The Structure of Human 15-Lipoxygenase-2 with a Substrate Mimic

Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques. We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic. 15-LOX-2 contains a long loop, composed of hydrophobic amino acids, which projects from the amino-terminal membrane-binding domain. The loop is flanked by two Ca²⁺-binding sites that confer Ca²⁺-dependent membrane binding. A comparison of the human 15-LOX-2 and 5-LOX structures reveals similarities at the active sites, as well striking differences that can be exploited for design of isoform-selective inhibitors.     

Membrane traffic in skeletal muscle

Skeletal muscle tissue is made up of highly organized multinuclear cells. The internal organization of the muscle cell is dictated by the necessary regular arrangement of repeated units within the protein myofibrils that mediate muscle contraction. Skeletal muscle cells have the usual membrane traffic pathways for partitioning newly synthesized proteins, internalizing cell surface receptors for hormones and nutrients, and mediating membrane repair. However, in muscle, these pathways must be further specialized to deal with targeting to and organizing muscle-specific membrane structures, satisfying the unique metabolic requirements of muscle and meeting the high demand for membrane repair in a tissue that is constantly under mechanical stress. Specialized membrane traffic pathways in muscle also play a role in the formation of muscle through fusion of myoblast membranes and the development of internal muscle-specific membrane structures during myogenesis and regeneration. It has recently become apparent that muscle-specific isoforms of proteins that are known to mediate ubiquitous membrane traffic pathways, as well as novel muscle-specific proteins, are involved in tissue-specific aspects of muscle membrane traffic. Here we describe the specialized membrane structures of skeletal muscle, how these are developed, maintained and repaired by specialized and generic membrane traffic pathways, and how defects in these pathways result in muscle disease.     

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30’s fusogenic activity depends on Mg²⁺ binding to IM30; yet, it still is unclear how Mg²⁺-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg²⁺ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg²⁺. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg²⁺. Thus, Mg²⁺-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.     

Ferrous iron oxidation by foam immobilized Acidithiobacillus ferrooxidans: Experiments and modeling

Ferrous iron bio‐oxidation by Acidithiobacillus ferrooxidans immobilized on polyurethane foam was investigated. Cells were immobilized on foams by placing them in a growth environment and fully bacterially activated polyurethane foams (BAPUFs) were prepared by serial subculturing in batches with partially bacterially activated foam (pBAPUFs). The dependence of foam density on cell immobilization process, the effect of pH and BAPUF loading on ferrous oxidation were studied to choose operating parameters for continuous operations. With an objective to have high cell densities both in foam and the liquid phase, pretreated foams of density 50 kg/m³ as cell support and ferrous oxidation at pH 1.5 to moderate the ferric precipitation were preferred. A novel basket‐type bioreactor for continuous ferrous iron oxidation, which features a multiple effect of stirred tank in combination with recirculation, was designed and operated. The results were compared with that of a free cell and a sheet‐type foam immobilized reactors. A fivefold increase in ferric iron productivity at 33.02 g/h/L of free volume in foam was achieved using basket‐type bioreactor when compared to a free cell continuous system. A mathematical model for ferrous iron oxidation by Acidithiobacillus ferrooxidans cells immobilized on polyurethane foam was developed with cell growth in foam accounted by an effectiveness factor. The basic parameters of simulation were estimated using the experimental data on free cell growth as well as from cell attachment to foam under nongrowing conditions. The model predicted the phase of both oxidation of ferrous in shake flasks by pBAPUFs as well as by fully activated BAPUFs for different cell loadings in foam. Model for stirred tank basket bioreactor predicted within 5% both transient and steady state of the experiments closely for the simulated dilution rates. Bio‐oxidation at high Fe²⁺ concentrations were simulated with experiments when substrate and product inhibition coefficients were factored into cell growth kinetics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn²⁺ deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn²⁺-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn²⁺-binding motifs. Here, we show that Zn²⁺ binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn²⁺ entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn²⁺ on membrane repair is abolished in mg53^−/− muscle fibers, suggesting that MG53 functions as a potential target for Zn²⁺ during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn²⁺-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn²⁺ interaction with MG53 in protection against injury to the cell membrane.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

Structural and functional properties of chromatophores and membrane vesicles from Rhodepseudomonas sphaeroides

Membrane vesicles have been isolated by a modified procedure from Rhodopseudomonas sphaeroides, grown phototrophically under high light intensity. In addition,chromatophores have been isolated from this organism grown phototrophically with low light intensities.Structural, chemical and functional properties of both preparations have been investigated and compared. The orientation of the membrane preparations has been studied by freeze-etch electron microscopy, the localization of cytochrome c₂, and light-driven active transport of amino acids and Ca²⁺. The results demonstrate that the orientation of the vesicle membrane is the same as the cytoplasmic membrane of intact cells; the membranes in chromatophores, however, have an inverted orientation.On a dry weight basis, the membrane vesicles contain less protein, carotenoids and bacteriochlorophyll and more lipids than do chromatophores. Qualitatively, however, the composition of both preparations is similar.It is concluded that the intracytoplasmic structures from which the chromatophores are derived are structurally and functionally similar to (and most likely continuous with) the cytoplasmic membranes from which the vesicles are derived.     

Interaction of DNA topoisomerase 1 with DNA intermediates and proteins of base excision repair

The interaction of human recombinant DNA topoisomerase 1 (Top1) with linear and circular DNA structures containing a nick or short gap but lacking a specific Top1 recognition site was studied. The effect of key excision repair proteins on formation of the Top1 covalent adduct with the DNA repair intermediates was shown. Partial inhibition of the Top1-DNA-adduct formation upon addition of poly(ADP-ribose) polymerase 1 in the absence of NAD⁺ was shown, whereas in the presence of NAD⁺ formation of a high molecular weight product, most likely corresponding to poly(ADP)-ribosylated Top1-DNA adduct, was observed. The data show that the key base excision repair proteins can influence formation of suicide Top1-DNA adducts. Top1 was identified by immunoprecipitation in the bovine testis nuclear extract as the protein forming the main modification product with nick-containing DNA.     

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Rapid plasma membrane resealing is essential for cellular survival. Earlier studies showed that plasma membrane repair requires Ca²⁺-dependent exocytosis of lysosomes and a rapid form of endocytosis that removes membrane lesions. However, the functional relationship between lysosomal exocytosis and the rapid endocytosis that follows membrane injury is unknown. In this study, we show that the lysosomal enzyme acid sphingomyelinase (ASM) is released extracellularly when cells are wounded in the presence of Ca²⁺. ASM-deficient cells, including human cells from Niemann-Pick type A (NPA) patients, undergo lysosomal exocytosis after wounding but are defective in injury-dependent endocytosis and plasma membrane repair. Exogenously added recombinant human ASM restores endocytosis and resealing in ASM-depleted cells, suggesting that conversion of plasma membrane sphingomyelin to ceramide by this lysosomal enzyme promotes lesion internalization. These findings reveal a molecular mechanism for restoration of plasma membrane integrity through exocytosis of lysosomes and identify defective plasma membrane repair as a possible component of the severe pathology observed in NPA patients.     

Lipid raft–dependent plasma membrane repair interferes with the activation of B lymphocytes

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca²⁺-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca²⁺-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR–lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte–mediated immune responses.     

海岸黑松和紫穗槐对间歇强净风和风沙流吹袭的生理响应机制

以海岸防风固沙优势树种紫穗槐(Amorpha fruticosa Linn)和黑松(Pinus thunbergii Parl)为研究对象,利用野外便携式沙风洞用间歇风吹模拟自然阵风,通过分析间歇强净风(18m/s)和强风沙流(172.93g cm^-1 min^-1)吹袭过程中和风后恢复中,两树种叶片膜脂过氧化产物含量、抗氧化酶活力、渗透调节物含量的变化,以探讨其对自然阵风吹袭响应机制及自愈修复生理机制。结果表明,自然状况下,紫穗槐和黑松叶片相对含水量(RWC)相近,但抗氧化酶活力及种类和渗透调节物含量及种类上存在差异。紫穗槐叶片丙二醛含量(MDA)、脯氨酸含量及过氧化氢酶(CAT)和过氧化物酶(POD)活力分别较黑松高93.3%、78.6%、118.8%、6.5倍。而黑松可溶糖含量和超氧化物歧化酶(SOD)活力较紫穗槐高111.5%和28.2%。在间歇净风和风沙流处理中,随着风吹袭次数增多,黑松叶片RWC趋于小幅降低,可溶性糖含量及POD、SOD、CAT活力呈小幅波动式变化;紫穗槐叶片RWC大幅下降,伴随着脯氨酸含量,POD、CAT、SOD活...     

S100 and annexin proteins identify cell membrane damage as the Achilles heel of metastatic cancer cells

Mechanical activity of cells and the stress imposed on them by extracellular environment is a constant source of injury to the plasma membrane (PM). In invasive tumor cells, increased motility together with the harsh environment of the tumor stroma further increases the risk of PM injury. The impact of these stresses on tumor cell plasma membrane and mechanism by which tumor cells repair the PM damage are poorly understood. Ca²⁺ entry through the injured PM initiates repair of the PM. Depending on the cell type, different organelles and proteins respond to this Ca²⁺ entry and facilitate repair of the damaged plasma membrane. We recently identified that proteins expressed in various metastatic cancers including Ca²⁺-binding EF hand protein S100A11 and its binding partner annexin A2 are used by tumor cells for plasma membrane repair (PMR). Here we will discuss the involvement of S100, annexin proteins and their regulation of actin cytoskeleton, leading to PMR. Additionally, we will show that another S100 member – S100A4 accumulates at the injured PM. These findings reveal a new role for the S100 and annexin protein up regulation in metastatic cancers and identify these proteins and PMR as targets for treating metastatic cancers.     

Effect of light and natural ventilation systems on the growth parameters and carvacrol content in the in vitro cultures of <Emphasis Type="Italic">Plectranthus amboinicus</Emphasis> (Lour.) Spreng

The aim of the current study is to investigate the influence of light intensity, quality of light and alternative membrane sytems on the growth and headspace-GC/MS chemical analysis of Plectranthus amboinicus cultivated in vitro. Nodal segments were grown under light intensities (26, 51, 69, 94 and 130 µmol m^?2 s^?1) provided by cool-white fluorescent lamps. Apical segments were grown under light-emitting diodes blue; red; 1 blue/2.5 red; 2.5 blue/1 red; 1 blue/1 red and white fluorescent lamps. Apical and nodal segments were grown under alternative membrane and membrane-free systems. One, two or four PTFE membranes were used on the lid of the culture vessel. The membranes provided natural ventilation and worked as filters. The results have shown significant differences in the growth and carvacrol content, as well as in the content of carvacrol precursors (γ-terpinene and p-cymene) in different treatments. Among all tested light intensities, the significant increase in the dry weight and in the carvacrol content of plantlets derived from the nodal segments was recorded at 69 µmol m^?2 s^?1. The monochromatic red led to greater shoot length and higher dry weight in plantlets derived from the apical segments, as well as to carvacrol accumulation greater than that provided by the fluorescent lamps. The culture vessel enclosure by one and two membranes led to higher dry weight in plantlets derived from the apical and nodal segments, respectively. They also showed higher carvacrol content. Thus, it is possible optimizing the growth and carvacrol content in P. amboinicus cultivated in vitro by adjusting these environmental parameters.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca2+-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

Synaptotagmin’s Role in Neurotransmitter Release Likely Involves Ca-induced Conformational Transition

Neuronal exocytosis is mediated by a Ca²⁺-triggered membrane fusion event that joins synaptic vesicles and presynaptic membrane. In this event, synaptotagmin I plays a key role as a Ca²⁺ sensor protein that binds to and bends the presynaptic membrane with its C2B domain, and thereby initiates membrane fusion. We report free energy calculations according to which C2B-induced membrane bending is preceded by a Ca²⁺- and membrane-dependent conformational transition. In this transition C2B attaches to the membrane, moves its C-terminal helix from the orientation seen in the available (but membrane-free) crystal/NMR structures as pointing away from the membrane (helix-up), to an orientation pointing toward the membrane (helix-down). In the C2B helix-down state, lipid tails in the proximal membrane bilayer leaflet interact with the moved helix and become disordered, whereas tails in the distal leaflet, to keep in contact with the proximal leaflet, become stretched and ordered. The difference in lipid tail packing between the two leaflets results in an imbalance of pressure across the membrane, and thereby causes membrane bending. The lipid-disordering monitored in the simulations is well suited to facilitate Ca²⁺-triggered membrane fusion.     

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca²⁺ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca²⁺-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca²⁺ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca²⁺-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca²⁺.     

Dimethylsulfoxide Enhances CNS Neuronal plasma membrane resealing after injury in low temperature or low calcium

The inability to repair the damaged membrane may be one of the key mechanisms underlying the severe neuronal degeneration and overall functional loss seen in in vivo spinal cord injury and traumatic axonal injury in blunt head trauma. Promoting membrane resealing following damage may therefore constitute a potential effective therapeutic intervention in treating head trauma and spinal cord injuries. In our previous studies, we have shown that the axolemma failed to reseal following transection in clinically related situations, such as low extracellular calcium and low temperature. Our current studies indicate that DMSO is capable of rendering significant improvement in guinea pig axonal membrane resealing following transection in both 0.5 mM [Ca²⁺]₀ and 25°C situations. This was demonstrated physiologically by monitoring membrane potential recovery and anatomically by conducting HRP-exclusion assays 60 minutes after injury. Further, we have shown that the addition of DMSO in normal Krebs' solution (2 mM [Ca²⁺]₀ and 37°C) resulted in a decrease in membrane repair following injury. This indicates that DMSO-mediated membrane repair is sensitive to temperature and calcium. This study suggests the role of DMSO in axonal membrane resealing in clinically relevant conditions and raises the possibility of using DMSO in combination with other more established therapies in spinal cord injury treatment.     

Changes of Transmembrane Ca2+ Gradient in the Formation of Macrophage-Derived Foam Cells

Macrophages from C57BL/6J mice, an animal susceptible toatherosclerosis, were chosen as target cells to study changes in thetransmembrane Ca²⁺adient during the formation ofmacrophage-derived foam cells. The transmembrane Ca²⁺adientsof single living cells were examined using Fura-2/AM combined withFluo-3/AM by laser scanning confocal microscopy. Exposure to OxidizedLow Density Lipoprotein, decreases the Ca²⁺adient acrossmacrophage plasma membrane, but increases that across the nuclearmembrane. The altered transmembrane Ca²⁺adients could inducethe up-regulation of scavenger receptor in macrophages, resulting inthe formation of foam cells.     

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

The pore-formation activity of monomeric and oligomeric forms of different Cry1 toxins (from Cry1A to Cry1G) was analyzed by monitoring ionic permeability across Manduca sexta brush border membrane vesicles. The membrane vesicles were isolated from microvilli structures, showing a high enrichment of apical membrane markers and low intrinsic K⁺ permeability. A fluorometric assay performed with 3,3′-dipropylthiodicarbocyanine fluorescent probe, sensitive to changes in membrane potential, was used. Previously, it was suggested that fluorescence determinations with this dye could be strongly influenced by the pH, osmolarity and ionic strength of the medium. Therefore, we evaluated these parameters in control experiments using the K⁺-selective ionophore valinomycin. We show here that under specific ionic conditions changes in fluorescence can be correlated with ionic permeability without effects on osmolarity or ionic strength of the medium. It is extremely important to attenuate the background response due to surface membrane potential and the participation of the endogenous permeability of the membrane vesicles. Under these conditions, we analyzed the pore-formation activity induced by monomeric and oligomeric structures of different Cry1 toxins. The Cry1 toxin samples containing oligomeric structures correlated with high pore activity, in contrast to monomeric samples that showed marginal pore-formation activity, supporting the hypothesis that oligomer formation is a necessary step in the mechanism of action of Cry toxins.     

Self-repairing filamentous actin hydrogel with hierarchical structure

A chemically cross-linked filamentous actin (F-actin) gel consisting of globular actin (G-actin) as repeating units was prepared. The F-actin gel was cross-linked by covalent bonds, and the main chain is represented by the self-assembly of G-actin with a high-ordered hierarchical structure. The gel exhibited good mechanical performance with a storage modulus >1 kPa and undergoes reversible sol-gel transitions in response to changes in the salt concentration (chemical-induced sol-gel transition) as well as to shear strain (mechanical-induced sol-gel transition). Therefore, the gel exhibits self-repairing ability through dynamic polymerization and depolymerization across the structure hierarchies under repeated shear stress.