Hélène Barelli

Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins

Bending the benefits of polyunsaturates We have often heard that it is beneficial to eat polyunsaturated fatty acids. We also know that some organelles such as synaptic vesicles are extremely rich in polyunsaturated lipids. However, what polyunsaturated lipids do in our body is unclear. Using cell biology, biochemical reconstitutions, and molecular dynamics, Pinot et al. show that polyunsaturated phospholipids can change the response of membranes to proteins involved in membrane curvature sensing, membrane shaping, and membrane fission. Polyunsaturated phospholipids make the plasma membrane more amenable to deformation; facilitate endocytosis; and, in reconstitution experiments, increased membrane fission by the dynamin-endophilin complex. Science, this issue p. 693 Certain membrane lipids adapt their conformation to membrane curvature, facilitating membrane deformation and fission. Phospholipids (PLs) with polyunsaturated acyl chains are extremely abundant in a few specialized cellular organelles such as synaptic vesicles and photoreceptor discs, but their effect on membrane properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of dynamin and endophilin to deform and vesiculate synthetic membranes. When cells incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to deformation by a pulling force and the rate of endocytosis was accelerated, in particular, under conditions in which cholesterol was limiting. Molecular dynamics simulations and biochemical measurements indicated that polyunsaturated PLs adapted their conformation to membrane curvature. Thus, by reducing the energetic cost of membrane bending and fission, polyunsaturated PLs may help to support rapid endocytosis.

A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment

Two parameters of biological membranes, curvature and lipid composition, direct the recruitment of many peripheral proteins to cellular organelles. Although these traits are often studied independently, it is their combination that generates the unique interfacial properties of cellular membranes. Here, we use a combination of in vivo, in vitro and in silico approaches to provide a comprehensive map of how these parameters modulate membrane adhesive properties. The correlation between the membrane partitioning of model amphipathic helices and the distribution of lipid-packing defects in membranes of different shape and composition explains how macroscopic membrane properties modulate protein recruitment by changing the molecular topography of the membrane interfacial region. Furthermore, our results suggest that the range of conditions that can be obtained in a cellular context is remarkably large because lipid composition and curvature have, under most circumstances, cumulative effects.

Kinetic Studies of the Arf Activator Arno on Model Membranes in the Presence of Arf Effectors Suggest Control by a Positive Feedback Loop

Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.

Neurotensin metabolism in various tissues of central and peripheral origins: ubiquitous involvement of a novel neurotensin degrading metalloendopeptidase

The metabolism of neurotensin in vitro, in various membrane preparations and cell lines of central and peripheral origins was studied. Neurotensin degradation products were separated by HPLC and identified by either amino acid analysis or by their retention times. Peptidases responsible for the cleavages were identified by means of specific fluorigenic substrates or inhibitors. Although the patterns of neurotensin inactivation varied according to the tissue source in all cases, a major primary cleavage occurred at the Pro10-Tyr11 bond, leading to the biologically inactive fragments NT1-10 and NT11-13. A novel neurotensin-degrading metallopeptidase was responsible for this cleavage. Interestingly, it was the only peptidase that was ubiquitously detected. In addition, endopeptidase 24.11 (EC 3.4.24.11) contributed to this cleavage in rat brain synaptic membranes as well as in circular and longitudinal smooth muscle plasma membranes from dog ileum.

Proteasome Contributes to the α‐Secretase Pathway of Amyloid Precursor Protein in Human Cells

Abstract: A major histopathological hallmark in Alzheimers disease consists of the extracellular deposition of the amyloid β‐peptide (Aβ) that is proteolytically derived from the β‐amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α‐secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C‐terminal‐truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic‐like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13‐dibutyrate‐sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild‐type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α‐secretase pathway in human cells.

Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum.

1 The degradation of tritiated and unlabelled neurotensin (NT) following close intra‐arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2 Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep‐Pak columns followed by reverse‐phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3 The half‐life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1–8), neurotensin (1–10) and neurotensin (1–11), were detected. 4 Neurotensin (1–11) was mainly generated by a phosphoramidon‐sensitive cleavage, probably elicited by endopeptidase 24–11. 5 Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro‐Ile, dose‐dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1–10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp‐AAY‐pAB neither protected neurotensin from degradation nor modifed the production of neurotensin (1–10). 6 Our study is the first evidence to indicate that endopeptidase 3.4.24.16 contributes to the catabolism of neurotensin, in vivo, in the dog intestine.

A filter at the entrance of the Golgi that selects vesicles according to size and bulk lipid composition

When small phosphatidylcholine liposomes are added to perforated cells, they bind preferentially to the Golgi suggesting an exceptional avidity of this organelle for curved membranes without stereospecific interactions. We show that the cis golgin GMAP-210 accounts for this property. First, the liposome tethering properties of the Golgi resembles that of the amphipathic lipid-packing sensor (ALPS) motif of GMAP-210: both preferred small (radius < 40 nm) liposomes made of monounsaturated but not saturated lipids. Second, reducing GMAP-210 levels or redirecting its ALPS motif to mitochondria decreased liposome capture by the Golgi. Extensive mutagenesis analysis suggests that GMAP-210 tethers authentic transport vesicles via the same mechanism whereby the ALPS motif senses lipid-packing defects at the vesicle surface through its regularly spaced hydrophobic residues. We conclude that the Golgi uses GMAP-210 as a filter to select transport vesicles according to their size and bulk lipid composition. DOI: http://dx.doi.org/10.7554/eLife.16988.001

Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment

The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.

Lipid unsaturation and organelle dynamics

The number of double bonds (=unsaturation) in the acyl chains of phospholipids (PL) influences the physical properties of cellular membranes. Here, we discuss disparate molecular processes, including vesicle budding, ion channel opening, and lipoprotein formation, which are greatly facilitated by PL polyunsaturation in membranes. Experimental and computer-based approaches for the structure and dynamics of PL suggest a common cause for these effects: the ability of the polyunsaturated acyl chain of PL to extend or bent along the membrane normal according to various constraints, thereby enabling a third dimension of motion in a structure that is essentially a 2D fluid.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).