全反式维甲酸及依托泊苷对急性早幼粒细胞白血病细胞磷脂酰丝氨酸暴露及促凝血活性的影响

目的：探讨磷脂酰丝氨酸（PS）暴露在急性早幼粒细胞白血病（APL）细胞促凝血活性中的作用及不同药物对其产生的影响。方法：实验共分为4组：新采集APL细胞组、APL细胞单纯培养组、APL细胞全反式维甲酸（ATRA）处理组及APL细胞依托泊苷（VP16）处理组。提取10名初发APL患者的骨髓APL细胞进行实验,提取10名健康成人外周血单个核细胞作为凝血实验的正常对照。分别用1μmol·L-1ATRA和1μmol·L-1VP16处理APL细胞24 h,利用共聚焦显微镜及流式细胞术检测各组细胞PS暴露情况。利用凝血实验检测各组细胞总的促凝活性及细胞表面磷脂的促凝血活性。利用PS特异结合蛋白乳粘素对各组细胞进行凝血抑制实验。结果：新采集的APL细胞存在一定量的PS外翻,并且与外周血单个核细胞相比,存在更高的促凝血活性（P〈0.05）,ATRA对APL细胞的PS外翻及促凝活性有抑制作用（P〈0.05）,VP16则对其有显著的促进作用（P〈0.001）。乳粘素可以拮抗APL细胞至少70%的促FXa和FIIa生成活性。结论：PS暴露在APL细胞促凝血过程中发挥着重要作用。分化治疗药物ATRA和化疗药物VP16分别通过减少和增加APL细胞表面PS的暴露来减轻和加重凝血紊乱。乳粘素通过与PS特异结合可以有效地阻断暴露的PS的促凝活性,是一种潜在的治疗APL凝血紊乱的抗凝剂。     

乳粘素与半胱氨酸蛋白酶3抑制剂检测舌鳞癌细胞CAL-27凋亡中差异性的比较研究

目的：比较荧光标记的乳粘素（Lactadherin）和半胱氨酸天冬肽酶3（caspase-3）在检测由三氧化二砷（As2O3）诱导舌鳞癌细胞CAL-27凋亡中的敏感性,以期对两种方法在检测结果方面的不同意义有进-步的了解。方法：将配置好终浓度的As20。分别作用舌鳞癌细胞CAL．270h、3h、6h、12h,用荧光标记的caspase-3抑制剂（FAM200—1）标记原位激活的caspase-3,荧光标记的Lactadherin647检测细胞凋亡,荧光倒置显微镜分析其形态学变化,流式细胞仪定量检测并分析其活性变化。结果：As20,诱导细胞3h后．98．5％细胞被FAM200．1标记,只有1．3％细胞被Lactadherin647标记。6h、12h后,被Lactadherin647标记的细胞分别增至2．5％、6．1％。结论：As203作用细胞3h后细胞发生凋亡。FAM200—1先于乳粘素检测出早期凋亡的细胞,caspase-3的激活早于PS翻转。     

Type IV P-type ATPases Distinguish Mono- versus Diacyl Phosphatidylserine Using a Cytofacial Exit Gate in the Membrane Domain

Type IV P-type ATPases (P4-ATPases) use the energy from ATP to “flip” phospholipid across a lipid bilayer, facilitating membrane trafficking events and maintaining the characteristic plasma membrane phospholipid asymmetry. Preferred translocation substrates for the budding yeast P4-ATPases Dnf1 and Dnf2 include lysophosphatidylcholine, lysophosphatidylethanolamine, derivatives of phosphatidylcholine and phosphatidylethanolamine containing a 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) group on the sn-2 C6 position, and were presumed to include phosphatidylcholine and phosphatidylethanolamine species with two intact acyl chains. We previously identified several mutations in Dnf1 transmembrane (TM) segments 1 through 4 that greatly enhance recognition and transport of NBD phosphatidylserine (NBD-PS). Here we show that most of these Dnf1 mutants cannot flip diacylated PS to the cytosolic leaflet to establish PS asymmetry. However, mutation of a highly conserved asparagine (Asn-550) in TM3 allowed Dnf1 to restore plasma membrane PS asymmetry in a strain deficient for the P4-ATPase Drs2, the primary PS flippase. Moreover, Dnf1 N550 mutants could replace the Drs2 requirement for growth at low temperature. A screen for additional Dnf1 mutants capable of replacing Drs2 function identified substitutions of TM1 and 2 residues, within a region called the exit gate, that permit recognition of dually acylated PS. These TM1, 2, and 3 residues coordinate with the “proline + 4” residue within TM4 to determine substrate preference at the exit gate. Moreover, residues from Atp8a1, a mammalian ortholog of Drs2, in these positions allow PS recognition by Dnf1. These studies indicate that Dnf1 poorly recognizes diacylated phospholipid and define key substitutions enabling recognition of endogenous PS.     

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.     

Exposure to Factor VIII Protein in the Presence of Phosphatidylserine Induces Hypo-responsiveness toward Factor VIII Challenge in Hemophilia A Mice

Administration of recombinant factor VIII (FVIII), an important co-factor in blood clotting cascade, elicits unwanted anti-FVIII antibodies in hemophilia A (HA) patients. Previously, FVIII associated with phosphatidylserine (PS) showed significant reduction in the anti-FVIII antibody response in HA mice. The reduction in the immune response to FVIII-PS could be due either to a failure of the immune system to recognize the antigen (i.e. immunological ignorance) or to an active induction of an antigen-specific nonresponsiveness (i.e. immunological tolerance). If it were a result of tolerance, one would predict that pre-exposure to FVIII-PS would render the mice hypo-responsive to a subsequent FVIII challenge. Here, we have demonstrated that naive HA mice that were pretreated with FVIII-PS showed a significantly reduced FVIII immune response to further challenge with native FVIII and that this decreased responsiveness could be adoptively transferred to other mice. An increase in number of FoxP3-expressing CD4⁺ regulatory T-cells (Treg) was observed for the FVIII-PS-immunized group as compared with animals that received FVIII alone, suggesting the involvement of Treg in PS-mediated hypo-responsiveness. The PS-mediated reduction in antibody response was reversed by the co-administration of function-blocking anti-TGF-β antibody with FVIII-PS. The decreased response to FVIII induced by FVIII-PS was determined to be antigen-specific because the immune response to another non-cross-reactive antigen (ovalbumin) was not altered. These results are consistent with the notion that FVIII-PS is tolerogenic and suggest that immunization with this tolerogenic form of the protein could be a useful treatment option to minimize immunogenicity of FVIII and other protein-based therapeutics.     

Phospholipid Flippases Lem3p-Dnf1p and Lem3p-Dnf2p Are Involved in the Sorting of the Tryptophan Permease Tat2p in Yeast

The type 4 P-type ATPases are flippases that generate phospholipid asymmetry in membranes. In budding yeast, heteromeric flippases, including Lem3p-Dnf1p and Lem3p-Dnf2p, translocate phospholipids to the cytoplasmic leaflet of membranes. Here, we report that Lem3p-Dnf1/2p are involved in transport of the tryptophan permease Tat2p to the plasma membrane. The lem3Δ mutant exhibited a tryptophan requirement due to the mislocalization of Tat2p to intracellular membranes. Tat2p was relocalized to the plasma membrane when trans-Golgi network (TGN)-to-endosome transport was inhibited. Inhibition of ubiquitination by mutations in ubiquitination machinery also rerouted Tat2p to the plasma membrane. Lem3p-Dnf1/2p are localized to endosomal/TGN membranes in addition to the plasma membrane. Endocytosis mutants, in which Lem3p-Dnf1/2p are sequestered to the plasma membrane, also exhibited the ubiquitination-dependent missorting of Tat2p. These results suggest that Tat2p is ubiquitinated at the TGN and missorted to the vacuolar pathway in the lem3Δ mutant. The NH₂-terminal cytoplasmic region of Tat2p containing ubiquitination acceptor lysines interacted with liposomes containing acidic phospholipids, including phosphatidylserine. This interaction was abrogated by alanine substitution mutations in the basic amino acids downstream of the ubiquitination sites. Interestingly, a mutant Tat2p containing these substitutions was missorted in a ubiquitination-dependent manner. We propose the following model based on these results; Tat2p is not ubiquitinated when the NH₂-terminal region is bound to membrane phospholipids, but if it dissociates from the membrane due to a low level of phosphatidylserine caused by perturbation of phospholipid asymmetry in the lem3Δ mutant, Tat2p is ubiquitinated and then transported from the TGN to the vacuole.     

Auto-inhibition of Drs2p,a Yeast Phospholipid Flippase,by Its Carboxyl-terminal Tail

Drs2p, a yeast type IV P-type ATPase (P4-ATPase), or flippase, couples ATP hydrolysis to phosphatidylserine translocation and the establishment of membrane asymmetry. A previous study has shown that affinity-purified Drs2p, possessing an N-terminal tandem affinity purification tag (TAP_N-Drs2), retains ATPase and translocase activity, but Drs2p purified using a C-terminal tag (Drs2-TAP_C) was inactive. In this study, we show that the ATPase activity of N-terminally purified Drs2p associates primarily with a proteolyzed form of Drs2p lacking the C-terminal cytosolic tail. Truncation of most of the Drs2p C-terminal tail sequence activates its ATPase activity by ∼4-fold. These observations are consistent with the hypothesis that the C-terminal tail of Drs2p is auto-inhibitory to Drs2p activity. Phosphatidylinositol 4-phosphate (PI(4)P) has been shown to positively regulate Drs2p activity in isolated Golgi membranes through interaction with the C-terminal tail. In proteoliposomes reconstituted with purified, N-terminally TAP-tagged Drs2p, both ATPase and flippase activity were significantly higher in the presence of PI(4)P. In contrast, PI(4)P had no significant effect on the activity of a truncated form of Drs2p, which lacked the C-terminal tail. This work provides the first direct evidence, in a purified system, that a phospholipid flippase is subject to auto-inhibition by its C-terminal tail, which can be relieved by a phosphoinositide to stimulate flippase activity.     

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation.Fish were fed 4 g kg^?1 MOS (Bio-Mos^® Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents.Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet.The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Structure, dynamics, lipid binding, and physiological relevance of the putative GTPase-binding domain of Dictyostelium formin C

Dictyostelium Formin C (ForC) is involved in the regulation of local actin cytoskeleton reorganization (e.g. during cellular adhesion or migration). ForC contains formin homology 2 and 3 (FH2 and -3) domains and an N-terminal putative GTPase-binding domain (GBD) but lacks a canonical FH1 region. To better understand the role of the GBD, its structure, dynamics, lipid-binding properties, and cellular functions were analyzed by NMR and CD spectroscopy and by in vivo fluorescence microscopy. Moreover, the program CS-Rosetta was tested for the structure prediction based on chemical shift data only. The ForC GBD adopts an ubiquitin-like α/β-roll fold with an unusually long loop between β-strands 1 and 2. Based on the lipid-binding data, the presence of DPC micelles induces the formation of α-helical secondary structure and a rearrangement of the tertiary structure. Lipid-binding studies with a mutant protein and a peptide suggest that the β1-β2 loop is not relevant for these conformational changes. Whereas small amounts of negatively charged phosphoinositides (1,2-dioctanoyl-sn-glycero-3-(phosphoinositol 4,5-bisphosphate) and 1,2-dihexanoyl-sn-glycero-3-(phosphoinositol 3,4,5-trisphosphate)) lower the micelle concentration necessary to induce the observed spectral changes, other negatively charged phospholipids (1,2-dihexanoyl-sn-glycero-3-(phospho-L-serine) and 1,2-dihexanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) had no such effect. Interestingly, bicelles and micelles composed of diacylphosphocholines had no effect on the GBD structure. Our data suggest a model in which part of the large positively charged surface area of the GBD mediates localization to specific membrane patches, thereby regulating interactions with signaling proteins. Our cellular localization studies show that both the GBD and the FH3 domain are required for ForC targeting to cell-cell contacts and early phagocytic cups and macropinosomes.