Surface behaviour and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-disrupting peptide

Amyloid aggregates, found in patients that suffer from Alzheimer's disease, are composed of fibril-forming peptides in a β-sheet conformation. One of the most abundant components in amyloid aggregates is the β-amyloid peptide 1–42 (Aβ 1–42). Membrane alterations may proceed to cell death by either an oxidative stress mechanism, caused by the peptide and synergized by transition metal ions, or through formation of ion channels by peptide interfacial self-aggregation. Here we demonstrate that Langmuir films of Aβ 1–42, either in pure form or mixed with lipids, develop stable monomolecular arrays with a high surface stability. By using micropipette aspiration technique and confocal microscopy we show that Aβ 1–42 induces a strong membrane destabilization in giant unilamellar vesicles composed of palmitoyloleoyl-phosphatidylcholine, sphingomyelin, and cholesterol, lowering the critical tension of vesicle rupture. Additionally, Aβ 1–42 triggers the induction of a sequential leakage of low- and high-molecular-weight markers trapped inside the giant unilamellar vesicles, but preserving the vesicle shape. Consequently, the Aβ 1–42 sequence confers particular molecular properties to the peptide that, in turn, influence supramolecular properties associated to membranes that may result in toxicity, including: 1), an ability of the peptide to strongly associate with the membrane; 2), a reduction of lateral membrane cohesive forces; and 3), a capacity to break the transbilayer gradient and puncture sealed vesicles.

Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.

The ß-amyloid peptide in Alzheimer's disease decreases adhesion of vascular muscle cells to the basement membrane

Cerebral amyloid angiopathy (CAA) is a major feature of Alzheimer's disease pathology. In CAA, degeneration of vascular smooth muscle cells (VSMCs) occurs close to regions of the basement membrane where the amyloid protein (Aβ) builds up. In this study, the possibility that Aβ disrupts adhesive interactions between VSMCs and the basement membrane was examined. VSMCs were cultured on a commercial basement membrane substrate (Matrigel). The presence of Aβ in the Matrigel decreased cell-substrate adhesion and cell viability. Full-length oligomeric Aβ was required for the effect, as N- and C-terminally truncated peptide analogues did not inhibit adhesion. Aβ that was fluorescently labelled at the N-terminus (fluo-Aβ) bound to Matrigel as well as to the basement membrane heparan sulfate proteoglycan (HSPG) perlecan and laminin. Adhesion of VSMCs to perlecan or laminin was decreased by Aβ. As perlecan influences VSMC viability through the extracellular signal-regulated kinase (ERK)1/2 signalling pathway, the effect of Aβ1–40 on ERK1/2 phosphorylation was examined. The level of phospho-ERK1/2 was decreased in cells following Aβ treatment. An inhibitor of ERK1/2 phosphorylation enhanced the effect of Aβ on cell adhesion. The studies suggest that Aβ can decrease VSMC viability by disrupting VSMC–extracellular matrix (ECM) adhesion.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell–cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II+ cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell–cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus

Natriuretic peptide receptors in the central vasculature of the toad, Bufo marinus, were characterized using autoradiographical, molecular, and physiological techniques. Specific ¹²⁵I-rat ANP binding sites were present in the carotid and pulmonary arteries, the lateral aorta, the pre- and post-cava, and the jugular vein, and generally occurred in each layer of the blood vessel. The ¹²5I-rat ANP binding was partially displaced by the specific natriuretic peptide receptor C ligand, C-ANF, which indicates the presence of two types of natriuretic peptide receptors in the blood vessels. This was confirmed by a RT-PCR study, which demonstrated that guanylyl cyclase receptor (NPR-GC) and NPR-C mRNAs are expressed in arteries and veins. An in vitro guanylyl cyclase assay showed that frog ANP stimulated the production of cGMP in arterial membrane fractions. Physiological recordings from isolated segments of the carotid and pulmonary arteries and the lateral aorta, which had been pre-constricted with arginine vasotocin, showed that rat ANP, frog ANP and porcine CNP relaxed the vascular smooth muscle with relatively similar potency. Together, the data show that the central vasculature contains two types of natriuretic peptide receptors (NPR-C and NPR-GC) and that the vasculature is a target for ANP and CNP.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

Plant natriuretic peptide active site determination and effects on cGMP and cell volume regulation

Natriuretic peptides (NP) were first identified in animals where they play a role in the regulation of salt and water balance. This regulation is partly mediated by intracellular changes in cyclic GMP (cGMP). NP immunoanalogues occur in many plants and have been isolated, with two NP encoding genes characterised in Arabidopsis thaliana L. (AtPNP-A and AtPNP-B). Part of AtPNP-A contains the region with homology to human atrial (A)NP. We report here on the effects of recombinant AtPNP-A and smaller synthetic peptides within the ANP-homologous region with a view to identifying the biologically active domain of the molecule. Furthermore, we investigated interactions between AtPNP-A and the hormone, abscisic acid (ABA). ABA does not significantly affect Arabidopsis mesophyll protoplast volume regulation, whereas AtPNP-A and synthetic peptides promote water uptake into the protoplasts causing swelling. This effect is promoted by the membrane permeable cGMP analogue, 8-Br-cGMP, and inhibited by guanylate cyclase inhibitors indicating that increases in cGMP are an essential component of the plant natriuretic peptides (PNP) signalling cascade. ABA does not induce cGMP transients and does not affect AtPNP-A dependent cGMP increases, hence the two regulators differ in their second messenger signatures. Interestingly, AtPNP-A significantly delays and reduces the extent of ABA stimulated stomatal closure that is also based on cell volume regulation. We conclude that a complex interplay between observed PNP effects (stomatal opening and protoplast swelling) and ABA is likely to be cell type specific.

Genomic analyses and cloning of novel chicken natriuretic peptide genes reveal new insights into natriuretic peptide evolution

The natriuretic peptide (NP) family consists of multiple subtypes in teleosts, including atrial, B-type, ventricular, and C-type NPs (ANP, BNP, VNP, CNP-1–4, respectively), but only ANP, BNP, CNP-3, and CNP-4 have been identified in tetrapods. As part of understanding the molecular evolution of NPs in the tetrapod lineage, we identified NP genes in the chicken genome. Previously, only BNP and CNP-3 have been identified in birds, but we characterized two new chicken NP genes by cDNA cloning, synteny and phylogenetic analyses. One gene is an orthologue of CNP-1, which has only ever been reported in teleostei and bichir. The second gene could not be assigned to a particular NP subtype because of high sequence divergence and was named renal NP (RNP) due to its predominant expression in the kidney. CNP-1 mRNA was only detected in brain, while CNP-3 mRNA was expressed in kidney, heart, and brain. In the developing embryo, BNP and RNP transcripts were most abundant 24 h post-fertilization, while CNP mRNA increased in a stage-dependant manner. Synthetic chicken RNP stimulated an increase in cGMP production above basal level in chicken kidney membrane preparations and caused a potent dose-dependant vasodilation of pre-constricted dorsal aortic rings. From conserved chromosomal synteny, we propose that the CNP-4 and ANP genes have been lost in chicken, and that RNP may have evolved from a VNP-like gene. Furthermore, we have demonstrated for the first time that CNP-1 is retained in the tetrapod lineage.

Neurotoxic, redox-competent Alzheimer's --amyloid is released from lipid membrane by methionine oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu₂₊ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimer's disease.

Clustering of Th cell epitopes on exposed regions of HIV envelope despite defects in antibody activity

A long-standing question in the field of immunology concerns the factors that contribute to Th cell epitope immunodominance. For a number of viral membrane proteins, Th cell epitopes are localized to exposed protein surfaces, often overlapping with Ab binding sites. It has therefore been proposed that Abs on B cell surfaces selectively bind and protect exposed protein fragments during Ag processing, and that this interaction helps to shape the Th cell repertoire. While attractive in concept, this hypothesis has not been thoroughly tested. To test this hypothesis, we have compared Th cell peptide immunodominance in normal C57BL/6 mice with that in C57BL/6MT/MT mice (lacking normal B cell activity). Animals were first vaccinated with DNA constructs expressing one of three different HIV envelope proteins, after which the CD4 T cell response profiles were characterized toward overlapping peptides using an IFN- ELISPOT assay. We found a striking similarity between the peptide response profiles in the two mouse strains. Profiles also matched those of previous experiments in which different envelope vaccination regimens were used. Our results clearly demonstrate that normal Ab activity is not required for the establishment or maintenance of Th peptide immunodominance in the HIV envelope response. To explain the clustering of Th cell epitopes, we propose that localization of peptide on exposed envelope surfaces facilitates proteolytic activity and preferential peptide shuttling through the Ag processing pathway.

Physical, chemical and biological characterization of membrane fouling

In this chapter, advanced characterization of membrane fouling as a diagnostic tool has been summarized to prevent membrane fouling. Physical, chemical and biological analyses as membrane autopsies are mainly utilized to better understand membrane foulant. The physical characterization gives structure, roughness, charge effect, strength and hydrophobicity of membrane fouling. The chemical methods provide qualitative and quantitative measurements of different inorganic and organic matter. The biological properties present the spatial biofilm distribution, structure of dominant microorganisms and isolation and identification of microorganisms. In addition, detailed membrane foulant types are reviewed in terms of structure, roughness, hydrophobicity, charge effect, strength, calcium, magnesium, aluminum, iron, silicate, particle, functional group, biopolymer, humic acid, polysaccharide, structural composition, biofilm structure, microorganism and foulant interaction.

Membrane bound members of the M1 family : more than aminopeptidases

In mammals the M1 aminopeptidase family consists of nine different proteins, five of which are integral membrane proteins. The aminopeptidases are defined by two motifs in the catalytic domain; a zinc binding motif HEXXH-(X18)-E and an exopeptidase motif GXMEN. Aminopeptidases of this family are able to cleave a broad range of peptides down to only to a single peptide. This ability to either generate or degrade active peptide hormones is the focus of this review. In addition to their capacity to degrade a range of peptides a number of these aminopeptidases have novel functions that impact on cell signalling and will be discussed.

Interaction of the Akt substrate, AS160, with the glucose transporter 4 vesicle marker protein, insulin-regulated aminopeptidase

Insulin-regulated aminopeptidase (IRAP), a marker of glucose transporter 4 (GLUT4) storage vesicles (GSVs), is the only protein known to traffic with GLUT4. In the basal state, GSVs are sequestered from the constitutively recycling endosomal system to an insulin-responsive, intracellular pool. Insulin induces a rapid translocation of GSVs to the cell surface from this pool, resulting in the incorporation of IRAP and GLUT4 into the plasma membrane. We sought to identify proteins that interact with IRAP to further understand this GSV trafficking process. This study describes our identification of a novel interaction between the amino terminus of IRAP and the Akt substrate, AS160 (Akt substrate of 160 kDa). The validity of this interaction was confirmed by coimmunoprecipitation of both overexpressed and endogenous proteins. Moreover, confocal microscopy demonstrated colocalization of these proteins. In addition, we demonstrate that the IRAP-binding domain of AS160 falls within its second phosphotyrosine-binding domain and the interaction is not regulated by AS160 phosphorylation. We hypothesize that AS160 is localized to GLUT4-containing vesicles via its interaction with IRAP where it inhibits the activity of Rab substrates in its vicinity, effectively tethering the vesicles intracellularly.

Long-distance delivery of bacterial virulence factors by Pseudomonas aeruginosa outer membrane vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including b-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.