Membrane binding and insertion of the predicted transmembrane domain of human scramblase 1.

Human phospholipid scramblase 1 (SCR) was originally described as an intrinsic membrane protein catalyzing transbilayer phospholipid transfer in the absence of ATP. More recently, a role as a nuclear transcription factor has been proposed for SCR, either in addition or alternatively to its capacity to facilitate phospholipid flip-flop. Uncertainties exist as well from the structural point of view. A predicted α-helix (aa residues 288-306) located near the C-terminus has been alternatively proposed as a transmembrane domain, or as a protein core structural element. This paper explores the possibilities of the above helical segment as a transmembrane domain. To this aim two peptides were synthesized, one corresponding to the 19 α-helical residues, and one containing both the helix and the subsequent 12-residues constituting the C-end of the protein. The interaction of these peptides with lipid monolayers and bilayers was tested with Langmuir balance surface pressure measurements, proteoliposome reconstitution and analysis, differential scanning calorimetry, tests of bilayer permeability, and fluorescence confocal microscopy. Bilayers of 28 different lipid compositions were examined in which lipid electric charge, bilayer fluidity and lateral heterogeneity (domain formation) were varied. All the results concur in supporting the idea that the 288-306 peptide of SCR becomes membrane inserted in the presence of lipid bilayers. Thus, the data are in agreement with the possibility of SCR as an integral membrane protein, without rejecting alternative cell locations.

Cytotoxicity of bovine α-lactalbumin: oleic acid complexes correlates with the disruption of lipid membranes.

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.