Binding steps of apolipoprotein A-I with phospholipid monolayers: adsorption and penetration.

In whole HDL particles, the arrangement of apoA-I relative to phospholipids is of crucial interest with respect to the physiological formations of HDL. We report here new data concerning the nature of the interaction of apoA-I with condensed phospholipid (PL) monolayers (phosphatidylcholine and phosphatidylserine). The use of alternative current polarography allowed the detection for the first time of different binding steps which are dependent on apoA-I concentration. At low concentration (below 10 micrograms/mL), apoA-I adsorbs on PL polar headgroups, through electrostatic interactions. Above this threshold concentration, apoA-I penetrates within the monolayer (i.e., part of apoA-I crosses the PL polar headgroup/hydrocarbon chain interface). The process of penetration described here brings experimental evidence supporting Segrest's "snorkel" model. Penetrated helices are lying at the interface, their apolar face in contact with PL hydrocarbon chains and their polar face in contact with PL polar headgroups. In the absence of cholesterol, a second level of penetration was detected at higher apoA-I concentrations. It was facilitated in the presence of phosphatidylserine in comparison to phosphatidylcholine and disappeared in the presence of cholesterol. It is proposed that the C-terminal domain is involved in the first binding steps and that hinged domains may also be implicated. Furthermore, we propose that the apoA-I binding states stabilize the protein/phospholipid layer complex. These different binding states are discussed with respect to their roles in HDL metabolism.

Opposite effects of tumor necrosis factor alpha on the sphingomyelin-ceramide pathway in two myeloid leukemia cell lines: role of transverse sphingomyelin distribution in the plasma membrane.

Tumor necrosis factor alpha (TNF alpha) mediates proliferation, functional activation, and apoptotic cell death depending on the target cell type. Although sphingomyelin (SPM) hydrolysis and ceramide generation may function as an important mediator in TNF alpha signaling, the molecular mechanisms of the signaling pathway(s) are still not well understood. The aim of the present study is to compare the effect of TNF alpha on SPM metabolism and cell growth in two myeloid leukemic cell lines (U937 and KG1a) that differ in their sensitivity to TNF alpha. Our results show that TNF alpha induced apoptosis in U937 but not in KG1a cells. TNF alpha triggered in KG1a cells neither SPM hydrolysis nor ceramide generation, but induced SPM synthesis and ceramide breakdown as well as dose-dependent cell proliferation. In contrast, TNF alpha induced in U937 SPM hydrolysis and ceramide generation as well as dose-dependent cell death. Synthetic cell permeant ceramide, as well as natural ceramide, generated by treatment with bacterial sphingomyelinase (SPMase), all induced apoptosis in both U937 and KG1a cells. These findings indicate that the SPM-ceramide pathway is altered in KG1a cells upstream of the ceramide generation. Analysis of the transverse distribution of SPM in the plasma membrane showed that the SPM pool involved in cell signaling (inner leaflet) was markedly reduced in KG1a cells; it is 7-fold lower than that found in the inner leaflet of U937 cells. Therefore, our study strongly suggests that the different responses induced by TNF alpha in myeloid cells are dependent on the SPM plasma membrane transverse asymmetry.