BBA - General Subjects (v.1820, #11)

Proteome analysis identified human neutrophil membrane tubulovesicular extensions (cytonemes, membrane tethers) as bactericide trafficking by Svetlana I. Galkina; Natalia V. Fedorova; Marina V. Serebryakova; Julia M. Romanova; Sergei A. Golyshev; Vladimir I. Stadnichuk; Ludmila A. Baratova; Galina F. Sud'ina; Thomas Klein (1705-1714).
Following adhesion to fibronectin neutrophils can develop membrane tubulovesicular extensions (TVEs) that can be 200 nm wide and several cell diameters long. TVEs attach neutrophils to the other cells, substrata or bacteria over distance. To understand the physiological significance of TVEs we performed proteome analysis of TVE content in neutrophils plated to fibronectin in the presence of compounds known to induce TVE formation (nitric oxide donor diethylamine NONOate, 4-bromophenacyl bromide, cytochalasin D).Development of TVEs was confirmed by scanning electron microscopy. TVEs were disrupted following removal of inductors and biochemical, high-performance liquid chromatography and mass spectrometry investigations were employed to characterize the proteins within the incubation media.TVE disruption released (a) the granular bactericides lactoferrin, lipocalin, myeloperoxidase, cathepsin G and defensins; (b) energy metabolism enzymes; (c) actin cytoskeleton proteins; (d) S100 proteins; and (e) annexin 1.The data confirm that TVEs represent a means of secretory bactericide trafficking, where the protrusions fuse with the plasma membrane upon neutrophil adhesion or extend from the cell surface when fusion is impaired. It is proposed that proteins abundantly presented in TVE (energy metabolism enzymes, actin cytoskeleton and S100 proteins, annexin 1) play an important role in fusion of TVE with the plasma membrane.Our study confirms TVEs as neutrophil secretory protrusions that make direct contacts with cells and bacteria over distance. The membrane-packed content and outstanding length of TVEs might allow targeted neutrophil secretion of aggressive bactericides over a long distance without dilution or injury to surrounding tissues.► Human neutrophils develop 200 nm wide and very long extensions (cytonemes). ► Cytonemes consist of lined up membrane tubules and vesicles of the same diameter. ► Neutrophils establish contacts with cells and bacteria over distance via cytonemes. ► Proteome analysis and HPLC revealed secretory granular bactericides in cytonemes. ► Cytonemes are bactericide trafficking establishing direct contacts with destination.
Keywords: Neutrophil; Secretion; Bactericides; Cytonemes; Membrane tubulovesicular extensions; Membrane tethers;

The human CD10 lacking an N-glycan at Asn628 is deficient in surface expression and neutral endopeptidase activity by Ban Sato; Yohko U. Katagiri; Kazutoshi Iijima; Hiroyuki Yamada; Satsuki Ito; Nana Kawasaki; Hajime Okita; Junichiro Fujimoto; Nobutaka Kiyokawa (1715-1723).
CD10, also known as neprilysin or enkephalinase exhibiting neutral endopeptidase (NEP) activity, is expressed by B-lineage hematopoietic cells as well as a variety of cells from normal tissues. It cleaves peptides such as cytokines to act for terminating inflammatory responses. Although CD10 molecules of the human pre-B-cell line NALM-6 have 6 consensus N-glycosylation sites, three of them are known to be N-glycosylated by X-ray crystallography.In order to investigate the role of N-glycans in the full expression of NEP activity, we modified N-glycans by treatment of NALM6 cells with various glycosidases or alter each of the consensus N-glycosylation sites by generating site-directed mutagenesis and compared the NEP activities of the sugar-altered CD10 with those of intact CD10.CD10 of the human B-cell line NALM-6 was dominantly localized in raft microdomains and heterogeneously N-glycosylated. Although neither desialylation nor further degalactosylation caused defective NEP activity, removal of only a small part of N-glycans by treatment with glycopeptidase F under non-denaturing conditions decreased NEP activity completely. All of the three consensus sites of CD10 in HEK293 cells introduced with wild type-CD10 were confirmed to be N-glycosylated. Surface expression of N-glycan at Asn628-deleted CD10 by HEK293 cells was greatly decreased as well as it lost entire NEP activities. N-glycosylation at Asn628 is essential not only for NEP activities, but also for surface expression.Quality control system does not allow dysfunctional ecto-type proteases to express on plasma membrane.► CD10 is a type II membrane glycoprotein exhibiting neutral endopeptidase activity. ► Removal of only a small part of N-glycans caused entire loss of the activity. ► CD10 lacking an N-glycan at Asn628 did not exhibit the activity. ► CD10 lacking an N-glycan at Asn628 was not expressed by the transfectants. ► An N-glycan at Asn628 is essential both for surface expression and NEP activities.
Keywords: CD10; N-glycan; Neutral endopeptidase; Glycopeptidase F; Site-directed mutagenesis; Common acute lymphoblastic leukemia antigen (CALLA);

The phytoestrogen 8-prenylnaringenin inhibits agonist-dependent activation of human platelets by Clara Di Vito; Alessandra Bertoni; Michela Nalin; Sara Sampietro; Manuela Zanfa; Fabiola Sinigaglia (1724-1733).
Phytoestrogens are plant-derived polyphenolic compounds that exert beneficial effects on human health, mostly related to their estrogen mimetic activity. In particular a strong correlation between phytoestrogens intake and a lower risk of cardiovascular diseases has been reported. The flavanone 8-prenylnaringenin, extracted from hop flowers, has been identified as a novel phytoestrogen, unique with respect to estrogen receptors specificity and potency. However, to date no investigations on the 8-prenylnaringenin role in modulating platelet function have been undertaken.We evaluated the effect of 8-prenylnaringenin on platelet aggregation, intracellular calcium mobilization and protein phosphorylation triggered by thrombin and collagen, and platelet adhesion and dense granule secretion triggered by collagen.8-Prenylnaringenin inhibited platelet aggregation induced by different agonists and platelet adhesion to collagen matrix. 8-Prenylnaringenin directly increased intracellular cAMP and cGMP levels and thus promoted VASP phosphorylation. However, these molecular events were not responsible for the inhibitory action of 8-prenylnaringenin on platelets. Moreover, 8-prenylnaringenin inhibited the phosphorylation of Pyk2, Akt, and ERK1/2. Finally, 8-prenylnaringenin suppressed the mobilization of calcium and the secretion of dense granules. All these effects were independent of estrogen receptors recruitment.8-Prenylnaringenin exerted anti-aggregatory and anti-adhesive effects on human platelets, independently of estrogen receptors, acting as an inhibitor of multiple proteins essential for the morphological and biochemical transformations that occur during platelet activation and aggregation.8-Prenylnaringenin may represent a useful tool in the therapy and prevention of vascular diseases associated with platelet aggregation, such as atherosclerosis, myocardial infarction, coronary artery disease, and thrombosis.► 8-PN inhibits platelet aggregation induced by thrombin, collagen and U46619. ► 8-PN impairs platelet adhesion to collagen. ► 8-PN inhibits calcium mobilization and dense granule release. ► 8-PN inhibits protein phosphorylation induced by thrombin and collagen. ► 8-PN directly activates the inhibitory cGMP/PGK/VASP pathway in platelets.
Keywords: Platelets; 8-Prenylnaringenin; Signal transduction; Thrombin; Collagen; Cyclic nucleotides;

TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells by Nandhini Muthukrishnan; Gregory A. Johnson; Jongdoo Lim; Eric E. Simanek; Jean-Philippe Pellois (1734-1743).
Lysis of endocytic organelles is a necessary step in many cellular delivery methodologies. This is achieved efficiently in the photochemical internalization approach but the cell death that accompanies this process remains a problem.We investigate the mechanisms of cell death that accompanies photochemical internalization of the fluorescent peptide TMR-TAT.TMR-TAT kills cells after endocytosis and light irradiation. The lysis of endocytic organelles by TMR-TAT causes a rapid increase in the concentration of calcium in the cytosol. TMR-TAT co-localizes with endocytic organelles containing calcium prior to irradiation and photochemical internalization leads to the release of the lumenal content of these organelles. Ruthenium red and cyclosporin A, inhibitors of calcium import in mitochondria and of the mitochondria permeability transition pore, inhibit cell death.TMR-TAT mediated photochemical internalization leads to a disruption of calcium homeostasis. The subsequent import of calcium in mitochondria is a causative factor of the cell death that accompanies photochemical internalization.General significanceUnderstanding how the lysis of endocytic organelles affects cellular physiology and causes cell death is crucial to the development of optimal delivery methodologies.► TMR-TAT mediated PCI is a promising delivery method but it is accompanied by cell death. ► TMR-TAT co-localizes with calcium containing endocytic vesicles in cells. ► Endosomal escape of TMR-TAT with light increases cytosolic calcium concentration. ► Calcium flux into the cytosol is followed by import of calcium into mitochondria. ► Release of calcium from endocytic vesicles is a causative factor of the cell death.
Keywords: Photochemical internalization; TAT; Cell-penetrating peptide; Calcium;

Tumor-associated NADH oxidase (tNOX; ENOX2) is a growth-related protein expressed in transformed cells. High concentrations of numerous chemotherapeutic agents have shown to inhibit tNOX activity and protein levels leading to a reduction in cell growth while little is known for the effects of low concentrations of chemotherapeutic agents on tNOX expression.Effects of chemotherapeutic agents on cell function were evaluated with traditional in vitro assays and the xCELLigence System. Western blot analyses were used to study protein expression profiles of the epithelial-to-mesenchymal transition.We showed that doxorubicin treatment transiently up-regulates tNOX expression in human lung carcinoma A549 cells in association with enhanced cell migration. Similar results were observed in tamoxifen-exposed A549 cells. Furthermore, protein marker analyses revealed that the enhanced migration induced by tamoxifen was correlated with epithelial-to-mesenchymal transition, as evidenced by down-regulation of epithelial markers and up-regulation of mesenchymal markers. Importantly, tNOX overexpression enhanced cell migration, confirming the essential role of tNOX in cell migration.Based on these findings, we conclude that doxorubicin and tamoxifen induce a transient up-regulation of tNOX expression, leading to enhanced cell migration and EMT.These findings establish an essential role for tNOX in cell migration and survival and may provide a rational framework for the further development of tNOX inhibitors as a novel class of antitumor agents.► Doxorubicin treatment transiently up-regulates tNOX expression in human lung carcinoma A549 cells. ► Tamoxifen treatment also transiently up-regulates tNOX expression in A549 cells. ► The up-regulation of tNOX expression results in enhanced cell migration. ► Tamoxifen-induced cell migration is correlated with epithelial-to-mesenchymal transition.
Keywords: Doxorubicin; Epithelial-to-mesenchymal transition (EMT); Migration; Tamoxifen; Tumor-associated NADH oxidase (tNOX ENOX2);

The PepT1-transportable soy tripeptide VPY reduces intestinal inflammation by Jennifer Kovacs-Nolan; Hua Zhang; Masahisa Ibuki; Toshihiro Nakamori; Keiko Yoshiura; Patricia V. Turner; Toshiro Matsui; Yoshinori Mine (1753-1763).
Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal tract. The peptide transporter PepT1 is responsible for the intestinal uptake of dietary peptides, and its expression in the gastrointestinal tract is up-regulated during intestinal inflammation, indicating that PepT1 may be a promising target for IBD therapeutics.The transport of soy-derived di- and tripeptides across Caco-2 intestinal epithelial cells was examined, and the anti-inflammatory effects of the transported peptide VPY were evaluated in vitro in Caco-2 and THP-1 macrophages, and in vivo in a mouse model of DSS-induced colitis.VPY inhibited the secretion of IL-8 and TNF-α, respectively, from Caco-2 and THP-1 cells. VPY transport and anti-inflammatory activity in Caco-2 cells was reduced in the presence of Gly-Sar, indicating this activity was mediated by PepT1. In mice, VPY treatment reduced DSS-induced colitis symptoms and weight loss, improved colon histology, reduced MPO activity, and decreased gene expression of the pro-inflammatory cytokines TNF-α, IL-6, IL-1β, IFN-γ and IL-17 in the colon.VPY is a novel PepT1 substrate that can inhibit the production of pro-inflammatory mediators in vitro in intestinal epithelial and immune cells, and reduce the severity of colitis in mice by down-regulating the expression of pro-inflammatory cytokines in the colon, suggesting that VPY may be promising for the treatment of IBD.► Transport of soy-derived peptides across intestinal epithelial cells was examined. ► The tripeptide VPY was identified as a novel substrate for the transporter PepT1. ► VPY reduced production of inflammatory cytokines in IECs and immune cells. ► VPY reduced the severity of DSS-induced colitis in mice. ► VPY may be a promising new therapeutic for the treatment of intestinal inflammation.
Keywords: Soy peptide; PepT1; Inflammatory bowel disease (IBD); Anti-inflammatory; Dextran sodium sulfate (DSS); Pro-inflammatory cytokine;

De novo fatty acid synthesis catalyzed by fatty acid synthase (FASN) is crucial for tumor cell survival. Thus therapeutic targeting of FASN is considered as a novel antineoplastic strategy. However, little is understood in this respect regarding malignancies of hematological origin. The present investigation was therefore, undertaken to study the molecular mechanisms of the antitumor action of FASN inhibitor orlistat (tetrahydrolipstatin) using a murine model of a T cell lymphoma.The antitumor efficacy of orlistat was investigated in vitro by estimating cell survival by MTT assay and apoptosis by Wright Giemsa, TUNEL, Annexin-V/PI staining and % DNA fragmentation. Generation of reactive oxygen species (ROS) in tumor cells was studied using fluorescence microscopy. Expression of genes and proteins was carried out by RT-PCR and western blot analyses respectively. FASN and CPT-1 activity was estimated by spectrophotometer. Cytokines expression was analyzed by ELISA.We report that inhibition of FASN with its specific inhibitor orlistat manifests tumor-specific inhibition of cell survival, accompanied by induction of apoptosis. Orlistat-treated tumor cells showed an altered ROS generation, shift in cytokine balance and modulated expression of cell survival regulatory molecules like HSP70, Bcl2, p53, PUMA, Caspase-3 and CAD. It was observed that IFN-γ mediates orlistat-dependent modulation of FASN expression.In this study, we report some of the so far unexplored novel aspects underlying the molecular mechanisms associated with orlistat-dependent modulation of tumor cell survival. These observations will help in designing antineoplastic therapeutic protocols using orlistat against malignancies of hematological origin.► Fatty acid synthase (FASN) inhibitor orlistat inhibits survival of tumor cells by inducing apoptosis. ► FASN inhibition causes modulation of cell survival regulatory molecules. ► 1. Shift of cytokines balance & ROS generation is implicated.
Keywords: T cell lymphoma; Fatty acid synthase; Orlistat; Apoptosis; Cell survival;

Curcumin's pre-incubation temperature affects its inhibitory potency toward amyloid fibrillation and fibril-induced cytotoxicity of lysozyme by Kuan-Nan Liu; Chia-Min Lai; Yi-Ting Lee; Sung-Ning Wang; Rita P.-Y. Chen; Jeng-Shiung Jan; Hwai-Shen Liu; Steven S.-S. Wang (1774-1786).
More than twenty-seven human proteins can fold abnormally to form amyloid deposits associated with a number of degenerative diseases. The research reported here is aimed at exploring the connection between curcumin's thermostability and its inhibitory activity toward the amyloid fibrillation of hen egg-white lysozyme (HEWL).ThT fluorescence spectroscopy, equilibrium thermal denaturation analysis, and transmission electron microscopy were employed for structural characterization. MTT reduction and flow cytometric analyses were used to examine cell viability.The addition of thermally pre-treated curcumin was found to attenuate the formation of HEWL fibrils and the observed fibrillation inhibition was dependent upon the pre-incubation temperature of curcumin. Our results also demonstrated that the cytotoxic effects of fibrillar HEWL species on PC 12 and SH-SY5Y cells were decreased and negatively correlated with curcumin's thermostability. Next, an enhanced stability of HEWL was perceived upon the addition of curcumin pre-incubated at lower temperature. Furthermore, we found that the alteration of curcumin's thermostability was associated with its inhibitory potency against HEWL fibrillation.We believe that the results from this research may contribute to the development of effective therapeutics for amyloidoses.► Fibrillogenesis of hen lysozyme is attenuated by thermally pre-treated curcumin. ► Thermally pre-treated curcumin enhances lysozyme’s structural stability. ► Thermally pre-treated curcumin protects cells against fibril-induced cell death. ► Curcumin’s protection toward fibril cytotoxicity relates to its thermostability.
Keywords: Amyloid; Curcumin; Cytotoxicity; Inhibitors; Thermostability; Pre-treatment;

In vivo role of aldehyde reductase by Motoko Takahashi; Satoshi Miyata; Junichi Fujii; Yoko Inai; Shigemitsu Ueyama; Motoko Araki; Tomoyoshi Soga; Reiko Fujinawa; Chiaki Nishitani; Shigeru Ariki; Takeyuki Shimizu; Tomomi Abe; Yoshito Ihara; Morimitsu Nishikimi; Yasunori Kozutsumi; Naoyuki Taniguchi; Yoshio Kuroki (1787-1796).
Aldehyde reductase (AKR1A; EC 1.1.1.2) catalyzes the reduction of various types of aldehydes. To ascertain the physiological role of AKR1A, we examined AKR1A knockout mice.Ascorbic acid concentrations in AKR1A knockout mice tissues were examined, and the effects of human AKR1A transgene were analyzed. We purified AKR1A and studied the activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis. Metabolomic analysis and DNA microarray analysis were performed for a comprehensive study of AKR1A knockout mice.The levels of ascorbic acid in tissues of AKR1A knockout mice were significantly decreased which were completely restored by human AKR1A transgene. The activities of glucuronate reductase and glucuronolactone reductase, which are involved in ascorbic acid biosynthesis, were suppressed in AKR1A knockout mice. The accumulation of d-glucuronic acid and saccharate in knockout mice tissue and the expression of acute-phase proteins such as serum amyloid A2 are significantly increased in knockout mice liver.AKR1A plays a predominant role in the reduction of both d-glucuronic acid and d-glucurono-γ-lactone in vivo. The knockout of AKR1A in mice results in accumulation of d-glucuronic acid and saccharate as well as a deficiency of ascorbic acid, and also leads to upregulation of acute phase proteins.AKR1A is a major enzyme that catalyzes the reduction of d-glucuronic acid and d-glucurono-γ-lactone in vivo, besides acting as an aldehyde-detoxification enzyme. Suppression of AKR1A by inhibitors, which are used to prevent diabetic complications, may lead to the accumulation of d-glucuronic acid and saccharate.► AKR1A (aldehyde reductase) knockout mice were examined. ► d-Glucuronic acid and saccharate are accumulated in AKR1A knockout mice tissue. ► The expression of serum amyloid A2 is increased in AKR1A knockout mouse liver.
Keywords: Glucose metabolism; Oxidoreductase; Transgenic rescue; Acute-phase protein;

Carbohydrate clearance receptors in transfusion medicine by Anne Louise Tølbøll Sørensen; Henrik Clausen; Hans H. Wandall (1797-1808).
Complex carbohydrates play important functions for circulation of proteins and cells. They provide protective shields and refraction from non-specific interactions with negative charges from sialic acids to enhance circulatory half-life. For recombinant protein therapeutics carbohydrates are especially important to enhance size and reduce glomerular filtration loss. Carbohydrates are, however, also ligands for a large number of carbohydrate-binding lectins exposed to the circulatory system that serve as scavenger receptors for the innate immune system, or have more specific roles in targeting of glycoproteins and cells.Here we provide an overview of the common lectin receptors that play roles for circulating glycoproteins and cells, and present a discussion of ways to engineer glycosylation of recombinant biologics and cells to improve therapeutic effects.While the pharmaceutical industry has learned how to exploit carbohydrates to improve pharmacokinetic properties of recombinant therapeutics, our understanding of how to improve cell-based therapies by manipulation of complex carbohydrates is still at its infancy. Progress with the latter has recently been achieved with cold-stored platelets, where exposure of uncapped glycans lead to rapid clearance from circulation by several lectin-mediated pathways.Understanding lectin-mediated clearance pathways is essential for progress in development of biological pharmaceuticals.► Complex carbohydrates influence the circulatory half-life of proteins and cells. ► Lectin-receptors participate in clearance of glycosylated blood components. ► Glycosylation of transfused cold-stored platelets improves circulation time. ► Glycoengineering of recombinant biologics improve therapeutic effects. ► Understanding lectin-mediated clearance underlies biopharmaceutical progress.
Keywords: Lectins; Carbohydrate; Sialic acid; Recombinant proteins; Platelet transfusion; Platelet storage;

BJ-PI2, A non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom by Igor Rapp Ferreira da Silva; Raquel Lorenzetti; André Lisboa Rennó; Lineu Baldissera; André Zelanis; Solange Maria de Toledo Serrano; Stephen Hyslop (1809-1821).
Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions.BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle.BJ-PI2 was a 23.08 kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and β-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells.BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo.BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis. ► Bothrops jararaca venom contains snake venom metalloproteinases (SVMPs). ► BJ-PI2 is a non-hemorrhagic, non-myotoxic, non-coagulant class P-I SVMP. ► BJ-PI2 increases vascular permeability and causes inflammatory cell migration. ► BJ-PI2 may contribute to venom-induced vascular permeability and inflammation.
Keywords: Bothrops jararaca; Coagulopathy; Hemorrhage; Myonecrosis; Snake venom metalloproteinase (SVMP); Vascular permeability;

Fifty years in the thioredoxin field and a bountiful harvest by Bob B. Buchanan; Arne Holmgren; Jean-Pierre Jacquot; Renate Scheibe (1822-1829).
Discovered 50 years ago as a hydrogen donor for the reduction of ribonucleotides, thioredoxin is currently recognized as a protein central to the regulation of multiple processes in the cell. Two meetings separated by a period of 30 years serve as benchmarks for assessing this transition—the first held in Berkeley (California) in 1981 and the other convened in 2011 in Sant Feliu de Guixols (Spain). The four of us contributing this article attended both meetings and thus have witnessed the development of the thioredoxin field and its notable extension in unanticipated new directions. In this Perspective we briefly recount the unfolding of this remarkable story.► Commemoration of the 50th birthday of protein thioredoxin. ► Development of field traced from discovery in 1964 to 2011. ► Function expands from hydrogen carrier to regulatory protein acting throughout biology. ► Stockholm, Berkeley and Saint Feliu de Guixols conferences serve as benchmarks.
Keywords: Thioredoxin; Redox regulation; Redox biology;