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 acids desaturation and cholesterol although some cells, such insect cells, are almost devoid of sterols synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. In order to investigate how both sterols and phospholipids control fluidity homeostasis we quantified the lipidic composition of insect SF9b and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared to mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (four 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 while 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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Allosteric regulation of G protein-coupled receptor activity by phospholipids.

Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

How lipids influence the activity of β2-adrenergic receptor

The lipidic composition of the membrane bilayer has been proven to be a critical player in the modulation of many membrane protein structure and function. Several studies have proposed a functional role for lipids, especially cholesterol, on the biological behavior of GPCRs. Our studies will focus on the human b2-adrenergic receptor (b2AR), which is involved mostly in the modulation of the sympathetic nervous system. Since b2AR is widely expressed in the organism, the question of the receptor regulation in different tissues arises. It has been suggested that a tissue-specific regulation of b2AR is possible in relation to its environment. The effect of cholesterol on b2AR stability and function has been clearly shown in some publications. However, very few studies tackle the role of other membrane lipids on GPCRs activity. Thus our work was aimed to understand the effect of lipids on b2AR function. We have developed a method to examine the interaction between lipids in biological membranes and the receptor using mass spectrometry. We were thus able to identify the lipid species bound to the purified receptor. We have also tested the effect of lipid environment on the functionality of the receptor. Using different lipid species individually, we have reconstituted b2AR in HDL particles of phosphatidylcholine (PC), phosphatydilethanolamine (PE), phosphatidylglycerol (PG), phosphatydylserine (PS), PC/Cholesteryl hemisuccinate (PC/CHS) and PC/sphingomyelin (PC/SM). We show the choice of lipids strongly modulate the affinity of the receptor towards its ligands as well as liguand-induced conformational changes. Some lipid species, like PE favor an inactive state of the receptor, whereas others like PG promote the activation of the protein. Cholesterol analogue CHS enhances the affinity of b2AR towards its ligands (agonist and antagonist). We have also monitored the effect of fatty acid chain length on the receptor activity.The results presented here constitute a significant step towards understanding the relationship between b2AR and the lipids in biological membrane.info:eu-repo/semantics/publishe

Nitrogen catabolite repressible GAP1 promoter, a new tool for efficient recombinant protein production in S. cerevisiae.

Decades of work requiring heterologous expression of eukaryotic proteins have shown that no expression system can be considered as the panacea and the appropriate expression strategy is often protein-dependent. In a large number of cases, yeasts have proven to be reliable organisms for heterologous protein expression by combining eukaryotic cellular organization with the ease of use of simpler microorganisms.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe