FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

In situ artificial contact sites (ISACS) between synthetic and endogenous organelle membranes allow for quantification of protein-tethering activities.

Membrane contact sites are specialized areas where the membranes of two distinct organelles are physically connected and allow for the exchange of molecules and for signaling processes. Understanding the mechanisms whereby proteins localize to and function in these structures is of special interest; however, methods allowing for reconstitution of these contact sites are few and only based on synthetic membranes and recombinant proteins. Here, we devised a strategy to create in situ artificial contact sites between synthetic and endogenous organelle membranes. Liposomes functionalized with a peptide containing a two phenylalanines in an acidic tract (FFAT) motif were added to adherent cells whose plasma membrane was perforated. Confocal and super-resolution microscopy revealed that these liposomes associated with the endoplasmic reticulum via the specific interaction of the FFAT motif with endoplasmic reticulum-resident vesicle-associated membrane protein-associated proteins. This approach allowed for quantification of the attachment properties of peptides corresponding to FFAT motifs derived from distinct proteins and of a protein construct derived from steroidogenic acute regulatory protein-related lipid transfer domain-3. Collectively, these data indicate that the creation of in situ artificial contact sites represents an efficient approach for studying the membrane-tethering activity of proteins and for designing membrane contact site reconstitution assays in cellular contexts.

Functional analyses of phosphatidylserine/PI(4)P exchangers with diverse lipid species and membrane contexts reveal unanticipated rules on lipid transfer.

BACKGROUND: Lipid species are accurately distributed in the eukaryotic cell so that organelle and plasma membranes have an adequate lipid composition to support numerous cellular functions. In the plasma membrane, a precise regulation of the level of lipids such as phosphatidylserine, PI(4)P, and PI(4,5)P2, is critical for maintaining the signaling competence of the cell. Several lipid transfer proteins of the ORP/Osh family contribute to this fine-tuning by delivering PS, synthesized in the endoplasmic reticulum, to the plasma membrane in exchange for PI(4)P. To get insights into the role of these PS/PI(4)P exchangers in regulating plasma membrane features, we question how they selectively recognize and transfer lipid ligands with different acyl chains, whether these proteins exchange PS exclusively for PI(4)P or additionally for PI(4,5)P2, and how sterol abundance in the plasma membrane impacts their activity. RESULTS: We measured in vitro how the yeast Osh6p and human ORP8 transported PS and PI(4)P subspecies of diverse length and unsaturation degree between membranes by fluorescence-based assays. We established that the exchange activity of Osh6p and ORP8 strongly depends on whether these ligands are saturated or not, and is high with representative cellular PS and PI(4)P subspecies. Unexpectedly, we found that the speed at which these proteins individually transfer lipid ligands between membranes is inversely related to their affinity for them and that high-affinity ligands must be exchanged to be transferred more rapidly. Next we determined that Osh6p and ORP8 cannot use PI(4,5)P2 for exchange processes, because it is a low-affinity ligand, and do not transfer more PS into sterol-rich membranes. CONCLUSIONS: Our study provides new insights into PS/PI(4)P exchangers by indicating the degree to which they can regulate the acyl chain composition of the PM, and how they control PM phosphoinositide levels. Moreover, we establish general rules on how the activity of lipid transfer proteins relates to their affinity for ligands.

Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites.

Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP-A and VAP-B, interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain-containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro MOSPD2 is an ER-anchored protein, and it interacts with several FFAT-containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle-bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.

The role of paraoxonase 1 in regulating high-density lipoprotein functionality during aging.

Pharmacological interventions to increase the concentration of high-density lipoprotein (HDL) have led to disappointing results and have contributed to the emergence of the concept of HDL functionality. The anti-atherogenic activity of HDLs can be explained by their functionality or quality. The capacity of HDLs to maintain cellular cholesterol homeostasis and to transport cholesterol from peripheral cells to the liver for elimination is one of their principal anti-atherogenic activities. However, HDLs possess several other attributes that contribute to their protective effect against cardiovascular diseases. HDL functionality is regulated by various proteins and lipids making up HDL particles. However, several studies investigated the role of paraoxonase 1 (PON1) and suggest a significant role of this protein in the regulation of the functionality of HDLs. Moreover, research on PON1 attracted much interest following several studies indicating that it is involved in cardiovascular protection. However, the mechanisms by which PON1 exerts these effects remain to be elucidated.

Advanced glycation end products affect cholesterol homeostasis by impairing ABCA1 expression on macrophages.

Reverse cholesterol transport (RCT), which is intimately linked to high-density lipoproteins (HDLs), plays a key role in cholesterol homeostasis and the prevention of atherosclerosis. The goal of the present study was to investigate the effect of aging and advanced glycation end products (AGEs) on RCT as well as on other factors that may affect the antiatherogenic property of HDLs. The transfer of macrophage-derived cholesterol to the plasma and liver and then to the feces for elimination was significantly lower in aged mice than in young mice. Chronic injection of d -galactose (D-gal) or AGEs also significantly reduced RCT (65.3% reduction in [3H]cholesterol levels in the plasma of D-gal-treated mice after 48 h compared with control mice, P < 0.01). The injection of both D-gal and aminoguanidine hydrochloride increased [3H]cholesterol levels in the plasma, although the levels were lower than those of control mice. The in vitro incubation of HDLs with dicarbonyl compounds increased the carbonyl and conjugated diene content of HDLs and significantly reduced PON1 paraoxonase activity (87.4% lower than control HDLs, P < 0.0001). Treating J774A.1 macrophages with glycated fetal bovine serum increased carbonyl formation (39.5% increase, P < 0.003) and reduced ABCA1 protein expression and the capacity of macrophages to liberate cholesterol (69.1% decrease, P < 0.0001). Our results showed, for the first time, that RCT is altered with aging and that AGEs contribute significantly to this alteration.

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes.

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found to represent a novel lipid transfer protein (LTP) group in yeast and human cells. They transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol 4-phosphate (PI(4)P) exchange cycles. This finding allows a better understanding of how PS, which is critical for signaling processes, is distributed throughout the cell and the investigation of the link between this process and phosphoinositide (PIP) metabolism. The development of new fluorescence-based protocols has been instrumental in the discovery and characterization of this new cellular mechanism in vitro at the molecular level. This paper describes the production and the use of two fluorescently labelled lipid sensors, NBD-C2Lact and NBD-PHFAPP, to measure the ability of a protein to extract PS or PI(4)P and to transfer these lipids between artificial membranes. First, the protocol describes how to produce, label, and obtain high-purity samples of these two constructs. Secondly, this paper explains how to use these sensors with a fluorescence microplate reader to determine whether a protein can extract PS or PI(4)P from liposomes, using Osh6p as a case study. Finally, this protocol shows how to accurately measure the kinetics of PS/PI(4)P exchange between liposomes of defined lipid composition and to determine lipid transfer rates by fluorescence resonance energy transfer (FRET) using a standard fluorometer.

Lipid Exchangers: Cellular Functions and Mechanistic Links With Phosphoinositide Metabolism.

Lipids are amphiphilic molecules that self-assemble to form biological membranes. Thousands of lipid species coexist in the cell and, once combined, define organelle identity. Due to recent progress in lipidomic analysis, we now know how lipid composition is finely tuned in different subcellular regions. Along with lipid synthesis, remodeling and flip-flop, lipid transfer is one of the active processes that regulates this intracellular lipid distribution. It is mediated by Lipid Transfer Proteins (LTPs) that precisely move certain lipid species across the cytosol and between the organelles. A particular subset of LTPs from three families (Sec14, PITP, OSBP/ORP/Osh) act as lipid exchangers. A striking feature of these exchangers is that they use phosphatidylinositol or phosphoinositides (PIPs) as a lipid ligand and thereby have specific links with PIP metabolism and are thus able to both control the lipid composition of cellular membranes and their signaling capacity. As a result, they play pivotal roles in cellular processes such as vesicular trafficking and signal transduction at the plasma membrane. Recent data have shown that some PIPs are used as energy by lipid exchangers to generate lipid gradients between organelles. Here we describe the importance of lipid counter-exchange in the cell, its structural basis, and presumed links with pathologies.

Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport.

This study was aimed to investigate the effect of human PON1 overexpression in mice on cholesterol efflux and reverse cholesterol transport. PON1 overexpression in PON1-Tg mice induced a significant 3-fold (p<0.0001) increase in plasma paraoxonase activity and a significant ~30% (p<0.0001) increase in the capacity of HDL to mediate cholesterol efflux from J774 macrophages compared to wild-type mice. It also caused a significant 4-fold increase (p<0.0001) in the capacity of macrophages to transfer cholesterol to apoA-1, a significant 2-fold (p<0.0003) increase in ABCA1 mRNA and protein expression, and a significant increase in the expression of PPARγ (p<0.0003 and p<0.04, respectively) and LXRα (p<0.0001 and p<0.01, respectively) mRNA and protein compared to macrophages from wild-type mice. Moreover, transfection of J774 macrophages with human PON1 also increased ABCA1, PPARγ and LXRα protein expression and stimulates macrophages cholesterol efflux to apo A1. In vivo measurements showed that the overexpression of PON1 significantly increases the fecal elimination of macrophage-derived cholesterol in PON1-Tg mice. Overall, our results suggested that the overexpression of PON1 in mice may contribute to the regulation of the cholesterol homeostasis by improving the capacity of HDL to mediate cholesterol efflux and by stimulating reverse cholesterol transport.

Paraoxonase 1-treated oxLDL promotes cholesterol efflux from macrophages by stimulating the PPARγ-LXRα-ABCA1 pathway.

Here, we investigate the mechanism through which paraoxonase 1 (PON1) may regulate cholesterol efflux. Pretreatment of oxLDL with PON1 (oxLDL-PON1) contributed to the formation of LysoPC. In J774 macrophages, oxLDL-PON1 increased cholesterol efflux by more than 47% compared to oxLDL alone. oxLDL-PON1 significantly increased mRNA and protein expression of ABCA1 and ABCG1, as well as of PPARγ and LXRα compared to oxLDL alone. Intraperitoneal injection of oxLDL-PON1- or LysoPC-treated J774 macrophages significantly increased the fecal elimination of macrophage-derived cholesterol in these mice. Our results suggest that PON1 stimulates cholesterol efflux via a mechanism that involves oxidized phospholipid hydrolysis.

Alteration of HDL functionality and PON1 activities in acute coronary syndrome patients.

OBJECTIVE: The functionality of HDL has been suggested as an important factor in the prevention of cardiovascular and coronary artery diseases. The objective of the present study was to investigate the functionality of HDL and the factors that may affect the anti-atherogenic properties of HDL in ACS patients. METHODS AND RESULTS: One hundred healthy subjects and 205 ACS patients were recruited. HDL functionality was evaluated by measuring their capacity to mediate cholesterol efflux from J774 macrophages. Oxidative stress status was determined by measuring plasma malondialdehyde (MDA), protein carbonyl, and vitamin E levels by HPLC. The PON1 Q192R polymorphism status and PON1 paraoxonase and arylesterase activities of the healthy subjects and ACS patients were also determined. The HDL of ACS patients displayed a limited capacity to mediate cholesterol efflux, especially via the ABCA1-pathway. MDA (7.06±0.29 µM) and protein carbonyl (9.29±0.26 µM) levels were significantly higher in ACS patients than in healthy subjects (2.29±0.21 µM and 3.07±0.17 µM, respectively, p<0.0001), while α- and γ-tocopherol (vitamin E) levels in ACS patients were 8-fold (p<0.001) and 2-fold (p<0.05) lower than in healthy subjects. Paraoxonase, arylesterase and HDL-corrected PON1 activities (PON1 activity/HDL ratio) were significantly lower in ACS patients. Logistic regression analyses showed that high PON1 paraoxonase and arylesterase activities had a significant protective effect (OR=0.413, CI 0.289-0.590, p<0.001; OR=0.232 CI 0.107-0.499, p<0.001, respectively) even when adjusted for HDL level, age, BMI, and PON1 polymorphism. CONCLUSION: The results of the present study showed that the functionality of HDL is impaired in ACS patients and that the impairment may be due to oxidative stress and an alteration of PON1 activities.