyA, together with the nonlytic precursor pro-HlyA, and of different mutants demonstrates that although some nonlytic variants (e.g. pro-HlyA) exhibit very low levels of insertion, others (e.g. the nonlytic mutant HlyA H859N) insert even more strongly than the lytic wild type. These results show that insertion does not necessarily lead to membrane lysis, i.e. that insertion and lysis are not "coupled" phenomena. Millimolar levels of Ca 2؉ , which are essential for the lytic activity, cause an extra degree of insertion but only in the case of the lytic forms of HlyA.