Coordinated regulation of phosphatidylinositol 4-phosphate and phosphatidylserine levels by Osh4p and Osh5p is an essential regulatory mechanism in autophagy.

Macroautophagy (hereafter autophagy) is an intracellular degradative pathway in budding yeast cells. Certain lipid types play essential roles in autophagy; yet the precise mechanisms regulating lipid composition during autophagy remain unknown. Here, we explored the role of the Osh family proteins in the modulating lipid composition during autophagy in budding yeast. Our results showed that osh1-osh7∆ deletions lead to autophagic dysfunction, with impaired GFP-Atg8 processing and the absence of autophagosomes and autophagic bodies in the cytosol and vacuole, respectively. Freeze-fracture electron microscopy (EM) revealed elevated phosphatidylinositol 4-phosphate (PtdIns(4)P) levels in cytoplasmic and luminal leaflets of autophagic bodies and vacuolar membranes in all deletion mutants. Phosphatidylserine (PtdSer) levels were significantly decreased in the autophagic bodies and vacuolar membranes in osh4∆ and osh5∆ mutants, whereas no significant changes were observed in other osh deletion mutants. Furthermore, we identified defects in autophagic processes in the osh4∆ and osh5∆ mutants, including rare autophagosome formation in the osh5∆ mutant and accumulation of autophagic bodies in the vacuole in the osh4∆ mutant, even in the absence of the proteinase inhibitor PMSF. These findings suggest that Osh4p and Osh5p play crucial roles in the transport of PtdSer to autophagic bodies and autophagosome membranes, respectively. The precise control of lipid composition in the membranes of autophagosomes and autophagic bodies by Osh4p and Osh5p represents an important regulatory mechanism in autophagy.

The Relevance, Predictability, and Utility of Annexin A5 for Human Physiopathology.

As an important functional protein molecule in the human body, human annexin A5 (hAnxA5) is widely found in human cells and body fluids. hAnxA5, the smallest type of annexin, performs a variety of biological functions by reversibly and specifically binding phosphatidylserine (PS) in a calcium-dependent manner and plays an important role in many human physiological and pathological processes. The free state hAnxA5 exists in the form of monomers and usually forms a polymer in a specific self-assembly manner when exerting biological activity. This review systematically discusses the current knowledge and understanding of hAnxA5 from three perspectives: physiopathological relevance, diagnostic value, and therapeutic utility. hAnxA5 affects the occurrence and development of many physiopathological processes. Moreover, hAnxA5 can be used independently or in combination as a biomarker of physiopathological phenomena for the diagnosis of certain diseases. Importantly, based on the properties of hAnxA5, many novel drug candidates have been designed and prepared for application in actual medical practice. However, there are also some gaps and shortcomings in hAnxA5 research. This in-depth study will not only expand the understanding of structural and functional relationships but also promote the application of hAnxA5 in the field of biomedicine.

Bending of a lipid membrane edge by annexin A5 trimers.

Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.

Bufalin reprograms erythrocyte lifespan through p38 MAPK and Rac1 GTPase.

Ample evidence indicates that bufalin (BFN), a cardiotonic steroid in Bufo toad toxin, possesses a potent anticancer activity mainly by stimulating apoptosis in cancer cells. Human red blood cells (RBCs) undergo eryptosis which contributes to a plethora of pathological conditions. No reports, however, have examined the potential toxicity of BFN to RBCs. This study aims to characterize the biochemical mechanisms governing the influence of BFN on the physiology and lifespan of RBCs. Isolated RBCs from healthy volunteers were exposed to anticancer concentrations of commercially available BFN from the skin of Bufo gargarizans (10-200 µM) for 24 h at 37 °C. Photometric assays were used to estimate hemolysis and hemolytic markers, and flow cytometry was used to detect eryptotic markers. Phosphatidylserine externalization was captured by fluorescein isothiocyante-labeled annexin V, cellular dimensions by light scatter patterns, and intracellular Ca2+ and reactive oxygen species (ROS) by fluorogenic dyes Fluo4/AM and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. BFN caused Ca2+-independent hemolysis and release of LDH, AST, CK, and K+, and increased annexin V-bound cells, cytosolic Ca2+, cell shrinkage, and ROS levels. BFN also disrupted Na+ and Mg2+ trafficking, and was sensitive to PEG 8000, sucrose, SB203580, and NSC 23766. In whole blood, BFN depleted hemoglobin stores, increased fragmented RBCs, and was selectively toxic to reticulocytes, lymphocytes, and platelets. In conclusion, BFN elicits premature RBC death, subject to regulation by p38 MAPK and Rac1 GTPase, and is detrimental to other peripheral blood cells. Altogether, these novel findings prompt cautious consideration of the toxin in anticancer therapy.

Smoking is associated with increased eryptosis, suicidal erythrocyte death, in a large population-based cohort.

Smoking has multiple detrimental effects on health, and is a major preventable cause of premature death and chronic disease. Despite the well-described effect of inhaled substances from tobacco smoke on cell toxicity, the association between smoking and suicidal erythrocyte death, termed eryptosis, is virtually unknown. Therefore, the blood samples of 2023 participants of the German National Cohort Study (NAKO) were analyzed using flow cytometry analysis to determine eryptosis from fluorescent annexin V-FITC-binding to phosphatidylserine-exposing erythrocytes. Blood analyses were complemented by the measurement of hematologic parameters including red blood cell count, hematocrit, hemoglobin, mean corpuscular cell volume (MCV) and mean corpuscular hemoglobin (MCH). Eryptosis was higher in smokers than in non- and ex-smokers, and positively associated with the number of cigarettes smoked daily (r = 0.08, 95% CI [0.03, 0.12]). Interestingly, despite increased eryptosis, smokers had higher red blood cell indices than non-smokers. To conclude, smokers were characterized by higher eryptosis than non-smokers, without showing any obvious detrimental effect on classic hematological parameters.

Redistribution of the glycocalyx exposes phagocytic determinants on apoptotic cells.

Phagocytes remove dead and dying cells by engaging "eat-me" ligands such as phosphatidylserine (PtdSer) on the surface of apoptotic targets. However, PtdSer is obscured by the bulky exofacial glycocalyx, which also exposes ligands that activate "don't-eat-me" receptors such as Siglecs. Clearly, unshielding the juxtamembrane "eat-me" ligands is required for the successful engulfment of apoptotic cells, but the mechanisms underlying this process have not been described. Using human and murine cells, we find that apoptosis-induced retraction and weakening of the cytoskeleton that anchors transmembrane proteins cause an inhomogeneous redistribution of the glycocalyx: actin-depleted blebs emerge, lacking the glycocalyx, while the rest of the apoptotic cell body retains sufficient actin to tether the glycocalyx in place. Thus, apoptotic blebs can be engaged by phagocytes and are targeted for engulfment. Therefore, in cells with an elaborate glycocalyx, such as mucinous cancer cells, this "don't-come-close-to-me" barrier must be removed to enable clearance by phagocytosis.

Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Phosphatidylserine turns the gears of phospholipids in B cell lymphoma.

Phosphatidylserine levels and distribution are tightly controlled by dedicated enzymes at the ER and plasma membrane. Nakatsu and Kawasaki discuss new work by Aoki and colleagues (https://doi.org/10.1083/jcb.202212074), which reveals an acute reliance on phosphatidylserine synthesis in B cell lymphomas needed to prevent aberrant B cell receptor activation and ensuing apoptosis.

Apoptosis and eryptosis: similarities and differences.

Eryptosis is a regulated cell death (RCD) of mature erythrocytes initially described as a counterpart of apoptosis for enucleated cells. However, over the recent years, a growing number of studies have emphasized certain differences between both cell death modalities. In this review paper, we underline the hallmarks of eryptosis and apoptosis and highlight resemblances and dissimilarities between both RCDs. We summarize and critically discuss differences in the impact of caspase-3, Ca2+ signaling, ROS signaling pathways, opposing roles of casein kinase 1α, protein kinase C, Janus kinase 3, cyclin-dependent kinase 4, and AMP-activated protein kinase to highlight a certain degree of divergence between apoptosis and eryptosis. This review emphasizes the crucial importance of further studies that focus on deepening our knowledge of cell death machinery and identifying novel differences between cell death of nucleated and enucleated cells. This might provide evidence that erythrocytes can be defined as viable entities capable of programmed cell destruction. Additionally, the revealed cell type-specific patterns in cell death can facilitate the development of cell death-modulating therapeutic agents.

Neuraminidase-induced externalization of phosphatidylserine activates ADAM17 and impairs insulin signaling in endothelial cells.

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously have shown that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We have also demonstrated that ADAM17 sheddase activity targets IRα; however, the mechanisms driving endothelial ADAM17 activity in T2D are largely unknown. Herein, we report that externalization of phosphatidylserine (PS) to the outer leaflet of the plasma membrane causes ADAM17-mediated shedding of IRα and blunting of insulin signaling in endothelial cells. Furthermore, we demonstrate that endothelial PS externalization is mediated by the phospholipid scramblase anoctamin-6 (ANO6) and that this process can be stimulated by neuraminidase, a soluble enzyme that cleaves sialic acid residues. Of note, we demonstrate that men and women with T2D display increased levels of neuraminidase activity in plasma, relative to age-matched healthy individuals, and this occurs in conjunction with increased ADAM17 activity and impaired leg blood flow responses to endogenous insulin. Collectively, this work reveals the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.NEW & NOTEWORTHY This work provides the first evidence that neuraminidase, an enzyme increased in the circulation of men and women with type 2 diabetes (T2D), promotes anoctamin-6 (ANO6)-dependent externalization of phosphatidylserine in endothelial cells, which in turn leads to activation of a disintegrin and metalloproteinase-17 (ADAM17) and consequent shedding of the insulin receptor-α from the cell surface. Hence, this work supports that consideration should be given to the neuraminidase-ANO6-ADAM17 axis as a novel potential target for restoring endothelial insulin sensitivity in T2D.

Mitofusin-2 Regulates Platelet Mitochondria and Function.

BACKGROUND: Single-nucleotide polymorphisms linked with the rs1474868 T allele (MFN2 [mitofusin-2] T/T) in the human mitochondrial fusion protein MFN2 gene are associated with reduced platelet MFN2 RNA expression and platelet counts. This study investigates the impact of MFN2 on megakaryocyte and platelet biology. METHODS: Mice with megakaryocyte/platelet deletion of Mfn2 (Mfn2-/- [Mfn2 conditional knockout]) were generated using Pf4-Cre crossed with floxed Mfn2 mice. Human megakaryocytes were generated from cord blood and platelets isolated from healthy subjects genotyped for rs1474868. Ex vivo approaches assessed mitochondrial morphology, function, and platelet activation responses. In vivo measurements included endogenous/transfused platelet life span, tail bleed time, transient middle cerebral artery occlusion, and pulmonary vascular permeability/hemorrhage following lipopolysaccharide-induced acute lung injury. RESULTS: Mitochondria was more fragmented in megakaryocytes derived from Mfn2-/- mice and from human cord blood with MFN2 T/T genotype compared with control megakaryocytes. Human resting platelets of MFN2 T/T genotype had reduced MFN2 protein, diminished mitochondrial membrane potential, and an increased rate of phosphatidylserine exposure during ex vivo culture. Platelet counts and platelet life span were reduced in Mfn2-/- mice accompanied by an increased rate of phosphatidylserine exposure in resting platelets, especially aged platelets, during ex vivo culture. Mfn2-/- also decreased platelet mitochondrial membrane potential (basal) and activated mitochondrial oxygen consumption rate, reactive oxygen species generation, calcium flux, platelet-neutrophil aggregate formation, and phosphatidylserine exposure following dual agonist activation. Ultimately, Mfn2-/- mice showed prolonged tail bleed times, decreased ischemic stroke infarct size after cerebral ischemia-reperfusion, and exacerbated pulmonary inflammatory hemorrhage following lipopolysaccharide-induced acute lung injury. Analysis of MFN2 SNPs in the iSPAAR study (Identification of SNPs Predisposing to Altered ALI Risk) identified a significant association between MFN2 and 28-day mortality in patients with acute respiratory distress syndrome. CONCLUSIONS: Mfn2 preserves mitochondrial phenotypes in megakaryocytes and platelets and influences platelet life span, function, and outcomes of stroke and lung injury.

Phosphatidylthreonine is a procoagulant lipid detected in human blood and elevated in coronary artery disease.

Aminophospholipids (aPL) such as phosphatidylserine are essential for supporting the activity of coagulation factors, circulating platelets, and blood cells. Phosphatidylthreonine (PT) is an aminophospholipid previously reported in eukaryotic parasites and animal cell cultures, but not yet in human tissues. Here, we evaluated whether PT is present in blood cells and characterized its ability to support coagulation. Several PT molecular species were detected in human blood, washed platelets, extracellular vesicles, and isolated leukocytes from healthy volunteers using liquid chromatography-tandem mass spectrometry. The ability of PT to support coagulation was demonstrated in vitro using biochemical and biophysical assays. In liposomes, PT supported prothrombinase activity in the presence and absence of phosphatidylserine. PT nanodiscs strongly bound FVa and lactadherin (nM affinity) but poorly bound prothrombin and FX, suggesting that PT supports prothrombinase through recruitment of FVa. PT liposomes bearing tissue factor poorly generated thrombin in platelet poor plasma, indicating that PT poorly supports extrinsic tenase activity. On platelet activation, PT is externalized and partially metabolized. Last, PT was significantly higher in platelets and extracellular vesicle from patients with coronary artery disease than in healthy controls. In summary, PT is present in human blood, binds FVa and lactadherin, supports coagulation in vitro through FVa binding, and is elevated in atherosclerotic vascular disease. Our studies reveal a new phospholipid subclass, that contributes to the procoagulant membrane, and may support thrombosis in patients at elevated risk.

Enhanced CD1d phosphatidylserine presentation using a single-domain antibody promotes immunomodulatory CD1d-TIM-3 interactions.

BACKGROUND: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. METHODS: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. RESULTS: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. CONCLUSION: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.

A missense mutant of ocrl1 promotes apoptosis of tubular epithelial cells and disrupts endocytosis and the cell cycle of podocytes in Dent-2 Disease.

BACKGROUND: This study aimed to identify an orcl1 mutation in a patient with Dent-2 Disease and investigate the underlying mechanisms. METHODS: The ocrl1 mutation was identified through exome sequencing. Knockdown of orcl1 and overexpression of the orcl1 mutant were performed in HK-2 and MPC5 cells to study its function, while flow cytometry measured reactive oxygen species (ROS), phosphatidylserine levels, and cell apoptosis. Scanning electron microscopy observed crystal adhesion, while transmission electron microscopy examined kidney tissue pathology. Laser scanning confocal microscopy was used to examine endocytosis, and immunohistochemical and immunofluorescence assays detected protein expression. Additionally, podocyte-specific orcl1 knockout mice were generated to investigate the role of orcl1 in vivo. RESULTS: We identified a mutation resulting in the replacement of Histidine with Arginine at position 318 (R318H) in ocrl1 in the proband. orcl1 was widely expressed in the kidney. In vitro experiments showed that knockdown of orcl1 and overexpression of ocrl1 mutant increased ROS, phosphatidylserine exocytosis, crystal adhesion, and cell apoptosis in HK-2 cells. Knockdown of orcl1 in podocytes reduced endocytosis and disrupted the cell cycle while increasing cell migration. In vivo studies in mice showed that conditional deletion of orcl1 in podocytes caused glomerular dysfunction, including proteinuria and fibrosis. CONCLUSION: This study identified an R318H mutation in orcl1 in a patient with Dent-2 Disease. This mutation may contribute to renal injury by promoting ROS production and inducing cell apoptosis in tubular cells, while disrupting endocytosis and the cell cycle, and promoting cell migration of podocytes. Video Abstract.

Deciphering Lipid Arrangement in Phosphatidylserine/Phosphatidylcholine Mixed Membranes: Simulations and Experiments.

Phosphatidylserine (PS) exposure on the plasma membrane is crucial for many cellular processes including apoptotic cell recognition, blood clotting regulation, cellular signaling, and intercellular interactions. In this study, we investigated the arrangement of PS headgroups in mixed PS/phosphatidylcholine (PC) bilayers, serving as a simplified model of the outer leaflets of mammalian cell plasma membranes. Combining atomistic-scale molecular dynamics (MD) simulations with Langmuir monolayer experiments, we unraveled the mutual miscibility of POPC and POPS lipids and the intricate intermolecular interactions inherent to these membranes as well as the disparities in position and orientation of PC and PS headgroups. Our experiments revealed micrometer-scale miscibility at all mole fractions of POPC and POPS, marked by modest deviations from ideal mixing with no apparent microscale phase separation. The MD simulations, meanwhile, demonstrated that these deviations were due to strong electrostatic interactions between like-lipid pairs (POPC-POPC and POPS-POPS), culminating in lateral segregation and nanoscale clustering. Notably, PS headgroups profoundly affect the ordering of the lipid acyl chains, leading to lipid elongation and subtle PS protrusion above the zwitterionic membrane. In addition, PC headgroups are more tilted with respect to the membrane normal, while PS headgroups align at a smaller angle, making them more exposed to the surface of the mixed PC/PS membranes. These findings provide a detailed molecular-level account of the organization of mixed PC/PS membranes, corroborated by experimental data. The insights gained here extend our comprehension of the physiological role of PSs.

Rosmarinic Acid Elicits Calcium-Dependent and Sucrose-Sensitive Eryptosis and Hemolysis through p38 MAPK, CK1α, and PKC.

BACKGROUND: Rosmarinic acid (RA) possesses promising anticancer potential, but further development of chemotherapeutic agents is hindered by their toxicity to off-target tissue. In particular, chemotherapy-related anemia is a major obstacle in cancer therapy, which may be aggravated by hemolysis and eryptosis. This work presents a toxicity assessment of RA in human RBCs and explores associated molecular mechanisms. METHODS: RBCs isolated from healthy donors were treated with anticancer concentrations of RA (10-800 µM) for 24 h at 37 °C, and hemolysis and related markers were photometrically measured. Flow cytometry was used to detect canonical markers of eryptosis, including phosphatidylserine (PS) exposure by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, cell size by FSC, and oxidative stress by H2DCFDA. Ions and pH were assessed by an ion-selective electrode, while B12 was detected by chemiluminescence. RESULTS: RA elicited concentration-dependent hemolysis with AST and LDH release but rescued the cells from hypotonic lysis at sub-hemolytic concentrations. RA also significantly increased annexin-V-positive cells, which was ameliorated by extracellular Ca2+ removal and isosmotic sucrose. Furthermore, a significant increase in Fluo4-positive cells and B12 content and a decrease in FSC and extracellular pH with KCl efflux were noted upon RA treatment. Hemolysis was augmented by blocking KCl efflux and was blunted by ATP, SB203580, staurosporin, D4476, isosmotic urea, and PEG 8000. CONCLUSIONS: RA stimulates Ca2+-dependent and sucrose-sensitive hemolysis and eryptosis characterized by PS exposure, Ca2+ accumulation, loss of ionic regulation, and cell shrinkage. These toxic effects were mediated through energy deprivation, p38 MAPK, protein kinase C, and casein kinase 1α.

A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Annexin A5 stabilizes matrix vesicle-biomimetic lipid membranes: unravelling a new role of annexins in calcification.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5-2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture.

MFG-E8: a model of multiple binding modes associated with ps-binding proteins.

Membrane-binding proteins often associate with lipid membranes through a singular binding interface which is generally modeled as a two-state system: bound or unbound. However, even a single interface can engage with more than one mode of binding since a variety of interactions can contribute to the binding event. Unfortunately, the ability to clearly delineate the different binding modes of a singular binding interface has been elusive with existing models. Here, we present a study on milk fat globule EGF factor 8 (MFG-E8), which belongs to a class of proteins that identifies and binds phosphatidylserine (PS). These proteins detect membrane dysregulation implicated in exposed PS in apoptosis and malignant cells. In order to elucidate the factors affecting the binding of MFG-E8, we used a model system consisting of a series of lipid vesicles with varying PS mole fraction to identify the sensitivity of MFG-E8's binding affinity to changes in electrostatics using a tryptophan fluorescence spectral shift assay. Using a newly developed model, we experimentally identified three binding modes, each associated with a different number of PS lipids, with its cooperativity for binding being enhanced by the availability of negatively charged lipids. X-ray reflectivity experiments additionally suggest that MFG-E8's binding modes are influenced by membrane packing. The protocols established for elucidating MFG-E8's interaction with lipid membranes under different membrane conditions can be applied to the study of other membrane-binding proteins that target specific membrane attributes, such as fluidity and electrostatics, and help elucidate these membrane targeting mechanisms and their subsequent binding events.

Selective targeting and clustering of phosphatidylserine lipids by RSV M protein is critical for virus particle production.

Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and antiviral drugs. RSV assembles and buds from the host cell plasma membrane by forming infectious viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of the matrix (M) protein with cell plasma membrane lipids forming a layer at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with cell membrane lipids, and with which one, to promote viral assembly. Here, we demonstrate that M proteins are able to cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as a dimer and upon M oligomerization, PS clustering is observed. In contrast, the presence of other negatively charged lipids like PI(4, 5)P2 does not enhance M binding beyond control zwitterionic lipids, while cholesterol negatively affects M interaction with membrane lipids. Moreover, we show that the initial binding of the RSV M protein with PS lipids is independent of the cytoplasmic tail of the fusion (F) glycoprotein (FCT). Here, we highlight that M binding on membranes occurs directly through PS lipids, this interaction is electrostatic in nature, and M oligomerization generates PS clusters.