Membrane Counterparts Research Articles

Cathepsin D and its newly identified transport receptor SEZ6L2 can modulate neurite outgrowth.

How, in the absence of a functional mannose 6-phosphate (Man-6-P)-signal-dependent transport pathway, some acid hydrolases remain sorted to endolysosomes in the brain is poorly understood. We demonstrate that cathepsin D binds to mouse SEZ6L2, a type 1 transmembrane protein predominantly expressed in the brain. Studies of the subcellular trafficking of SEZ6L2, and its silencing in a mouse neuroblastoma cell line reveal that SEZ6L2 is involved in the trafficking of cathepsin D to endosomes. Moreover, SEZ6L2 can partially correct the cathepsin D hypersecretion resulting from the knockdown of UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase in HeLa cells (i.e. in cells that are unable to synthesize Man-6-P signals). Interestingly, cleavage of SEZ6L2 by cathepsin D generates an N-terminal soluble fragment that induces neurite outgrowth, whereas its membrane counterpart prevents this. Taken together, our findings highlight that SEZ6L2 can serve as receptor to mediate the sorting of cathepsin D to endosomes, and suggest that proteolytic cleavage of SEZ6L2 by cathepsin D modulates neuronal differentiation.

Decreased plasma soluble erythropoietin receptor in high-altitude excessive erythrocytosis and Chronic Mountain Sickness.

Excessive erythrocytosis (EE) is the hallmark of chronic mountain sickness (CMS), a prevalent syndrome in high-altitude Andean populations. Although hypoxemia represents its underlying stimulus, why some individuals develop EE despite having altitude-normal blood erythropoietin (Epo) concentration is still unclear. A soluble form of the Epo receptor (sEpoR) has been identified in human blood and competes directly for Epo with its membrane counterpart (mEpoR). Thus, reduced levels of circulating sEpoR could lead to higher Epo availability and ultimately to EE. We characterized the relationship between Epo and sEpoR, with hematocrit and hemoglobin concentration in healthy highlanders and CMS patients at 4,340 m in Cerro de Pasco, Peru. Our results show that EE patients show decreased plasma sEpoR levels and can be subdivided into two subgroups of normal and high plasma Epo concentration for the altitude of residence, with hemoglobin concentration rising exponentially with an increasing Epo-to-sEpoR ratio (Epo/sEpoR). Also, we showed that the latter varies as an inverse exponential function of arterial pulse O2 saturation. Our findings suggests that EE is strongly associated with higher Epo/sEpoR values, leading to elevated plasma Epo availability to bind mEpoR, and thereby a stronger stimulus for augmented erythropoiesis. Differences in the altitude normal and high Epo CMS patients with a progressively higher Epo/sEpoR supports the hypothesis of the existence of two genetically different subgroups suffering from EE and possibly different degrees of adaptation to chronic high-altitude hypoxia.

Proton Transport Property in Supported Nafion Nanothin Films by Electrochemical Impedance Spectroscopy

The proton transport property of Nafion nanothin films (4–300 nm) has been investigated by electrochemical impedance spectroscopy (EIS) using interdigitated array (IDA) of gold electrodes on SiO2 substrate. The proton conductivity has been investigated as a function of film drying/heating protocol, relative humidity, temperature and film thickness. It is found that the film treatment protocol makes a difference in film transport property. Ultimately, the proton conductivity and capacitance of the Nafion nanothin film is thickness-dependent, where the former decreased and the latter increased exponentially with decreasing film thickness. Moreover, the activation energy increased exponentially with decreasing film thickness. The proton conductivity of the thin films of Nafion is significantly lower than that of the membrane counterpart regardless of the thickness, which is consistent with the high activation energy found in the thin films. These differences are likely related to the polymer confinement and morphological differences between the thin films and the freestanding membrane.

Nuclear inositide specific phospholipase C signalling - interactions and activity

Evidence accumulated over the past 20years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signalling molecules are important components of signalling pathways operating within the nucleus. Nuclear polyphosphoinositide (PI) signalling relies on the synthesis and metabolism of phosphatidylinositol4,5-bisphosphate, which can modulate the activity of effector proteins and is a substrate of signalling enzymes. The regulation of the nuclear PI pool is totally independent from the plasma membrane counterpart, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. Among the nuclear enzymes involved in PI metabolism, inositide specific phospholipase C (PI-PLC) has been one of the most extensively studied. Several isoforms of PI-PLCs have been identified in the nucleus, namely PI-PLC-β1, γ1, δ1 and ζ; however, the β1 isozyme is the best characterized. In the present review, we focus on the signal transduction-related metabolism of nuclear PI-PLC and review the most convincing evidence for PI-PLC expression and activity being involved in differentiation and proliferation programmes in several cell systems. Moreover, nuclear PI-PLC is an intermediate effector and interactor for nuclear inositide signalling. The inositide cycle exists and shows a biological role inside the nucleus. It is an autonomous lipid-dependent signalling system, independently regulated with respect to the one at the plasma membrane counterpart, and is involved in cell cycle progression and differentiation.

Separation Performance and Stability of PVDF-co-HFP/Alkylphosphonium Dicyanamide Ionic Liquid Gel-Based Membrane in Pervaporative Separation of 1-Butanol

In this study, a membrane prepared by gelling trihexyl(tetradecyl)phosphonium dicyanamide ionic liquid (IL) with poly(vinylidene fluoride-co-hexafluoropropylene) copolymer is tested for performance and stability in pervaporative separation of 1-butanol. Feed concentration and temperature affected separation performance mainly through plasticizing and swelling effects in the membrane which promoted liquid-like transport behavior. Microscale IL-in-polymer networks formed within the gel matrix where the neat IL possibly functioned as conventional liquid membrane. IL gelling has significantly improved the membrane operational lifetime to ∼80 h, a significant improvement on the ∼10 h shown by a simple supported IL membrane counterpart. The instability was consequent to polymer-IL incompatibility and so further investigations are underway to resolve this issue.

Interactions between Ether Phospholipids and Cholesterol As Determined by Scattering and Molecular Dynamics Simulations

Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol's molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data. From analysis of the data the various bilayer structural parameters were obtained. Surface area constrained MD simulations were also performed to reproduce the experimental data. This iterative analysis approach resulted in good agreement between the experimental and simulated form factors. The molecular interactions taking place between cholesterol and ether lipids were then determined from the validated MD simulations. We found that in ether membranes cholesterol primarily hydrogen bonds with the lipid headgroup phosphate oxygen, while in their ester membrane counterparts cholesterol hydrogen bonds with the backbone ester carbonyls. This different mode of interaction between ether lipids and cholesterol induces cholesterol to reside closer to the bilayer surface, dehydrating the headgroup's phosphate moiety. Moreover, the three-dimensional lipid chain spatial density distribution around cholesterol indicates anisotropic chain packing, causing cholesterol to tilt. These insights lend a better understanding of ether lipid-mediated cholesterol trafficking and the roles that the different lipid species have in determining the structural and dynamical properties of membrane associated biomolecules.

Identification of tissue-specific targeting peptide

Using phage display technique, we identified tissue-targeting peptide sets that recognize specific tissues (bone-marrow dendritic cell, kidney, liver, lung, spleen and visceral adipose tissue). In order to rapidly evaluate tissue-specific targeting peptides, we performed machine learning studies for predicting the tissue-specific targeting activity of peptides on the basis of peptide sequence information using four machine learning models and isolated the groups of peptides capable of mediating selective targeting to specific tissues. As a representative liver-specific targeting sequence, the peptide "DKNLQLH" was selected by the sequence similarity analysis. This peptide has a high degree of homology with protein ligands which can interact with corresponding membrane counterparts. We anticipate that our models will be applicable to the prediction of tissue-specific targeting peptides which can recognize the endothelial markers of target tissues.

Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can alter the critical membrane tension at which they undergo a transition from a closed to an open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.

Entropic Tension in Crowded Membranes

Unlike their model membrane counterparts, biological membranes are richly decorated with a heterogeneous assembly of membrane proteins. These proteins are so tightly packed that their excluded area interactions can alter the free energy landscape controlling the conformational transitions suffered by such proteins. For membrane channels, this effect can completely change the critical driving force (such as membrane tension) needed to induce the transition from the closed to the open state, and therefore influence protein function in vivo. Despite their obvious importance, crowding phenomena in membranes are much less well studied than in the cytoplasm. Using statistical mechanics results for hard disk liquids, we show that crowding induces an entropic tension in the membrane, which influences transitions that alter the projected area and circumference of a membrane protein. As a specific case study in this effect, we consider the impact of crowding on the gating properties of bacterial mechanosensitive membrane channels, which are thought to confer osmoprotection when these cells are subjected to osmotic shock. We find that crowding can alter the gating energies by more than ≈ 2 kB T in physiological conditions, a substantial fraction of the total gating energies in some cases. Given the ubiquity of membrane crowding, the nonspecific nature of excluded volume interactions, and the fact that the function of many membrane proteins involve significant conformational changes, this specific case study highlights a general aspect in the function of membrane proteins.

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca2+/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

Solute and Ion Transport: Outer Membrane Pores and Receptors.

Two membranes enclose Gram-negative bacteria-an inner membrane consisting of phospholipid and an outer membrane having an asymmetric structure in which the inner leaflet contains phospholipid and the outer leaflet consists primarily of lipopolysaccharide. The impermeable nature of the outer membrane imposes a need for numerous outer membrane pores and transporters to ferry substances in and out of the cell. These outer membrane proteins have structures distinct from their inner membrane counterparts and most often function without any discernable energy source. In this chapter, we review the structures and functions of four classes of outer membrane protein: general and specific porins, specific transporters, TonB-dependent transporters, and export channels. While not an exhaustive list, these classes exemplify small-molecule transport across the outer membrane and illustrate the diversity of structures and functions found in Gram-negative bacteria.

Parameter Governing Thin Film Adhesion-Delamination in the Transition from DMT- to JKR-Limit

Professor David A. Dillard and his collaborators invented the constrained blister and the related punch test for characterizing thin film adhesion-delamination and the disjoining pressure at the interface of a clamped membrane and a rigid substrate. A new dimensionless parameter, ψs = [6(1 − ν)a 4/γ3 Eh]1/4 p, relating the membrane dimensions (radius, a, and thickness, h), materials properties (elastic modulus, E, and Poisson's ratio, ν), and adhesion (interface energy, γ, and disjoining pressure, p), is shown to account for the transition from the long range surface force limit with small ψ (Derjaguin-Muller-Toporov, DMT) to the short range limit with large ψ (Johnson-Kendall-Roberts, JKR). This parameter serves as a flexible membrane counterpart to the classical Tabor's parameter for adhesion in bulk solids. An alternative form is defined for thick circular plate under bending, ψb = [6(1 − ν)a 4/γEh 3]1/2 p. Three case studies, namely, the standard pressurized blister, the constrained blister, and the punch tests are investigated for membranes under either pure bending or pure stretching deformations.

Delamination Mechanics of a Clamped Rectangular Membrane in the Presence of Long-Range Intersurface Forces: Transition from JKR to DMT Limits

A 1-dimensional rectangular freestanding membrane clamped at opposite ends adheres to the planar surface of a rectangular punch. A tensile load applied to the punch causes the membrane to deform and gradually delaminate from the substrate. At equilibrium, the applied load is balanced by the disjoining pressure at the membrane-punch interface with range, y, and magnitude, p. Applying the Dugdale-Barenblatt-Maugis cohesive zone approximation, the disjoining pressure is taken to be uniform and confined to a finite cohesive length at the contact edge. For a fixed adhesion energy, γ = p y, we investigate the following: (i) the Derjaguin-Muller-Toporov (DMT) limit where y → ∞ and p → 0, (ii) the Johnson-Kendall-Roberts (JKR) limit where y → 0 and p → ∞, and (iii) the general case for intermediate but finite y and p. Delamination continues until the contact area shrinks to a line prior to “pinch-off”. The results are compared with the 2-dimensional axisymmetric membrane counterpart.

Subcellular localization of marine bacterial alkaline phosphatases

Bacterial alkaline phosphatases (APases) are important enzymes in organophosphate utilization in the ocean. The subcellular localization of APases has significant ecological implications for marine biota but is largely unknown. The extensive metagenomic sequence databases from the Global Ocean Sampling Expedition provide an opportunity to address this question. A bioinformatics pipeline was developed to identify marine bacterial APases from the metagenomic databases, and a consensus classification algorithm was designed to predict their subcellular localizations. We identified 3,733 bacterial APase sequences (including PhoA, PhoD, and PhoX) and found that cytoplasmic (41%) and extracellular (30%) APases exceed their periplasmic (17%), outer membrane (12%), and inner membrane (0.9%) counterparts. The unexpectedly high abundance of cytoplasmic APases suggests that the transport and intracellular hydrolysis of small organophosphate molecules is an important mechanism for bacterial acquisition of phosphorus (P) in the surface ocean. On average, each marine bacterium possessed at least one suite of uptake of glycerol phosphate (ugp) genes (e.g., ugpA, ugpB, ugpC, ugpE) for dissolved organic phosphorus (DOP) transport, but only half of them had ugpQ, which hydrolyzes transported DOP, indicating that cytoplasmic APases play a role in hydrolyzing transported DOP. The most abundant heterotrophic marine bacteria, alpha- and gamma-Proteobacteria, might hydrolyze DOP outside the cytoplasmic membrane, but the former could also transport and hydrolyze DOP in the cytoplasm. The abundant extracellular APases could provide bioavailable P for organisms that cannot directly access organophosphates, and thereby increase marine biological productivity and diversity.

A splice variant of the SLC28A3 gene encodes a novel human concentrative nucleoside transporter‐3 (hCNT3) protein localized in the endoplasmic reticulum

Nucleoside transporters are plasma membrane proteins essential for nucleoside salvage. Among them, human concentrative nucleoside transporter 3 (hCNT3, SLC28A3) plays an essential role in this process because of its broader substrate selectivity and higher concentrative ability than the other members of the SLC28 protein family, hCNT1 and hCNT2. The aim of this study was to characterize an isoform of hCNT3, encoded by an alternatively spliced SLC28A3-related mRNA, the first identified for a CNT protein. This variant, named hCNT3ins, is the result of the insertion of 176 bp corresponding to an intron located between exons 2 and 3 of the gene. This insertion results in a shift of the reading frame, yielding a protein lacking 69 residues of the N terminus. hCNT3 and hCNT3ins mRNAs are simultaneously expressed both in normal and transformed cells and are differentially regulated by activation and differentiation. Because of the N-terminal deletion, hCNT3ins is retained in the endoplasmic reticulum, where it shows a typical hCNT3-related activity. hCNT3ins exhibits a shorter half-life than its plasma membrane counterpart, being degraded via a proteasome-dependent pathway. We suggest that this novel hCNT3 isoform would be involved in the salvage of intracellular nucleosides from the lumen of the endoplasmic reticulum to the cytoplasm.

Nuclear phospholipase C beta1 and cellular differentiation

Phosphoinositides (PI) are the most extensively studied lipids involved in cell signaling pathways. The bulk of PI is found in membranes where they are substrates for enzymes, such as kinases, phosphatases and phospholipases, which respond to the activation by cell-surface receptors. The outcome of the majority of signaling pathways involving lipid second messengers results in nuclear responses finally driving the cell into differentiation, proliferation or apoptosis. Some of these pathways are well established, such as that of PI-specific phospholipase C (PI-PLC), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) into the two second messengers diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3). Two independent cycles of PI are present inside the cell. One is localized at the plasma membrane, while the most recently discovered PI cycle is found inside the nuclear compartment. The regulation of the nuclear PI pool is totally independent from the plasma membrane counterpart, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. In this report we will focus on the signal transduction-related metabolism of nuclear PI and review the most convincing evidence that the PI cycle is involved in differentiation programs in several cell systems.

Kinetics of Phosphorus Release and Uptake in a Membrane-Assisted Biological Phosphorus Removal Process

A long-term comparative study on the kinetics of enhanced biological phosphorus removal (EBPR) was carried out in pilot scale membrane-assisted and conventional biological phosphorus removal processes, by monitoring system performance, phosphorus mass balances, and maximum specific rates in off-line batch tests. The two systems exhibited similar performance in the removal of soluble phosphorus (P) from the influent wastewater, in the specific P release observed in the anaerobic zone, and in the maximum specific P release and volatile fatty acid (VFA) uptake rates. However, when the VFA in the influent was limiting, the conventional EBPR (CEBPR) process performed significantly better than the membrane (MEBPR) counterpart, and this behavior was also reflected in the kinetics of P release. Denitrifying dephosphatation was observed to be significant in both processes during periods of satisfactory P removal. When the aerobic recycle ratio was reduced to a minimum level, the anoxic P uptake activity in the CEBPR sludge was lower than that of the MEBPR sludge. Finally, the biomass decay rates of the two sludge types were estimated to be comparable, with significant reduction of the decay under unaerated conditions.

Chloride‐Selective Electrodes with Poly(n‐butyl acrylate) Based Membranes

AbstractChloride‐selective electrodes with tridodecylmethylammonium chloride ion‐exchanger and poly(n‐butyl acrylate) membrane were prepared. A significant advantage of these electrodes, compared to poly(vinyl chloride) based membranes is the lack of plasticizer which can contaminate samples due to leaching during long term measurements. Analytical parameters: linear response range, slope of dependence and detection limit were comparable to those characterizing poly(vinyl chloride) membrane sensors. However, the analytical characteristic of poly(n‐butyl acrylate) membrane sensors was largely independent of the ion‐exchanger content in the membrane and of the galvanostatic polarization applied. The selectivity pattern obtained showed that interferences for highly lipophilic anions were slightly more pronounced than for PVC membrane counterparts. Laser ablation with inductively coupled plasma mass spectrometry (LA‐ICP‐MS) studies of poly(n‐butyl acrylate) membranes free from tridodecylmethylammonium chloride revealed a relatively high background content of chlorine – comparable with the amount purposely introduced to the membrane containing ion‐exchanger. These impurities, regardless their chemical state, are affecting the response of the sensors obtained. The LA‐ICP‐MS data showed that while contact with Cl− containing solutions did not induce the coextraction of cations, salicylate interference is accompanied by high uptake of solution cations.

Tic21 Is an Essential Translocon Component for Protein Translocation across the Chloroplast Inner Envelope Membrane

An Arabidopsis thaliana mutant defective in chloroplast protein import was isolated and the mutant locus, cia5, identified by map-based cloning. CIA5 is a 21-kD integral membrane protein in the chloroplast inner envelope membrane with four predicted transmembrane domains, similar to another potential chloroplast inner membrane protein-conducting channel, At Tic20, and the mitochondrial inner membrane counterparts Tim17, Tim22, and Tim23. cia5 null mutants were albino and accumulated unprocessed precursor proteins. cia5 mutant chloroplasts were normal in targeting and binding of precursors to the chloroplast surface but were defective in protein translocation across the inner envelope membrane. Expression levels of CIA5 were comparable to those of major translocon components, such as At Tic110 and At Toc75, except during germination, at which stage At Tic20 was expressed at its highest level. A double mutant of cia5 At tic20-I had the same phenotype as the At tic20-I single mutant, suggesting that CIA5 and At Tic20 function similarly in chloroplast biogenesis, with At Tic20 functioning earlier in development. We renamed CIA5 as Arabidopsis Tic21 (At Tic21) and propose that it functions as part of the inner membrane protein-conducting channel and may be more important for later stages of leaf development.

Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction.

Several studies have demonstrated the existence of an autonomous intranuclear phospho-inositide cycle that involves the activation of nuclear PI-PLC and the generation of diacylglycerol (DG) within the nucleus. Although several distinct isozymes of PI-PLC have been detected in the nucleus, the isoform that has been most consistently highlighted as being nuclear is PI-PLC-beta1. Nuclear PI-PLC-beta1 has been linked with either cell proliferation or differentiation. Remarkably, the activation mechanism of nuclear PI-PLC-beta1 has been shown to be different from its plasma membrane counterpart, being dependent on phosphorylation effected by p44/42 mitogen activated protein (MAP) kinase. In this review, we report the most up-dated findings about nuclear PI-PLC-beta1, such as the localization in nuclear speckles, the activity changes during the cell cycle phases, and the possible involvement in the progression of myelodisplastic syndrome to acute myeloid leukemia.