Science.gov

Sample records for adam protein mimics

  1. Polymeric ADAM protein mimics interrogate mammalian sperm-egg binding.

    PubMed

    Lee, Younjoo; Sampson, Nicole S

    2009-03-23

    The sperm proteins ADAM2 and ADAM3, members of the ADAM family of proteins, have been implicated in mammalian sperm-egg binding. However, elucidating their roles is complex because of the interdependence of ADAM protein expression in the testis. Hence, multivalent probes containing the three-amino acid binding sequence of ADAM2, glutamate-cysteine-aspartate (ECD), and ADAM3, glutamine-cysteine-aspartate (QCD), were designed, synthesized, and tested to investigate gamete interactions. In this work, ECD polymer mimics were synthesized by ring-opening metathesis polymerization with a faster initiating ruthenium catalyst than previously used. Polymers containing 100 copies of the ECD peptide mimic were found to be the best inhibitors of fertilization. The multivalent QCD polymers were also tested as inhibitors of fertilization. The structure-activity profile was the same as ECD polymers, but the overall potency was lower. Both ECD and QCD polymers require the presence of beta(1) integrin to inhibit fertilization. Next, triblock ABA and ABC copolymers containing both ECD and QCD ligands were synthesized with 96 monomer spacers as their B blocks. Although these polymers had lower densities of ECD and QCD peptides, their potencies correlated with the potencies of their corresponding homopolymers. In addition, no synergy between ECD and QCD mimics was observed. All the data suggest that QCD and ECD bind to the same complex of proteins that includes beta(1) integrin.

  2. Synthetic Peptides as Protein Mimics

    PubMed Central

    Groß, Andrea; Hashimoto, Chie; Sticht, Heinrich; Eichler, Jutta

    2016-01-01

    The design and generation of molecules capable of mimicking the binding and/or functional sites of proteins represents a promising strategy for the exploration and modulation of protein function through controlled interference with the underlying molecular interactions. Synthetic peptides have proven an excellent type of molecule for the mimicry of protein sites because such peptides can be generated as exact copies of protein fragments, as well as in diverse chemical modifications, which includes the incorporation of a large range of non-proteinogenic amino acids as well as the modification of the peptide backbone. Apart from extending the chemical and structural diversity presented by peptides, such modifications also increase the proteolytic stability of the molecules, enhancing their utility for biological applications. This article reviews recent advances by this and other laboratories in the use of synthetic protein mimics to modulate protein function, as well as to provide building blocks for synthetic biology. PMID:26835447

  3. Sexual selection and the molecular evolution of ADAM proteins.

    PubMed

    Finn, Scott; Civetta, Alberto

    2010-09-01

    Rapid evolution has been identified for many reproductive genes and recent studies have combined phylogenetic tests and information on species mating systems to test sexual selection. Here we examined the molecular evolution of the ADAM gene family, a diverse group of 35 proteins capable of adhesion to and cleavage of other proteins, using sequence data from 25 mammalian genes. Out of the 25 genes analyzed, all those expressed in male reproductive tissue showed evidence of positive selection. Positively selected amino acids within the protein adhesion domain were only found in sperm surface ADAM proteins (ADAMs 1, 2, 3, 4, and 32) suggesting selection driven by male x female interactions. We tested heterogeneity in rates of evolution of the adhesion domain of ADAM proteins by using sequence data from Hominidae and macaques. The use of the branch and branch-site models (PAML) showed evidence of higher d (N)/d (S) and/or positive selection linked to branches experiencing high postmating selective pressures (chimpanzee and macaque) for Adams 2, 18, and 23. Moreover, we found consistent higher proportion of nonsynonymous relative to synonymous and noncoding sequence substitutions in chimpanzee and/or macaque only for Adams 2, 18, and 23. Our results suggest that lineage-specific sexual selection bouts might have driven the evolution of the adhesion sperm protein surface domains of ADAMs 2 and 18 in primates. Adams 2 and 18 are localized in chromosome 8 of primates and adjacent to each other, so their evolution might have also been influenced by their common genome localization.

  4. DNA mimic proteins: functions, structures, and bioinformatic analysis.

    PubMed

    Wang, Hao-Ching; Ho, Chun-Han; Hsu, Kai-Cheng; Yang, Jinn-Moon; Wang, Andrew H-J

    2014-05-13

    DNA mimic proteins have DNA-like negative surface charge distributions, and they function by occupying the DNA binding sites of DNA binding proteins to prevent these sites from being accessed by DNA. DNA mimic proteins control the activities of a variety of DNA binding proteins and are involved in a wide range of cellular mechanisms such as chromatin assembly, DNA repair, transcription regulation, and gene recombination. However, the sequences and structures of DNA mimic proteins are diverse, making them difficult to predict by bioinformatic search. To date, only a few DNA mimic proteins have been reported. These DNA mimics were not found by searching for functional motifs in their sequences but were revealed only by structural analysis of their charge distribution. This review highlights the biological roles and structures of 16 reported DNA mimic proteins. We also discuss approaches that might be used to discover new DNA mimic proteins.

  5. Altered cell-matrix associated ADAM proteins in Alzheimer disease.

    PubMed

    Gerst, J L; Raina, A K; Pirim, I; McShea, A; Harris, P L; Siedlak, S L; Takeda, A; Petersen, R B; Smith, M A

    2000-03-01

    Alterations in cell-matrix 'contact' are often related to a disruption of cell cycle regulation and, as such, occur variously in neoplasia. Given the recent findings showing cell cycle alterations in Alzheimer disease, we undertook a study of ADAM-1 and 2 (A Disintegrin And Metalloprotease), developmentally-regulated, integrin-binding, membrane-bound metalloproteases. Our results show that whereas ADAM-1 and 2 are found in susceptible hippocampal neurons in Alzheimer disease, these proteins were not generally increased in similar neuronal populations in younger or age-matched controls except in association with age-related neurofibrillary alterations. This increase in both ADAM-1 and 2 in cases of Alzheimer disease was verified by immunoblot analysis (P < 0.05). An ADAM-induced loss of matrix integration would effectively "reset" the mitotic clock and thereby stimulate re-entry into the cell cycle in neurons in Alzheimer disease. Furthermore, given the importance of integrins in maintaining short-term memory, alterations in ADAM proteins or their proteolytic activity could also play a proximal role in the clinico-pathological manifestations of Alzheimer disease. Copyright 2000 Wiley-Liss, Inc.

  6. Identification of ADAM 31: a protein expressed in Leydig cells and specialized epithelia.

    PubMed

    Liu, L; Smith, J W

    2000-06-01

    A family of proteins containing a disintegrin and metalloproteinase domain (ADAMs) has been identified recently. Here, we report the identification of a novel member of the ADAM protein family from mouse. This protein is designated ADAM 31. The complementary DNA sequence of ADAM 31 predicts a transmembrane protein with metalloproteinase, disintegrin, cysteine-rich, and cytoplasmic domains. Messenger RNA encoding ADAM 31 was most abundant in testes, but was also detected in many other tissues. More significantly, the antibodies raised against ADAM 31 reveal that the protein has a unique and restricted expression pattern. ADAM 31 is expressed in Leydig cells of the testes, but unlike many other ADAMs, it is not found on developing sperm. Furthermore, ADAM 31 is highly expressed on four types of specialized epithelia: the cauda epididymidis, the vas deferens, the convoluted tubules of the kidney, and the parietal cells of the stomach.

  7. [The importance of ADAM family proteins in malignant tumors].

    PubMed

    Walkiewicz, Katarzyna; Gętek, Monika; Muc-Wierzgoń, Małgorzata; Kokot, Teresa; Nowakowska-Zajdel, Ewa

    2016-02-11

    Increasing numbers of reports about the role of adamalysins (ADAM) in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2), vascular growth factor (VEGF), tumor necrosis factor α (TNFα) and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  8. ADAM12 transmembrane and secreted isoforms promote breast tumor growth: a distinct role for ADAM12-S protein in tumor metastasis.

    PubMed

    Roy, Roopali; Rodig, Scott; Bielenberg, Diane; Zurakowski, David; Moses, Marsha A

    2011-06-10

    Increased levels of ADAM12 have been reported in a variety of human cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. On the basis of these findings, the goal of this study was to elucidate the contribution of ADAM12 in breast tumor growth and progression. Overexpression of both the ADAM12-L (transmembrane) and ADAM12-S (secreted) isoforms in human breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. Cells expressing the enzymatically inactive form of the secreted isoform, ADAM12-S, had tumor take rates and tumor volumes similar to those of wild-type cells, suggesting that the tumor-promoting activity of ADAM12-S was a function of its proteolytic activity. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo. This stimulatory effect of ADAM12-S on migration and invasion was dependent on its catalytic activity. Expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue. Taken together, our results suggest that ADAM12 overexpression results in increased tumor take, tumor size, and metastasis in vivo. These findings suggest that ADAM12 may represent a potential therapeutic target in breast cancer.

  9. Species Specificity of ADAM10 and ADAM17 Proteins in Interleukin-6 (IL-6) Trans-signaling and Novel Role of ADAM10 in Inducible IL-6 Receptor Shedding*

    PubMed Central

    Garbers, Christoph; Jänner, Nathalie; Chalaris, Athena; Moss, Marcia L.; Floss, Doreen M.; Meyer, Dörte; Koch-Nolte, Friedrich; Rose-John, Stefan; Scheller, Jürgen

    2011-01-01

    Hypomorphic ADAM17ex/ex mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17ex/ex mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice. PMID:21454673

  10. Evidence for Functional Redundancy Between C. elegans ADAM Proteins SUP-17/Kuzbanian and ADM-4/TACE

    PubMed Central

    Jarriault, Sophie; Greenwald, Iva

    2005-01-01

    The ectodomain of LIN-12/Notch proteins is cleaved and shed upon ligand binding. In C. elegans, genetic evidence has implicated SUP-17, the ortholog of Drosophila Kuzbanian and mammalian ADAM10, as the protease that mediates this event. In mammals, however, biochemical evidence has implicated TACE, a different ADAM protein. We have investigated potential functional redundancy of sup-17 and the C. elegans ortholog of TACE, adm-4, by exploring their roles in cell fate decisions mediated by lin-12/Notch genes. We found that reduced adm-4 activity, like reduced sup-17 activity, suppresses an allele of glp-1 that encodes a constitutively active receptor. Furthermore, concomitant reduction of adm-4 and sup-17 activity causes the production of two anchor cells in the hermaphrodite gonad, instead of one--a phenotype associated with loss of lin-12 activity. We also identified a novel fertility defect in the somatic gonad when both sup-17 and adm-4 are depleted. Expression of a truncated form of LIN-12 that mimics the product of ectodomain shedding rescues this fertility defect, suggesting that sup-17 and adm-4 may mediate ectodomain shedding of LIN-12 and/or GLP-1. Our results are consistent with the possibility that sup-17 and adm-4 are functionally redundant for at least a subset of LIN-12/Notch mediated decisions in C. elegans. PMID:16197940

  11. Peptoid mimics of agouti related protein.

    PubMed

    Thompson, Darren A; Chai, Biao-Xin; Rood, Hilary L E; Siani, Michael A; Douglas, Nicholai R; Gantz, Ira; Millhauser, Glenn L

    2003-04-17

    The Agouti Related Protein (AGRP) is an endogenous antagonist of melanocortin-3 and -4 receptors, each of which plays a key role in body weight homeostasis. We designed a peptoid trimer based on AGRP 111-113 in which a single chiral atom is used to partially restrain the backbone structure. Peptoid 5 displaced both radiolabeled Nle4-alpha-MSH (IC(50)=3.1 microM) and AGRP (86-132) (IC(50)=1.9 microM) from the human melanocortin-4 receptor and functioned as an antagonist of alpha-MSH stimulated cAMP generation, thus providing an important lead in the development of AGRP mimetics.

  12. Conservation and divergence of ADAM family proteins in the Xenopus genome

    PubMed Central

    2010-01-01

    Background Members of the disintegrin metalloproteinase (ADAM) family play important roles in cellular and developmental processes through their functions as proteases and/or binding partners for other proteins. The amphibian Xenopus has long been used as a model for early vertebrate development, but genome-wide analyses for large gene families were not possible until the recent completion of the X. tropicalis genome sequence and the availability of large scale expression sequence tag (EST) databases. In this study we carried out a systematic analysis of the X. tropicalis genome and uncovered several interesting features of ADAM genes in this species. Results Based on the X. tropicalis genome sequence and EST databases, we identified Xenopus orthologues of mammalian ADAMs and obtained full-length cDNA clones for these genes. The deduced protein sequences, synteny and exon-intron boundaries are conserved between most human and X. tropicalis orthologues. The alternative splicing patterns of certain Xenopus ADAM genes, such as adams 22 and 28, are similar to those of their mammalian orthologues. However, we were unable to identify an orthologue for ADAM7 or 8. The Xenopus orthologue of ADAM15, an active metalloproteinase in mammals, does not contain the conserved zinc-binding motif and is hence considered proteolytically inactive. We also found evidence for gain of ADAM genes in Xenopus as compared to other species. There is a homologue of ADAM10 in Xenopus that is missing in most mammals. Furthermore, a single scaffold of X. tropicalis genome contains four genes encoding ADAM28 homologues, suggesting genome duplication in this region. Conclusions Our genome-wide analysis of ADAM genes in X. tropicalis revealed both conservation and evolutionary divergence of these genes in this amphibian species. On the one hand, all ADAMs implicated in normal development and health in other species are conserved in X. tropicalis. On the other hand, some ADAM genes and ADAM protease

  13. RACK1, a new ADAM12 interacting protein. Contribution to liver fibrogenesis.

    PubMed

    Bourd-Boittin, Katia; Le Pabic, Hélène; Bonnier, Dominique; L'Helgoualc'h, Annie; Théret, Nathalie

    2008-09-19

    ADAM12 belongs to a disintegrin-like and metalloproteinase-containing protein family that possesses multidomain structures composed of a pro-domain, a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, and transmembrane domains, and a cytoplasmic tail. Overexpression of several ADAMs has been reported in human cancer, and we recently described the involvement of ADAM12 in liver injury (Le Pabic, H., Bonnier, D., Wewer, U. M., Coutand, A., Musso, O., Baffet, G., Clement, B., and Theret, N. (2003) Hepatology 37, 1056-1066). In this study, we used a yeast two-hybrid screening of a cDNA library from human hepatocellular carcinoma to analyze binding partners of ADAM12. We identify RACK1, a receptor for activated protein kinase C (PKC), as a new ADAM12 interacting protein. RACK1 is up-regulated in patients with hepatocellular carcinoma and is highly expressed by activated hepatic stellate cells. We demonstrate the involvement of RACK1 in mediating the PKC-dependent translocation of ADAM12 to membranes of activated hepatic stellate cells. In particular, treatment of cells with phorbol esters enhances ADAM12 immunostaining in the membrane fractions and the co-immunoprecipitation of ternary complexes containing RACK1, ADAM12, and PKC. By using RNA interference, we demonstrate that inhibition of RACK1 expression diminishes the phorbol 12-myristate 13-acetate-dependent translocation of ADAM12 to membranes of hepatic stellate cells. Finally, hepatic stellate cells cultured on coated type I collagen induces relocalization of ADAM12 in the membrane, suggesting that this major matrix component in liver cancer and fibrogenesis might stimulate ADAM12 translocation to the cell membrane where its shedding activity takes place.

  14. ADAM and ADAMTS Family Proteins and Snake Venom Metalloproteinases: A Structural Overview

    PubMed Central

    Takeda, Soichi

    2016-01-01

    A disintegrin and metalloproteinase (ADAM) family proteins constitute a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell-surface protein ectodomains, including the latent forms of growth factors, cytokines, receptors and other molecules. Snake venom metalloproteinases (SVMPs) are major components in most viper venoms. SVMPs are primarily responsible for hemorrhagic activity and may also interfere with the hemostatic system in envenomed animals. SVMPs are phylogenetically most closely related to ADAMs and, together with ADAMs and related ADAM with thrombospondin motifs (ADAMTS) family proteinases, constitute adamalysins/reprolysins or the M12B clan (MEROPS database) of metalloproteinases. Although the catalytic domain structure is topologically similar to that of other metalloproteinases such as matrix metalloproteinases, the M12B proteinases have a modular structure with multiple non-catalytic ancillary domains that are not found in other proteinases. Notably, crystallographic studies revealed that, in addition to the conserved metalloproteinase domain, M12B members share a hallmark cysteine-rich domain designated as the “ADAM_CR” domain. Despite their name, ADAMTSs lack disintegrin-like structures and instead comprise two ADAM_CR domains. This review highlights the current state of our knowledge on the three-dimensional structures of M12B proteinases, focusing on their unique domains that may collaboratively participate in directing these proteinases to specific substrates. PMID:27196928

  15. ADAM and ADAMTS family proteins and their role in the colorectal cancer etiopathogenesis

    PubMed Central

    Przemyslaw, Leszczynski; Boguslaw, Hendrich Andrzej; Elzbieta, Szmida; Malgorzata, Sasiadek Maria

    2013-01-01

    The ADAM and ADAMTS families, also called adamalysins belong to an important group of extracellular matrix proteins. The ADAMs family belong to both the transmembrane and secreted proteins, while ADAMTS family only contains secreted forms. Adamalysins play an important role in the cell phenotype regulation via their activities in signaling pathways, cell adhesion and migration. The human proteome contains 21 ADAM, and 19 ADAMTS proteins, which are involved in extracellular matrix remodeling, shedding of various substrates such as: adhesion ligands, growth factors, their receptors and diverse cytokines. Recent studies provide evidence that adamalysins play a crucial role in colorectal cancer (CRC) etiopathogenesis. It seems possible that adamalysins might be used as CRC prediction markers or potential pharmaceutical targets. [BMB Reports 2013; 46(3): 139-150] PMID:23527857

  16. Association of ADAM12-S protein with radiographic features of knee osteoarthritis and bone and cartilage markers.

    PubMed

    Kerna, I; Kisand, K; Laitinen, P; Tamm, A E; Kumm, J; Lintrop, M; Tamm, A O

    2012-02-01

    ADAM12 (A disintegrin and metalloprotease) is one of the candidate genes demonstrating susceptibility to osteoarthritis. The purpose of this study was to investigate the relationship between ADAM12-S protein and radiographic knee osteoarthritis (KOA) and its correlation to several bone and cartilage biomarkers. The ADAM12-S protein was measured in 276 subjects (60% women, aged 32-60 years), including 181 individuals with and 95 without radiographic KOA features. The radiographs were obtained from both tibiofemoral (TF) and patellofemoral (PF) joints. The serum levels of ADAM12-S protein were measured by DELFIA1/AutoDELFIA research kit. The ADAM12-S protein was found in detectable ranges in 43 subjects (16 men), without statistical difference between the two genders. In the whole group, the ADAM12-S was related to radiographic KOA grades in TF (P = 0.004) as well in PF joint (P = 0.003). We also found a correlation between ADAM12-S protein and osteophytes in TF and/or PF joints (P = 0.003). No correlations were found between serum levels of S-CTx-I (C-terminal cross-linked telopeptides of type I collagen) or S-PINP (type I procollagen N-terminal propeptide) and ADAM12-S. Similarly, in the whole group, the ADAM12-S protein was not correlated with U-CTx-II (urinary C-telopeptide fragments of type II collagen); however, in the female group, trend to positive correlation between the investigated biomarkers (P = 0.019) was observed. The ADAM12-S protein could be elevated in some KOA cases, and this elevation correlates with the grades of the disease, mostly owning to development of osteophytes. This finding suggests the possible involvement of the ADAM12-S protein in the pathogenesis of KOA.

  17. Identification of SH3 Domain Proteins Interacting with the Cytoplasmic Tail of the A Disintegrin and Metalloprotease 10 (ADAM10)

    PubMed Central

    Ebsen, Henriette; Lettau, Marcus; Kabelitz, Dieter; Janssen, Ottmar

    2014-01-01

    The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology. PMID:25036101

  18. The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17

    PubMed Central

    Dombernowsky, Sarah Louise; Samsøe-Petersen, Jacob; Petersen, Camilla Hansson; Instrell, Rachael; Hedegaard, Anne-Mette Bornhardt; Thomas, Laurel; Atkins, Katelyn Mae; Auclair, Sylvain; Albrechtsen, Reidar; Mygind, Kasper Johansen; Fröhlich, Camilla; Howell, Michael; Parker, Peter; Thomas, Gary; Kveiborg, Marie

    2015-01-01

    The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth, and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology. PMID:26108729

  19. ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

    PubMed

    Tanaka, Motonari; Nanba, Daisuke; Mori, Seiji; Shiba, Fumio; Ishiguro, Hiroshi; Yoshino, Koichiro; Matsuura, Nariaki; Higashiyama, Shigeki

    2004-10-01

    A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

  20. Epidermal growth factor (EGF) ligand release by substrate-specific a disintegrin and metalloproteases (ADAMs) involves different protein kinase C (PKC) isoenzymes depending on the stimulus.

    PubMed

    Dang, Michelle; Dubbin, Karen; D'Aiello, Antonio; Hartmann, Monika; Lodish, Harvey; Herrlich, Andreas

    2011-05-20

    The dysregulation of EGF family ligand cleavage has severe consequences for the developing as well as the adult organism. Therefore, their production is highly regulated. The limiting step is the ectodomain cleavage of membrane-bound precursors by one of several a disintegrin and metalloprotease (ADAM) metalloproteases, and understanding the regulation of cleavage is an important goal of current research. We have previously reported that in mouse lung epithelial cells, the pro-EGF ligands TGFα, neuregulin 1β (NRG), and heparin-binding EGF are differentially cleaved depending on the cleavage stimulus (Herrlich, A., Klinman, E., Fu, J., Sadegh, C., and Lodish, H. (2008) FASEB J.). In this study in mouse embryonic fibroblasts that lack different ADAMs, we show that induced cleavage of EGF ligands can involve the same substrate-specific metalloprotease but does require different stimulus-dependent signaling pathways. Cleavage was stimulated by phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA), a mimic of diacylglycerol and PKC activator), hypertonic stress, lysophosphatidic acid (LPA)-induced G protein-coupled receptor activation, or by ionomycin-induced intracellular calcium release. Although ADAMs showed substrate preference (ADAM17, TGFα and heparin-binding EGF; and ADAM9, NRG), substrate cleavage differed substantially with the stimulus, and cleavage of the same substrate depended on the presence of different, sometimes multiple, PKC isoforms. For instance, classical PKC was required for TPA-induced but not hypertonic stress-induced cleavage of all EGF family ligands. Inhibition of PKCζ enhanced NRG release upon TPA stimulation, but it blocked NRG release in response to hypertonic stress. Our results suggest a model in which substantial regulation of ectodomain cleavage occurs not only on the metalloprotease level but also on the level of the substrate or of a third protein.

  1. The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function.

    PubMed

    Lovero, Kathryn L; Fukata, Yuko; Granger, Adam J; Fukata, Masaki; Nicoll, Roger A

    2015-07-28

    Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

  2. Regulation of Alpha-Secretase ADAM10 In vitro and In vivo: Genetic, Epigenetic, and Protein-Based Mechanisms

    PubMed Central

    Endres, Kristina; Deller, Thomas

    2017-01-01

    ADAM10 (A Disintegrin and Metalloproteinase 10) has been identified as the major physiological alpha-secretase in neurons, responsible for cleaving APP in a non-amyloidogenic manner. This cleavage results in the production of a neuroprotective APP-derived fragment, APPs-alpha, and an attenuated production of neurotoxic A-beta peptides. An increase in ADAM10 activity shifts the balance of APP processing toward APPs-alpha and protects the brain from amyloid deposition and disease. Thus, increasing ADAM10 activity has been proposed an attractive target for the treatment of neurodegenerative diseases and it appears to be timely to investigate the physiological mechanisms regulating ADAM10 expression. Therefore, in this article, we will (1) review reports on the physiological regulation of ADAM10 at the transcriptional level, by epigenetic factors, miRNAs and/or protein interactions, (2) describe conditions, which change ADAM10 expression in vitro and in vivo, (3) report how neuronal ADAM10 expression may be regulated in humans, and (4) discuss how this knowledge on the physiological and pathophysiological regulation of ADAM10 may help to preserve or restore brain function. PMID:28367112

  3. ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins.

    PubMed

    Fröhlich, Camilla; Klitgaard, Marie; Noer, Julie B; Kotzsch, Alexander; Nehammer, Camilla; Kronqvist, Pauliina; Berthelsen, Jens; Blobel, Carl; Kveiborg, Marie; Albrechtsen, Reidar; Wewer, Ulla M

    2013-05-15

    ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.

  4. Snake venom metalloproteinases: structure, function and relevance to the mammalian ADAM/ADAMTS family proteins.

    PubMed

    Takeda, Soichi; Takeya, Hiroyuki; Iwanaga, Sadaaki

    2012-01-01

    Metalloproteinases are among the most abundant toxins in many Viperidae venoms. Snake venom metalloproteinases (SVMPs) are the primary factors responsible for hemorrhage and may also interfere with the hemostatic system, thus facilitating loss of blood from the vasculature of the prey. SVMPs are phylogenetically most closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif) family of proteins and, together with them, constitute the M12B clan of metalloendopeptidases. Large SVMPs, referred to as the P-III class of SVMPs, have a modular architecture with multiple non-catalytic domains. The P-III SVMPs are characterized by higher hemorrhagic and more diverse biological activities than the P-I class of SVMPs, which only have a catalytic domain. Recent crystallographic studies of P-III SVMPs and their mammalian counterparts shed new light on structure-function properties of this class of enzymes. The present review will highlight these structures, particularly the non-catalytic ancillary domains of P-III SVMPs and ADAMs that may target the enzymes to specific substrates. This article is part of a Special Issue entitled: Proteolysis 50years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein.

    PubMed

    Seipold, Lisa; Damme, Markus; Prox, Johannes; Rabe, Björn; Kasparek, Petr; Sedlacek, Radislav; Altmeppen, Hermann; Willem, Michael; Boland, Barry; Glatzel, Markus; Saftig, Paul

    2017-01-01

    Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

  6. ADAM-9 is an insulin-like growth factor binding protein-5 protease produced and secreted by human osteoblasts.

    PubMed

    Mohan, Subburaman; Thompson, Garrett R; Amaar, Yousef G; Hathaway, Gary; Tschesche, Harald; Baylink, David J

    2002-12-24

    IGF binding protein-5 (BP-5) is an important bone formation regulator. Therefore, elucidation of the identity of IGF binding protein-5 (BP-5) protease produced by osteoblasts is important for our understanding of the molecular pathways that control the action of BP-5. In this regard, BP-5 protease purified by various chromatographic steps from a conditioned medium of U2 human osteosarcoma cells migrated as a single major band, which comigrated with the protease activity in native PAGE and yielded multiple bands in SDS-PAGE under reducing conditions. N-Terminal sequencing of these bands revealed that three of the bands yielded amino acid sequences that were identical to that of alpha2 macroglobulin (alpha2M). Although alpha2M was produced by human osteoblasts (OBs), it was not found to be a BP-5 protease. Because alpha2M had been shown to complex with ADAM proteases and because ADAM-12 was found to cleave BP-3 and BP-5, we evaluated if one of the members of ADAM family was the BP-5 protease. On the basis of the findings that (1) purified preparations of BP-5 protease from U2 cell CM contained ADAM-9, (2) ADAM-9 is produced and secreted in high abundance by various human OB cell types, (3) purified ADAM-9 cleaved BP-5 effectively while it did not cleave other IGFBPs or did so with less potency, and (4) purified ADAM-9 bound to alpha2M, we conclude that ADAM-9 is a BP-5 protease produced by human OBs.

  7. Scaffolded multiple cyclic peptide libraries for protein mimics by native chemical ligation.

    PubMed

    van de Langemheen, H; van Hoeke, M; Quarles van Ufford, H C; Kruijtzer, J A W; Liskamp, R M J

    2014-07-07

    The accessibility to collections, libraries and arrays of cyclic peptides is increasingly important since cyclic peptides may provide better mimics of the loop-like structures ubiquitously present in and - especially - on the surface of proteins. The next important step is the preparation of libraries of ensembles of scaffolded cyclic peptides, which upon screening may lead to promising protein mimics. Here we describe the synthesis of a tri-cysteine containing scaffold as well as the simultaneous native chemical ligation of three cyclic peptides thereby affording a clean library of multiple cyclic peptides on this scaffold, representing potential mimics of gp120. Members of this collection of protein mimics showed a decent inhibition of the gp120-CD4 interaction.

  8. The Role of the anti-amyloidogenic secretase ADAM10 in shedding the APP-like proteins.

    PubMed

    Endres, Kristina; Fahrenholz, Falk

    2012-02-01

    ADAM10 (A disintegrin and metalloproteinase 10) has been demonstrated as an enzyme with protective properties in Alzheimer's disease: in mouse models it not only lowered generation of toxic A-beta peptides and formation of senile plaques but also alleviated learning deficits and enhanced synaptic density. This is due to cleavage of the amyloid precursor protein (APP) within its A-beta stretch and to the release of the extracellular domain of APP with neuroprotective function. Aside from cleaving APP, ADAM10 has been linked to over 40 putative substrates at least in cell culture. These substrates are connected with important cellular functions such as cell migration, stress response and transport. For this contribution we focussed on ADAM10 acting on the APP-like proteins since for some of their representatives - in particular APLP2 and APP-L - a shedding by ADAM10 has been demonstrated in vivo. In addition, the importance of these proteins, especially of APLP2, has been repeatedly shown by intense analysis of double and triple transgenic mice which lack APP in combination with one or both APLPs: several phenotypes such as defects in migration of neuroblasts, in formation of synapses and synaptic transmission have been reported. However, the specific contribution of either the uncleaved full-length proteins or their extracellular domains secreted upon ADAM10 activity has not been elucidated. In this review, we report on recent findings concerning shedding of APP-like proteins and resulting functional consequences.

  9. Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis.

    PubMed

    Bertram, Anna; Lovric, Svjetlana; Engel, Alissa; Beese, Michaela; Wyss, Kristin; Hertel, Barbara; Park, Joon-Keun; Becker, Jan U; Kegel, Johanna; Haller, Hermann; Haubitz, Marion; Kirsch, Torsten

    2015-11-01

    ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease.

  10. Circulating ADAM17 Level Reflects Disease Activity in Proteinase-3 ANCA-Associated Vasculitis

    PubMed Central

    Bertram, Anna; Lovric, Svjetlana; Engel, Alissa; Beese, Michaela; Wyss, Kristin; Hertel, Barbara; Park, Joon-Keun; Becker, Jan U.; Kegel, Johanna; Haller, Hermann; Haubitz, Marion

    2015-01-01

    ANCA-associated vasculitides are characterized by inflammatory destruction of small vessels accompanied by enhanced cleavage of membrane-bound proteins. One of the main proteases responsible for ectodomain shedding is disintegrin and metalloproteinase domain-containing protein 17 (ADAM17). Given its potential role in aggravating vascular dysfunction, we examined the role of ADAM17 in active proteinase-3 (PR3)-positive ANCA-associated vasculitis (AAV). ADAM17 concentration was significantly increased in plasma samples from patients with active PR3-AAV compared with samples from patients in remission or from other controls with renal nonvascular diseases. Comparably, plasma levels of the ADAM17 substrate syndecan-1 were significantly enhanced in active AAV. We also observed that plasma-derived ADAM17 retained its specific proteolytic activity and was partly located on extracellular microparticles. Transcript levels of ADAM17 were increased in blood samples of patients with active AAV, but those of ADAM10 or tissue inhibitor of metalloproteinases 3, which inhibits ADAMs, were not. We also performed a microRNA (miR) screen and identified miR-634 as significantly upregulated in blood samples from patients with active AAV. In vitro, miR-634 mimics induced a proinflammatory phenotype in monocyte-derived macrophages, with enhanced expression and release of ADAM17 and IL-6. These data suggest that ADAM17 has a prominent role in AAV and might account for the vascular complications associated with this disease. PMID:25788529

  11. Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

    PubMed

    Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

    2013-04-28

    A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

  12. ADAM-10-Mediated N-cadherin Cleavage is Protein Kinase C-α–Dependent and Promotes Glioblastoma Cell Migration

    PubMed Central

    Kohutek, Zachary A.; diPierro, Charles G.; Redpath, Gerard T.; Hussaini, Isa M.

    2009-01-01

    Matrix metalloproteinases (MMPs) and the related ‘a disintegrin and metalloproteinases’ (ADAMs) promote tumorigenesis by cleaving extracellular matrix and protein substrates, including N-cadherin. While N-cadherin is thought to regulate cell adhesion, migration and invasion, its role has not been characterized in glioblastomas (GBMs). In this study, we investigated the expression and function of post-translational N-cadherin cleavage in GBM cells as well as its regulation by protein kinase C (PKC). N-cadherin cleavage occurred at a higher level in glioblastoma cells than in non-neoplastic astrocytes. Treatment with the PKC-activator phorbol 12-myristate 13-acetate (PMA) increased N-cadherin cleavage, which was reduced by pharmacological inhibitors and siRNA specific for ADAM-10 or PKC-α. Furthermore, treatment of GBM cells with PMA induced the translocation of ADAM-10 to the cell membrane, the site where N-cadherin was cleaved, and this translocation was significantly reduced by the PKC-α inhibitor Gö6976 or PKC-α shRNA. In functional studies, N-cadherin cleavage was required for GBM cell migration, as depletion of N-cadherin cleavage by N-cadherin siRNA, ADAM-10 siRNA, or a cleavage-site mutant N-cadherin, decreased GBM cell migration. Taken together, these results suggest that N-cadherin cleavage is regulated by a PKC-α-ADAM-10 cascade in GBM cells and may be involved in mediating GBM cell migration. PMID:19357285

  13. Catalysis of Protein Folding by Protein Disulfide Isomerase and Small-Molecule Mimics

    PubMed Central

    KERSTEEN, ELIZABETH A.; RAINES, RONALD T.

    2010-01-01

    Protein disulfide isomerase (PDI) catalyzes the formation of native disulfide pairings in secretory proteins. The ability of PDI to act as a disulfide isomerase makes it an essential enzyme in eukaryotes. PDI also fulfills other important roles. Recent studies have emphasized the importance of PDI as an oxidant in the endoplasmic reticulum. Intriguing questions remain regarding how PDI is able to catalyze both isomerization and oxidation in vivo. Studies of PDI and its homologs have led to the development of small-molecule folding catalysts that are able to accelerate disulfide isomerization in vitro and in vivo. PDI will continue to provide both an inspiration for the design of such artificial foldases and a benchmark with which to gauge the success of those designs. Here, we review current understanding of the chemistry and biology of PDI, its homologs, and small molecules that mimic its catalytic activity. PMID:13678529

  14. Peptide-Based Molecular Hydrogels as Supramolecular Protein Mimics.

    PubMed

    Singh, Nishant; Kumar, Mohit; Miravet, Juan F; Ulijn, Rein V; Escuder, Beatriu

    2017-01-23

    This Minireview concerns recent advances in the design, synthesis, and application of low molecular-weight peptidic hydrogelators. The sequence-specific combinations of amino acid side chain functionalities combined with hydrogen bonding of amide backbones and hydrophobic (aromatic) capping groups give these peptidic molecules the intrinsic tendency to self-assemble. The most prevalent designs include N-capped amino acid residues, bolamphiphilic peptides, and amphipathic peptides. Factors such as hydrophobic effects, the Hofmeister effect, and tunable ionization influence their aggregation properties. The self-assembly of simple bio-inspired building blocks into higher organized structures allows comparisons to be drawn with proteins and their complex functionalities, providing preliminary insights into complex biological functions and also enabling their application in a wide range of fields including catalysis, biomedical applications, and mimicry of natural dissipative systems. The Minireview is concluded by a short summary and outlook, highlighting the advances and steps required to bridge the gaps in the understanding of such systems.

  15. ADAM function in embryogenesis

    PubMed Central

    Alfandari, Dominique; McCusker, Catherine; Cousin, Hélène

    2009-01-01

    Cleavage of proteins inserted into the plasma membrane (shedding) is an essential process controlling many biological functions including cell signaling, cell adhesion and migration as well as proliferation and differentiation. ADAM surface metalloproteases have been shown to play an essential role in these processes. Gene inactivation during embryonic development have provided evidence of the central role of ADAM proteins in nematodes, flies, frogs, birds and mammals. The relative contribution of four subfamilies of ADAM proteins to developmental processes is the focus of this review. PMID:18935966

  16. Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation

    PubMed Central

    Aguer, Céline; Fiehn, Oliver; Seifert, Erin L.; Bézaire, Véronic; Meissen, John K.; Daniels, Amanda; Scott, Kyle; Renaud, Jean-Marc; Padilla, Marta; Bickel, David R.; Dysart, Michael; Adams, Sean H.; Harper, Mary-Ellen

    2013-01-01

    Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5–10%) and decreased oxidative stress (∼15–20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.—Aguer, C., Fiehn, O., Seifert, E. L., Bézaire, V., Meissen, J. K., Daniels, A., Scott, K., Renaud, J.-M., Padilla, M., Bickel, D. R., Dysart, M., Adams, S. H., Harper, M.-E. Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation. PMID:23825224

  17. ADAMs 10 and 17 Represent Differentially Regulated Components of a General Shedding Machinery for Membrane Proteins Such as Transforming Growth Factor α, L-Selectin, and Tumor Necrosis Factor α

    PubMed Central

    Le Gall, Sylvain M.; Bobé, Pierre; Reiss, Karina; Horiuchi, Keisuke; Niu, Xiao-Da; Lundell, Daniel; Gibb, David R.; Conrad, Daniel; Saftig, Paul

    2009-01-01

    Protein ectodomain shedding is a critical regulator of many membrane proteins, including epidermal growth factor receptor-ligands and tumor necrosis factor (TNF)-α, providing a strong incentive to define the responsible sheddases. Previous studies identified ADAM17 as principal sheddase for transforming growth factor (TGF)-α and heparin-binding epidermal growth factor, but Ca++ influx activated an additional sheddase for these epidermal growth factor receptor ligands in Adam17−/− cells. Here, we show that Ca++ influx and stimulation of the P2X7R signaling pathway activate ADAM10 as sheddase of many ADAM17 substrates in Adam17−/− fibroblasts and primary B cells. Importantly, although ADAM10 can shed all substrates of ADAM17 tested here in Adam17−/− cells, acute treatment of wild-type cells with a highly selective ADAM17 inhibitor (SP26) showed that ADAM17 is nevertheless the principal sheddase when both ADAMs 10 and 17 are present. However, chronic treatment of wild-type cells with SP26 promoted processing of ADAM17 substrates by ADAM10, thus generating conditions such as in Adam17−/− cells. These results have general implications for understanding the substrate selectivity of two major cellular sheddases, ADAMs 10 and 17. PMID:19158376

  18. Fe-TAML encapsulated inside mesoporous silica nanoparticles as peroxidase mimic: femtomolar protein detection.

    PubMed

    Kumari, Sushma; Dhar, Basab B; Panda, Chakadola; Meena, Abhishek; Sen Gupta, Sayam

    2014-08-27

    Peroxidase, such as horseradish peroxidase (HRP), conjugated to antibodies are routinely used for the detection of proteins via an ELISA type assay in which a critical step is the catalytic signal amplification by the enzyme to generate a detectable signal. Synthesis of functional mimics of peroxidase enzyme that display catalytic activity which far exceeds the native enzyme is extremely important for the precise and accurate determination of very low quantities of proteins (fM and lower) that is necessary for early clinical diagnosis. Despite great advancements, analyzing proteins of very low abundance colorimetrically, a method that is most sought after since it requires no equipment for the analysis, still faces great challenges. Most reported HRP mimics that show catalytic activity greater than native enzyme (∼10-fold) are based on metal/metal-oxide nanoparticles such as Fe3O4. In this paper, we describe a second generation hybrid material developed by us in which approximately 25,000 alkyne tagged biuret modified Fe-tetraamido macrocyclic ligand (Fe-TAML), a very powerful small molecule synthetic HRP mimic, was covalently attached inside a 40 nm mesoporous silica nanoparticle (MSN). Biuret-modified Fe-TAMLs represent one of the best small molecule functional mimics of the enzyme HRP with reaction rates in water close to the native enzyme and operational stability (pH, ionic strength) far exceeding the natural enzyme. The catalytic activity of this hybrid material is around 1000-fold higher than that of natural HRP and 100-fold higher than that of most metal/metal oxide nanoparticle based HRP mimics reported to date. We also show that using antibody conjugates of this hybrid material it is possible to detect and, most importantly, quantify femtomolar quantities of proteins colorimetrically in an ELISA type assay. This represents at least 10-fold higher sensitivity than other colorimetric protein assays that have been reported using metal/metal oxide

  19. Physiological functions of the amyloid precursor protein secretases ADAM10, BACE1, and presenilin.

    PubMed

    Prox, Johannes; Rittger, Andrea; Saftig, Paul

    2012-04-01

    Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aβ) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aβ) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, β-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the β-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.

  20. Protocadherin-12 Cleavage Is a Regulated Process Mediated by ADAM10 Protein

    PubMed Central

    Bouillot, Stéphanie; Tillet, Emmanuelle; Carmona, Guillaume; Prandini, Marie-Hélène; Gauchez, Anne-Sophie; Hoffmann, Pascale; Alfaidy, Nadia; Cand, Francine; Huber, Philippe

    2011-01-01

    Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E2, or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity. PMID:21402705

  1. DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores.

    PubMed

    Feng, Guangfu; Luo, Chao; Yi, Haibo; Yuan, Lin; Lin, Bin; Luo, Xingyu; Hu, Xiaoxiao; Wang, Honghui; Lei, Chunyang; Nie, Zhou; Yao, Shouzhuo

    2017-09-13

    Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583-668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. De novo designed protein transduction domain mimics from simple synthetic polymers.

    PubMed

    Tezgel, A Özgül; Telfer, Janice C; Tew, Gregory N

    2011-08-08

    Protein transduction domains (PTDs) that readily transverse cellular membranes are of great interest and are attractive tools for the intracellular delivery of bioactive molecules. Learning to program synthetic polymers and oligomers with the appropriate chemical information to capture adequately the biological activity of proteins is critical to our improved understanding of how these natural molecules work. In addition, the versatility of these synthetic mimics provides the opportunity to discover analogs with superior properties compared with their native sequences. Here we report the first detailed structure-activity relationship of a new PTD family of polymers based on a completely abiotic backbone. The synthetic approach easily allows doubling the density of guanidine functional groups, which increases the transduction efficiency of the sequences. Cellular uptake studies on three different cell lines (HEK 293T, CHO, and Jurkat T cells) confirm that these synthetic analogs are highly efficient novel protein transduction domain mimics (PTDMs), which are more effective than TAT(49-57) and nonaarginine (R9) and also highlight the usefulness of polymer chemistry at the chemistry-biology interface.

  3. Structural basis for activity of highly efficient RNA mimics of green fluorescent protein

    PubMed Central

    Warner, Katherine Deigan; Chen, Michael C.; Song, Wenjiao; Strack, Rita L.; Thorn, Andrea; Jaffrey, Samie R.; Ferré-D’Amaré, Adrian R.

    2014-01-01

    Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs. PMID:25026079

  4. Structure of Ocr from bacteriophage T7, a protein that mimics B-form DNA.

    PubMed

    Walkinshaw, M D; Taylor, P; Sturrock, S S; Atanasiu, C; Berge, T; Henderson, R M; Edwardson, J M; Dryden, D T F

    2002-01-01

    We have solved, by X-ray crystallography to a resolution of 1.8 A, the structure of a protein capable of mimicking approximately 20 base pairs of B-form DNA. This ocr protein, encoded by gene 0.3 of bacteriophage T7, mimics the size and shape of a bent DNA molecule and the arrangement of negative charges along the phosphate backbone of B-form DNA. We also demonstrate that ocr is an efficient inhibitor in vivo of all known families of the complex type I DNA restriction enzymes. Using atomic force microscopy, we have also observed that type I enzymes induce a bend in DNA of similar magnitude to the bend in the ocr molecule. This first structure of an antirestriction protein demonstrates the construction of structural mimetics of long segments of B-form DNA.

  5. ADAM12 — EDRN Public Portal

    Cancer.gov

    ADAM12 is a member of the ADAM (a disintegrin and metalloprotease) protein family. ADAM family members are membrane-anchored proteins structurally related to snake venom disintegrins, and have been implicated in a variety of biological processes involving cell-cell and cell-matrix interactions. ADAM12 has two alternatively spliced transcripts: a shorter secreted form and a longer membrane-bound form. The shorter form is found to stimulate myogenesis.

  6. Macrocyclic beta-sheet peptides that mimic protein quaternary structure through intermolecular beta-sheet interactions.

    PubMed

    Khakshoor, Omid; Demeler, Borries; Nowick, James S

    2007-05-02

    This paper reports the design, synthesis, and characterization of a family of cyclic peptides that mimic protein quaternary structure through beta-sheet interactions. These peptides are 54-membered-ring macrocycles comprising an extended heptapeptide beta-strand, two Hao beta-strand mimics [JACS 2000, 122, 7654] joined by one additional alpha-amino acid, and two delta-linked ornithine beta-turn mimics [JACS 2003, 125, 876]. Peptide 3a, as the representative of these cyclic peptides, contains a heptapeptide sequence (TSFTYTS) adapted from the dimerization interface of protein NuG2 [PDB ID: 1mio]. 1H NMR studies of aqueous solutions of peptide 3a show a partially folded monomer in slow exchange with a strongly folded oligomer. NOE studies clearly show that the peptide self-associates through edge-to-edge beta-sheet dimerization. Pulsed-field gradient (PFG) NMR diffusion coefficient measurements and analytical ultracentrifugation (AUC) studies establish that the oligomer is a tetramer. Collectively, these experiments suggest a model in which cyclic peptide 3a oligomerizes to form a dimer of beta-sheet dimers. In this tetrameric beta-sheet sandwich, the macrocyclic peptide 3a is folded to form a beta-sheet, the beta-sheet is dimerized through edge-to-edge interactions, and this dimer is further dimerized through hydrophobic face-to-face interactions involving the Phe and Tyr groups. Further studies of peptides 3b-3n, which are homologues of peptide 3a with 1-6 variations in the heptapeptide sequence, elucidate the importance of the heptapeptide sequence in the folding and oligomerization of this family of cyclic peptides. Studies of peptides 3b-3g show that aromatic residues across from Hao improve folding of the peptide, while studies of peptides 3h-3n indicate that hydrophobic residues at positions R3 and R5 of the heptapeptide sequence are important in oligomerization.

  7. mimicMe: a web server for prediction and analysis of host-like proteins in microbial pathogens.

    PubMed

    Petrenko, Pavel; Doxey, Andrew C

    2015-02-15

    mimicMe is a web server for prediction and analysis of host-like proteins (mimics) encoded by microbial pathogens. Users select a host species and any set of pathogen and control proteomes (bacterial, fungal, protozoan or viral) and mimicMe reports host-like proteins that are unique to or enriched among pathogens. Additional server features include visualization of structural similarities between pathogen and host proteins as well as function-enrichment analysis. mimicMe is available at http://mimicme.uwaterloo.ca acdoxey@uwaterloo.ca. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  9. The Role of ADAM9 in Tumor-Stromal Interactions in Breast Cancer

    DTIC Science & Technology

    2010-04-01

    ADAM family members in cancer. ADAM12 is expressed in carcinoma and promotes breast cancer progression by inducing the apoptosis of surrounding...stromal cells (13). Consequently, ADAM12 protein levels correlate with advanced breast cancer (14, 15). In contrast, the disintegrin domain of ADAM15...subgroup of ADAMs that also contains ADAM12 and ADAM15 (18). The ADAM9 metalloprotease activity cleaves heparin-binding epidermal growth factor (HB

  10. ADAM17/EGFR axis promotes transglutaminase-dependent skin barrier formation through phosholipase C γ1 and protein kinase C pathways

    PubMed Central

    Wolf , Cristina; Qian, Yawen; Brooke, Matthew A.; Kelsell, David P.; Franzke, Claus-Werner

    2016-01-01

    The vitally important skin barrier is formed by extensive cross-linking activity of transglutaminases (TGs) during terminal epidermal differentiation. We have previously shown that epidermal deficiency of a disintegrin and metalloproteinase 17 (ADAM17), the principal EGFR ligand sheddase, results in postnatal skin barrier defects in mice due to impeded TG activity. However, the mechanism by which ADAM17/EGFR signalling maintains TG activity during epidermal differentiation remains elusive. Here we demonstrate that ADAM17-dependent EGFR signalling promotes TG activity in keratinocytes committed to terminal differentiation by direct induction of TG1 expression. Restored TG1 expression of EGF-stimulated differentiated Adam17−/− keratinocytes was strongly repressed by inhibitors for PLCγ1 or protein kinase C (PKC) pathways, while treatment with the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate restored TG activity in the epidermis of keratinocyte-specific Adam17−/− (AD17ΔKC) mice. Further investigations emphasized the expression of PKCη, a mediator of TGM1 transcription, to be sensitive to EGFR activation. In agreement, topical skin application of cholesterol sulfate, an activator of PKCη, significantly improved TG activity in epidermis of AD17ΔKC mice. Our results suggest ADAM17/EGFR-driven PLCγ1 and PKC pathways as important promoters of TG1 expression during terminal keratinocyte differentiation. These findings may help to identify new therapeutic targets for inflammatory skin diseases related to epidermal barrier defects. PMID:28004780

  11. Amyloid β-sheet mimics that antagonize protein aggregation and reduce amyloid toxicity

    NASA Astrophysics Data System (ADS)

    Cheng, Pin-Nan; Liu, Cong; Zhao, Minglei; Eisenberg, David; Nowick, James S.

    2012-11-01

    The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of β-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the β-structures presents a challenge to developing a model system of β-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust β-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid β-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-β peptide associated with Alzheimer's disease, β2-microglobulin associated with dialysis-related amyloidosis, α-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.

  12. Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly.

    PubMed

    Sampath, Srihari C; Marazzi, Ivan; Yap, Kyoko L; Sampath, Srinath C; Krutchinsky, Andrew N; Mecklenbräuker, Ingrid; Viale, Agnes; Rudensky, Eugene; Zhou, Ming-Ming; Chait, Brian T; Tarakhovsky, Alexander

    2007-08-17

    Epigenetic gene silencing in eukaryotes is regulated in part by lysine methylation of the core histone proteins. While histone lysine methylation is known to control gene expression through the recruitment of modification-specific effector proteins, it remains unknown whether nonhistone chromatin proteins are targets for similar modification-recognition systems. Here we show that the histone H3 methyltransferase G9a contains a conserved methylation motif with marked sequence similarity to H3 itself. As with methylation of H3 lysine 9, autocatalytic G9a methylation is necessary and sufficient to mediate in vivo interaction with the epigenetic regulator heterochromatin protein 1 (HP1), and this methyl-dependent interaction can be reversed by adjacent G9a phosphorylation. NMR analysis indicates that the HP1 chromodomain recognizes methyl-G9a through a binding mode similar to that used in recognition of methyl-H3K9, demonstrating that the chromodomain functions as a generalized methyl-lysine binding module. These data reveal histone-like modification cassettes - or "histone mimics" - as a distinct class of nonhistone methylation targets and directly extend the principles of the histone code to the regulation of nonhistone proteins.

  13. MD-simulations of Beta-Amyloid Protein Insertion Efficiency and Kinetics into Neuronal Membrane Mimics

    NASA Astrophysics Data System (ADS)

    Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

    2011-03-01

    Early interaction events of beta-amyloid (A β) peptides with the neuronal membranes play a key role in the pathogenesis of Alzheimer's disease. We have used all-atom MD simulations to study the protein insertion efficiency and kinetics of monomeric A β40 and A β42 into phosphatidylcholine lipid bilayers (PC) with and without 40 mole% cholesterol (CHOL) that mimic the cholesterol-enriched and depleted lipid nanodomains of the neuronal plasma membranes. Independent replicates of 200-ns simulations of each protein pre-inserted in the upper lipid layer were generated. In PC bilayers, only 25% of A β40 and 50% of A β42 in the replicates showed complete insertion into the lower lipid layer, whereas the percentages increased to 50% and 100%, respectively, in PC/CHOL bilayers, providing evidence that cholesterol improves the protein insertion efficiency into the bilayers. The rate of protein insertion was proportional to the hydrophobic, transmembrane helix length of the inserted peptide and depended on the cholesterol content. We propose that the lysine snorkeling and C-terminus anchoring of A β to the PC headgroups at the upper and lower lipid/water interfaces represent the dual-transmembrane stabilization mechanisms of A β in the neuronal membrane domains.

  14. ROMP- and RAFT-Based Guanidinium-Containing Polymers as Scaffolds for Protein Mimic Synthesis.

    PubMed

    Sarapas, Joel M; Backlund, Coralie M; deRonde, Brittany M; Minter, Lisa M; Tew, Gregory N

    2017-05-17

    Cell-penetrating peptides are an important class of molecules with promising applications in bioactive cargo delivery. A diverse series of guanidinium-containing polymeric cell-penetrating peptide mimics (CPPMs) with varying backbone chemistries was synthesized and assessed for delivery of both GFP and fluorescently tagged siRNA. Specifically, we examined CPPMs based on norbornene, methacrylate, and styrene backbones to determine how backbone structure impacted internalization of these two cargoes. Either charge content or degree of polymerization was held constant at 20, with diguanidinium norbornene molecules being polymerized to both 10 and 20 repeat units. Generally, homopolymer CPPMs delivered low amounts of siRNA into Jurkat T cells, with no apparent backbone dependence; however, by adding a short hydrophobic methyl methacrylate block to the guanidinium-rich methacrylate polymer, siRNA delivery to nearly the entire cell population was achieved. Protein internalization yielded similar results for most of the CPPMs, though the block polymer was unable to deliver proteins. In contrast, the styrene-based CPPM yielded the highest internalization for GFP (≈40 % of cells affected), showing that indeed backbone chemistry impacts protein delivery, specifically through the incorporation of an aromatic group. These results demonstrate that an understanding of how polymer structure affects cargo-dependent internalization is critical to designing new, more effective CPPMs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Breast cancer-associated mutations in metalloprotease disintegrin ADAM12 interfere with the intracellular trafficking and processing of the protein.

    PubMed

    Dyczynska, Emilia; Syta, Emilia; Sun, Danqiong; Zolkiewska, Anna

    2008-06-01

    ADAM12 has recently emerged as a Candidate Cancer Gene in a comprehensive genetic analysis of human breast cancers. Three somatic mutations in ADAM12 were observed at significant frequencies in breast cancers: D301H, G479E and L792F. The first 2 of these mutations involve highly conserved residues in ADAM12, and our computational sequence analysis confirms that they may be cancer-related. We show that the corresponding mutations in mouse ADAM12 inhibit the proteolytic processing and activation of ADAM12 in NIH3T3, COS-7, CHO-K1 cells and in MCF-7 breast cancer cells. The D/H and G/E ADAM12 mutants exert a dominant-negative effect on the processing of the wild-type ADAM12. Immunofluorescence analysis and cell surface biotinylation experiments demonstrate that the D/H and G/E mutants are retained inside the cell and are not transported to the cell surface. Consequently, the D/H and G/E mutants, unlike the wild-type ADAM12, are not capable of shedding Delta-like l, a ligand for Notch receptor, at the cell surface, or of stimulating cell migration. Our results suggest that the breast cancer-associated mutations interfere with the intracellular trafficking of ADAM12 and result in loss of the functional ADAM12 at the cell surface.

  16. Single honeybee silk protein mimics properties of multi-protein silk.

    PubMed

    Sutherland, Tara D; Church, Jeffrey S; Hu, Xiao; Huson, Mickey G; Kaplan, David L; Weisman, Sarah

    2011-02-02

    Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk.

  17. Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and γ-Secretase

    PubMed Central

    Hogl, Sebastian; Kuhn, Peer-Hendrik; Colombo, Alessio; Lichtenthaler, Stefan F.

    2011-01-01

    Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities α-, β- and γ-secretase controls the generation of the neurotoxic amyloid β peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid β domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the β-secretase BACE1 and additionally by an α-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 α-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive α-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 α-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. γ-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development. PMID:21695060

  18. Expanding The Scope Of Oligo-pyrrolinone-pyrrolidines As Protein-protein Interface Mimics

    PubMed Central

    Raghuraman, Arjun; Xin, Dongyue; Perez, Lisa M.; Burgess, Kevin

    2013-01-01

    Oligo-pyrrolinone-pyrrolidines (generic structure 1) have the potential to interfere with protein-protein interactions (PPIs), but to reduce this to practice it is necessary to be able to synthesize these structures with a variety of different side-chains corresponding to genetically encoded proteins. This paper describes expansion of the synthetic scope of 1, the difficulties encountered in this process, particularly issues with epimerization and slow coupling rates, and methods to overcome them. Finally, spectroscopic and physicochemical properties as well as proteolytic stabilities of molecules in this series were measured; these data highlight the suitability of oligo-pyrrolinone-pyrrolidines for the development of pharmacological probes or pharmaceutical leads. PMID:23654284

  19. Identification of ADAM10 and ADAM17 with potential roles in the spermatogenesis of the Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Li, Qing; Xie, Jing; He, Lin; Wang, Yuanli; Duan, Zelin; Yang, Hongdan; Wang, Qun

    2015-05-10

    The ADAM (a disintegrin and metalloprotease) family plays an important role in sperm and egg fusion, development, inflammation, adhesion and migration. ADAM10 and ADAM17 are involved in the spermatogenesis. To better understand the role of ADAM10 and ADAM17 in the Chinese mitten crab, Eriocheir sinensis, the full-length cDNAs of ADAM10 and ADAM17 were cloned, and named Es-ADAM10 and Es-ADAM17, respectively. Sequence and structural analysis showed that Es-ADAM10 and Es-ADAM17 have the typical structure of the ADAM family. Quantitative real-time reverse transcription polymerase chain reaction analysis showed that Es-ADAM10 and Es-ADAM17 mRNAs were distributed in the heart, hepatopancreas, intestines, brain, muscle, thoracic ganglia, hemolymph, stomach, testis, ovary, gill and accessory gland. Both mRNAs were highly expressed in the muscles, and relatively high in the testis, ovary and accessory gland. In addition, the Es-ADAM17 mRNA level was detected in every stage of testis development, being relatively high from July to September, the lowest during October and November, increasing from December to January, and reached a peak in January. By contrast, the expression of Es-ADAM10 mRNA was constant during testis development. Immunofluorescence further showed that Es-ADAM10 and Es-ADAM17 proteins were present in the cytoplasm and cytomembrane of spermatocytes, and both detected in the sperm. Furthermore, etoposide induced upregulation of Es-ADAM17 and Es-ADAM10 at both the mRNA and protein levels. This study first showed that Es-ADAM10 and Es-ADAM17 were also involved in the spermatogenesis and mainly participated in the later germ cell apoptosis in E. sinensis.

  20. Molecular recognition of oxygen by protein mimics: dynamics on the femtosecond to microsecond time scale.

    PubMed

    Zou, Shouzhong; Baskin, J Spencer; Zewail, Ahmed H

    2002-07-23

    Molecular recognition by biological macromolecules involves many elementary steps, usually convoluted by diffusion processes. Here we report studies of the dynamics, from the femtosecond to the microsecond time scale, of the different elementary processes involved in the bimolecular recognition of a protein mimic, cobalt picket-fence porphyrin, with varying oxygen concentration at controlled temperatures. Electron transfer, bond breakage, and thermal "on" (recombination) and "off" (dissociation) reactions are the different processes involved. The reaction on-rate is 30 to 60 times smaller than that calculated from standard Smoluchowski theory. Introducing a two-step recognition model, with reversibility being part of both steps, removes the discrepancy and provides consistency for the reported thermodynamics, kinetics, and dynamics. The transient intermediates are configurations defined by the contact between oxygen (diatomic) and the picket-fence porphyrin (macromolecule). This intermediate is critical in the description of the potential energy landscape but, as shown here, both enthalpic and entropic contributions to the free energy are important. In the recognition process, the net entropy decrease is -33 cal mol(-1) K(-1); Delta H is -13.4 kcal mol(-1).

  1. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    PubMed

    Hausammann, Stefanie; Vogel, Monique; Kremer Hovinga, Johanna A; Lacroix-Desmazes, Sebastien; Stadler, Beda M; Horn, Michael P

    2013-01-01

    Inhibitory antibodies directed against coagulation factor VIII (FVIII) can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins) mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  2. Differential Surface Expression of ADAM10 and ADAM17 on Human T Lymphocytes and Tumor Cells

    PubMed Central

    Kabelitz, Dieter; Janssen, Ottmar

    2013-01-01

    A disintegrin and metalloproteases (ADAMs) have been implicated in many processes controlling organismic development and integrity. Important substrates of ADAM proteases include growth factors, cytokines and their receptors and adhesion proteins. The inducible but irreversible cleavage of their substrates alters cell-cell communication and signaling. The crucial role of ADAM proteases (e.g. ADAM10 and 17) for mammalian development became evident from respective knockout mice, that displayed pre- or perinatal lethality with severe defects in many organs and tissues. Although many substrates for these two ADAM proteases were identified over the last decade, the regulation of their surface appearance, their enzymatic activity and their substrate specificity are still not well understood. We therefore analyzed the constitutive and inducible surface expression of ADAM10 and ADAM17 on a variety of human T cell and tumor cell lines. We demonstrate that ADAM10 is constitutively present at comparably high levels on the majority of the tested cell types. Stimulation with phorbol ester and calcium ionophore does not significantly alter the amount of surface ADAM10, except for a slight down-regulation from T cell blasts. Using FasL shedding as a readout for ADAM10 activity, we show that PKC activation and calcium mobilization are both prerequisite for activation of ADAM10 resulting in a production of soluble FasL. In contrast to ADAM10, the close relative ADAM17 is detected at only low levels on unstimulated cells. ADAM17 surface expression on T cell blasts is rapidly induced by stimulation. Since this inducible mobilization of ADAM17 is sensitive to inhibitors of actin filament formation, we propose that ADAM17 but not ADAM10 is prestored in a subcellular compartment that is transported to the cell surface in an activation- and actin-dependent manner. PMID:24130797

  3. Increased expression of ADAM12 and ADAM17 genes in laser-capture microdissected breast cancers and correlations with clinical and pathological characteristics.

    PubMed

    Narita, Diana; Seclaman, Edward; Ursoniu, Sorin; Anghel, Andrei

    2012-02-01

    ADAMs (a desintegrin and metalloprotease) are transmembrane glycoproteins involved in cell growth, differentiation, motility, and respectively, tumor growth and progression. Our aim was to evaluate ADAM12 spliced variants (ADAM12L - long membrane-bound and ADAM12S - secreted-short variant) and ADAM17 genes expression in breast cancers and to correlate their level of expression with clinical and pathological characteristics. Expression of ADAMs was analyzed using quantitative reverse-transcription polymerase chain reaction in laser-capture microdissected specimens of breast cancers and corresponding non-neoplastic breast tissues from 92 patients. The proteins' expression was confirmed by immunohistochemistry. Significantly elevated amounts of ADAM12L, ADAM12S and ADAM17 transcripts were found in malignant breast cells compared with normal breast tissue and both ADAMs proteins showed moderate to strong immunoexpression in tumor cells and peritumoral fibroblasts. ADAM12L and ADAM12S expressions were correlated with age, younger patients having higher expression of ADAM12L and ADAM12S; ductal cancers had higher expression of ADAM12L compared with lobular types, whereas ADAM12S was higher expressed in lobular cancers; higher expressions were found for both ADAM12 and ADAM17 in HER2/neu positive and highly proliferative cancers. High-grade cancers showed significantly increased expression of ADAM17. Our study on laser-capture microdissected specimens confers motivation for future work on development of ADAM-selective inhibitors for treatment of breast cancers.

  4. Lysine pyrrolation is a naturally-occurring covalent modification involved in the production of DNA mimic proteins.

    PubMed

    Miyashita, Hiroaki; Chikazawa, Miho; Otaki, Natsuki; Hioki, Yusuke; Shimozu, Yuki; Nakashima, Fumie; Shibata, Takahiro; Hagihara, Yoshihisa; Maruyama, Shoichi; Matsumi, Noriyoshi; Uchida, Koji

    2014-06-18

    Covalent modification of proteins exerts significant effects on their chemical properties and has important functional and regulatory consequences. We now report the identification and verification of an electrically-active form of modified proteins recognized by a group of small molecules commonly used to interact with DNA. This previously unreported property of proteins was initially discovered when the γ-ketoaldehydes were identified as a source of the proteins stained by the DNA intercalators. Using 1,4-butanedial, the simplest γ-ketoaldehyde, we characterized the structural and chemical criteria governing the recognition of the modified proteins by the DNA intercalators and identified N(ε)-pyrrolelysine as a key adduct. Unexpectedly, the pyrrolation conferred an electronegativity and electronic properties on the proteins that potentially constitute an electrical mimic to the DNA. In addition, we found that the pyrrolated proteins indeed triggered an autoimmune response and that the production of specific antibodies against the pyrrolated proteins was accelerated in human systemic lupus erythematosus. These findings and the apparent high abundance of N(ε)-pyrrolelysine in vivo suggest that protein pyrrolation could be an endogenous source of DNA mimic proteins, providing a possible link connecting protein turnover and immune disorders.

  5. ERK1/2 Mediate Wounding- and G-protein-Coupled Receptor Ligands–Induced EGFR Activation via Regulating ADAM17 and HB-EGF Shedding

    PubMed Central

    Yin, Jia; Yu, Fu-Shin X.

    2013-01-01

    Purpose Previous studies have shown that wounding of human corneal epithelial cells (HCECs) results in the release of G-protein-coupled receptor ligands such as ATP and lysophosphatidic acid (LPA), which in turn transactivate epidermal growth factor (EGF) receptor (EGFR) through ectodomain shedding of heparin-binding EGF-like growth factor (HB-EGF). In the present study, the role of extracellular signal-regulated kinases 1/2 (ERK1/2) in regulating EGFR transactivation was investigated. Methods SV40-immortalized HCECs were wounded or stimulated with ATP and LPA. EGFR and ADAM17 activation was analyzed by immunoprecipitation followed by Western blot analysis with phospho-tyrosine or phospho-serine antibodies, respectively. Phosphorylation of ERK and AKT was analyzed by Western blot analysis. HB-EGF shedding was assessed by measuring the release of alkaline phosphatase (AP) in a stably transfected human corneal epithelial (THCE) cell line expressing HB-EGF-AP. ADAM17 and ERK interaction was determined by coimmunoprecipitation. Results Early, but not late, ERK1/2 phosphorylation in response to wounding, LPA, and ATP was EGFR independent, but sensitive to the inhibitors of calcium influx, protein kinase C and Src kinase. Wounding-, LPA-, and ATP-induced HB-EGF shedding and EGFR activation were attenuated by the MAPK/ERK kinase (MEK) inhibitors PD98059 and U0126, as well as by ADAM10 and -17 inhibitors. ADAM17 was found to be physically associated with active ERK and phosphorylated at serine residues in an ERK-dependent manner in wounded cells. Conclusions Taken together, our data suggest that in addition to functioning as an EGFR downstream effector, ERK1/2 also mediates ADAM-dependent HB-EGF shedding and subsequent EGFR transactivation in response to a variety of stimuli, including wounding and GPCR ligands. PMID:18658095

  6. Molecular profiling of ADAM12 and ADAM17 genes in human malignant melanoma.

    PubMed

    Cireap, Natalia; Narita, Diana

    2013-10-01

    ADAM12 and ADAM17 proteins belong to a family of transmembrane disintegrin-containing metalloproteinases (ADAMs) involved in the proteins ectodomain shedding and cell-cell and cell-matrix interactions. However, the specific biological functions of ADAMs are still unclear and, until now, these proteins were not investigated yet in melanoma. The aim of this study was to analyze the splicing variants of ADAM12 (L and S) and ADAM17 gene expression in melanoma at transcriptional and translational level in comparison with control (non-tumor) tissues. Taking in account that ADAM17 sheddase is involved in the modulation of TNF-α (tumor necrosis factor alpha), we analyzed also this cytokine in the plasma of the same patients before any treatment, and we compared the results with healthy controls. Quantitative-RT-PCR and immunohistochemistry were used to analyze ADAM12 and ADAM17 genes expression and the analysis of TNF-α expression was carried out in the plasma using ELISA. We demonstrated that ADAM12L splicing variant together with ADAM17 gene are strongly overexpressed in melanomas, whereas ADAM12S, although up-regulated when compared with the non-tumor controls, the difference was not statistically significant. When we compared the levels of expression for the ADAMs genes according to the tumor stage, we observed that all three investigated genes were significantly overexpressed in advanced stage in comparison with early stage melanomas. In the plasma of the same patients, the expression of TNF-α was up-regulated and significantly correlated with the expression of ADAM17 and respectively, with the advanced tumor stage.

  7. An amphiphilic selenide catalyst behaves like a hybrid mimic of protein disulfide isomerase and glutathione peroxidase 7.

    PubMed

    Arai, Kenta; Moriai, Kenji; Ogawa, Akinobu; Iwaoka, Michio

    2014-12-01

    Protein disulfide isomerase (PDI) and glutathione peroxidase 7 (GPx7) cooperatively promote the oxidative folding of disulfide (SS)-containing proteins in endoplasmic reticulum by recognizing the nascent proteins to convert them into the native folds by means of SS formation and SS isomerization and by catalyzing reoxidation of reduced PDI with H2O2, respectively. In this study, new amphiphilic selenides with a long-chain alkyl group were designed as hybrid mimics of PDI and GPx7 and were applied to the refolding of reduced hen egg-white lysozyme (HEL-R). Competitive SS formation at pH 4 using HEL-R and glutathione (GSH) in the presence of the selenide catalyst and H2O2 showed that the amphiphilic selenides can preferentially catalyze SS formation of HEL-R, probably on account of hydrophobic interactions between the protein and the catalyst. In contrast, simple water-soluble selenides did not exhibit such behavior. In addition, when the pH of the solution was adjusted to 8.5 after the SS formation, surviving GSH promoted the SS isomerization of misfolded HEL to recover the native SS linkages. Thus, the amphiphilic selenides designed here could mimic the function of the PDI-GPx7 system. The combination of a water-soluble selenide and a long-chain alkyl group would be a useful motif in designing medicines for both protein misfolding diseases and antioxidant therapy.

  8. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGES

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  9. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  10. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

    PubMed

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Gutierrez, Manuel Cantu; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen J T; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-03-31

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  11. Simulation of carbohydrate-protein interactions: Computer-aided design of a second generation GM1 mimic

    NASA Astrophysics Data System (ADS)

    Bernardi, Anna; Galgano, Marta; Belvisi, Laura; Colombo, Giorgio

    2001-02-01

    The oligosaccharide of ganglioside GM1 [Gal β1-3GalNAc β1-4(NeuAc α2-3)Gal β1-4Glc β1-1Cer] is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E.coli (LT). We recently reported that the pseudosaccharide 2[Gal β1-3GalNAc β1-4(NeuAc α2-3)DCCHD] is a high-affinity ligand for CT, and thus a functional mimic of GM1 (Bernardi, A., Checchia, A., Brocca, P., Sonnino, S. and Zuccotto, F., J. Am. Chem. Soc., 121 (1999) 2032-2036). In this paper we describe the design of a second-generation mimic, formally obtained from 2 by inverting the configuration of a single stereocenter, thus transforming a N-acetyl galactosamine into a N-acetyl glucosamine. The design process involved modeling of the free ligand and its LT complex, followed by qualitative and quantitative comparison with the corresponding structures of 2. The protocol employed relied on both conformational search and molecular dynamics methodologies to account for the flexibility of both the ligand and the protein receptor. The conformational search of the LT:inhibitor complex showed that, compared to 2, the new compound can insert one more hydroxy group within the protein binding site. Molecular dynamics simulations showed that, in turn, this may trigger a series of rearrangements and reorientations of side chains and crystallographic water molecules in the toxin, leading to new H-bond contacts which may result in enhanced affinity of the new inhibitor. FEP calculations were performed by mutating the structure of 2 in solution and in the protein complex, and the prediction was made that the second-generation mimic should be a stronger binder than its parent compound.

  12. Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

    PubMed Central

    Baños-Sanz, José Ignacio; Mojardín, Laura; Sanz-Aparicio, Julia; Lázaro, José M.; Villar, Laurentino; Serrano-Heras, Gemma; González, Beatriz; Salas, Margarita

    2013-01-01

    Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme. PMID:23671337

  13. Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage Φ29 DNA mimic protein p56.

    PubMed

    Baños-Sanz, José Ignacio; Mojardín, Laura; Sanz-Aparicio, Julia; Lázaro, José M; Villar, Laurentino; Serrano-Heras, Gemma; González, Beatriz; Salas, Margarita

    2013-07-01

    Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage 29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

  14. Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes.

    PubMed

    Lee, Hee Doo; Koo, Bon-Hun; Kim, Yeon Hyang; Jeon, Ok-Hee; Kim, Doo-Sik

    2012-07-01

    A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.

  15. LRP1 shedding in human brain: roles of ADAM10 and ADAM17.

    PubMed

    Liu, Qiang; Zhang, Juan; Tran, Hien; Verbeek, Marcel M; Reiss, Karina; Estus, Steven; Bu, Guojun

    2009-04-16

    The low-density lipoprotein receptor-related protein 1 (LRP1) plays critical roles in lipid metabolism, cell survival, and the clearance of amyloid-beta (Abeta) peptide. Functional soluble LRP1 (sLRP1) has been detected in circulating human placenta; however, whether sLRP1 is also present in the central nervous system is unclear. Here we show that abundant sLRP1 capable of binding its ligands is present in human brain tissue and cerebral spinal fluid (CSF). Interestingly, the levels of sLRP1 in CSF are significantly increased in older individuals, suggesting that either LRP1 shedding is increased or sLRP1 clearance is decreased during aging. To examine potential effects of pathological ligands on LRP1 shedding, we treated MEF cells with Abeta peptide and found that LRP1 shedding was increased. ADAM10 and ADAM17 are key members of the ADAM family that process membrane-associated proteins including amyloid precursor protein and Notch. We found that LRP1 shedding was significantly decreased in MEF cells lacking ADAM10 and/or ADAM17. Furthermore, forced expression of ADAM10 increased LRP1 shedding, which was inhibited by ADAM-specific inhibitor TIMP-3. Our results demonstrate that LRP1 is shed by ADAM10 and ADAM17 and functional sLRP1 is abundantly present in human brain and CSF. Dysregulated LRP1 shedding during aging could alter its function and may contribute to the pathogenesis of AD.

  16. Inhibition of Cellular Protein Secretion by Norwalk Virus Nonstructural Protein p22 Requires a Mimic of an Endoplasmic Reticulum Export Signal

    PubMed Central

    Sharp, Tyler M.; Guix, Susana; Katayama, Kazuhiko; Crawford, Sue E.; Estes, Mary K.

    2010-01-01

    Protein trafficking between the endoplasmic reticulum (ER) and Golgi apparatus is central to cellular homeostasis. ER export signals are utilized by a subset of proteins to rapidly exit the ER by direct uptake into COPII vesicles for transport to the Golgi. Norwalk virus nonstructural protein p22 contains a YXΦESDG motif that mimics a di-acidic ER export signal in both sequence and function. However, unlike normal ER export signals, the ER export signal mimic of p22 is necessary for apparent inhibition of normal COPII vesicle trafficking, which leads to Golgi disassembly and antagonism of Golgi-dependent cellular protein secretion. This is the first reported function for p22. Disassembly of the Golgi apparatus was also observed in cells replicating Norwalk virus, which may contribute to pathogenesis by interfering with cellular processes that are dependent on an intact secretory pathway. These results indicate that the ER export signal mimic is critical to the antagonistic function of p22, shown herein to be a novel antagonist of ER/Golgi trafficking. This unique and well-conserved human norovirus motif is therefore an appealing target for antiviral drug development. PMID:20976190

  17. A minimalist chemical model of matrix metalloproteinases--can small peptides mimic the more rigid metal binding sites of proteins?

    PubMed

    Árus, Dávid; Nagy, Nóra Veronika; Dancs, Ágnes; Jancsó, Attila; Berkecz, Róbert; Gajda, Tamás

    2013-09-01

    In order to mimic the active center of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-visible, CD, EPR, NMR, mass spectrometric and kinetic studies are aimed to explore the usefulness of such flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute to the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since the multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH 6-7 the peptide, similarly to MMPs, offers a {3Nim} coordination binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH abolished the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH7 (ZnH2L) possesses a {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH7 and 9.

  18. A supersandwich electrochemiluminescence immunosensor based on mimic-intramolecular interaction for sensitive detection of proteins.

    PubMed

    He, Ying; Chai, Yaqin; Yuan, Ruo; Wang, Haijun; Bai, Lijuan; Liao, Ni

    2014-10-21

    An electrochemiluminescence (ECL) immunoassay protocol was developed based on mimic-intramolecular interaction for sensitive detection of prostate specific antigen (PSA). It was constructed by integrating the ECL luminophore (tris(4,4'-dicarboxylicacid-2,2'-bipyridyl)-ruthenium(ii)dichloride (Ru(dcbpy)3(2+))) and coreactant (histidine) into the supersandwich DNA structure. This strategy was more effective in amplifying the ECL signal by shortening the electronic transmission distance, improving the ECL luminous stability and enhancing the ECL luminous efficiency. The ECL matrices denoted as MWCNTs@PDA-AuNPs were fabricated through spontaneous oxidative polymerization of dopamine (DA) on multiwalled carbon nanotubes (MWCNTs) and reducing HAuCl4 to produce gold nanoparticles (AuNPs) by DA simultaneously. Then, the prepared matrices were applied to bind capture antibodies. Moreover, supersandwich Ab2 bioconjugate was designed using a PAMAM dendrimer to immobilize the detection antibody and supersandwich DNA structure. The PAMAM dendrimer, with a plurality of secondary and tertiary amine groups, not only facilitated high-density immobilization of the detection antibody and supersandwich DNA structure, but also greatly amplified the ECL signal of Ru(dcbpy)3(2+). The supersandwich DNA structure contained multiple Ru(dcbpy)3(2+) and histidine, further amplifying the ECL signal. The proposed supersandwich immunosensor showed high sensitivity with a detection limit of 4.2 fg mL(-1) and a wide linear range of 0.01 pg mL(-1)-40.00 ng mL(-1). With the excellent stability, satisfying precision and reproducibility, the proposed immunosensor indicates promising practicability for clinical diagnosis.

  19. Peptide mimics of the M13 coat protein transmembrane segment. Retention of helix-helix interaction motifs.

    PubMed

    Wang, C; Deber, C M

    2000-05-26

    Sequence-specific noncovalent helix-helix interactions between transmembrane (TM) segments in proteins are investigated by incorporating selected TM sequences into synthetic peptides using the construct CKKK-TM-KKK. The peptides are of suitable hydrophobicity for spontaneous membrane insertion, whereas formation of an N-terminal S-S bond can bring pairs of TM helices into proximity and promote their parallel orientation. Using the propensity of the protein to undergo thermally induced alpha-helix --> beta-sheet transitions as a parameter for helix stability, we compared the wild type and mutant (V29A and V31A) bacteriophage M13 coat proteins with their corresponding TM peptide constructs (M13 residues 24-42). Our results demonstrated that the relevant helix-helix tertiary contacts found in the intact proteins persist in the peptide mimics. Molecular dynamics simulations support the tight "two in-two out" dimerization motif for V31A consistent with mutagenesis data. The overall results reinforce the notion of TM segments as autonomous folding domains and suggest that the generic peptide construct provides a viable reductionist system for membrane protein structural and computational analysis.

  20. Myositis Mimics.

    PubMed

    Michelle, E Harlan; Mammen, Andrew L

    2015-10-01

    Patients with autoimmune myositis typically present with muscle weakness, elevated serum levels of muscle enzymes, and abnormal muscle biopsies. However, patients with other acquired myopathies or genetic muscle diseases may have remarkably similar presentations. Making the correct diagnosis of another muscle disease can prevent these patients from being exposed to the risks of immunosuppressive medications, which benefit those with myositis, but not those with other types of muscle disease. Here, we review some of the most common acquired and inherited muscle diseases that can mimic autoimmune myositis, including inclusion body myositis, limb girdle muscular dystrophies, metabolic myopathies, mitochondrial myopathies, and endocrine myopathies. We emphasize aspects of the medical history, physical exam, laboratory evaluation, and muscle biopsy analysis that can help clinicians distinguish myositis mimics from true autoimmune myositis.

  1. Samuel Adams' tremor.

    PubMed

    Louis, E D

    2001-05-08

    Samuel Adams (1722--1803), the American Revolutionist and brewer, is said to have had a tremor. There has not been a formal study of the clinical characteristics and progression of his tremor. To use historical accounts and original materials to document the clinical characteristics and progression of Adams' tremor. In addition to historical accounts of Adams, the following materials were reviewed: 1) Samuel Adams' known writings, including personal and political letters, published posthumously in a four-volume book; and 2) original letters and speeches (handwritten by Adams) that are held in collection in the Manuscripts and Archives Division of the New York Public Library. Adams had a tremor that affected his hands, head, and voice. Although mild, the tremor was already manifest when Adams was in his early 40s. Adams, who was very prolific, experienced progressive difficulty with his ability to write while in his 50s and early 60s. His letters contain frequent references to his illness and reveal a penmanship that became more compromised with time. By age 71, he was forced to dictate all his correspondence. His tremor was familial, affecting his daughter Hannah and her children. Samuel Adams' progressive tremor rendered him unable to write his own letters for the last 10 years of his life. Adams had one of the earliest documented cases of essential tremor.

  2. Approved drug mimics of short peptide ligands from protein interaction motifs.

    PubMed

    Parthasarathi, Laavanya; Casey, Fergal; Stein, Amelie; Aloy, Patrick; Shields, Denis C

    2008-10-01

    Most biological functions are regulated through complex networks of transient protein interactions, and, thus, finding effective ways to modulate them would represent an important step towards defining the next generation of drugs. In this study, we set out to determine if existing approved drugs may represent a good source of compounds from which initial lead inhibitors of protein-protein interactions mediated by short peptide regions may be drawn. Peptide structures were defined in terms of pharmacophores and searched against U.S. Food and Drug Administration (FDA)-approved drugs to identify similar compounds. The top ranking matches (using a score that corrects root-mean-square deviation (rmsd) for the number of matched pharmacophores and for the number of drug rotatable bonds) included a number of nuclear receptor ligands that matched allosterically to the corepressor binding site of peroxisome proliferator-activated receptor alpha (PPARalpha). The top ranking drug matches were docked to the peptide-binding site using AUTODOCK. The majority of the top-ranking matches showed a negative estimated free energy change upon binding that is comparable to, or greater than, that of the original peptide. We conclude that the usage of certain approved drugs may represent a useful strategy in inhibiting specific protein-protein interactions. Such a strategy may benefit from the increased likelihood that developed compounds might have favorable bioactivity and safety profiles in clinical use.

  3. Stimulation of non-amyloidogenic processing of amyloid-β protein precursor by cryptotanshinone involves activation and translocation of ADAM10 and PKC-α.

    PubMed

    Durairajan, Siva Sundara Kumar; Liu, Liang-Feng; Lu, Jia-Hong; Koo, Irene; Maruyama, Kei; Chung, Sookja K; Huang, Jian-Dong; Li, Min

    2011-01-01

    Cerebral deposition of amyloid-β peptide (Aβ) plaques is now considered the central feature of Alzheimer's disease. Recent studies suggest that cryptotanshinone (CTS) extracted from the root of Salvia miltiorrhiza Bunge could be used for the prevention and treatment of Alzheimer's disease. In this study, we investigated the role of CTS on non-amyloidogenic processing of amyloid-β protein precursor (AβPP) as well as its regulation by protein kinase C (PKC). Treatment with CTS dose-dependently and significantly reduced both intracellular and secreted levels of Aβ40 and Aβ42 in N2a mouse neuroblastoma cells stably expressing human SwedishAβPP (N2a-SwedAβPP). Using N2a-SwedAβPP and human neuroblastoma SHSY5Y cells, it was demonstrated that CTS significantly and dose-dependently increased the production of sAβPPα and C-terminal fragment-α (CTF-α) from AβPP. At the same time, CTS specifically increased the maturation of "a disintegrin and metalloproteinase-10" (ADAM10), an α-secretase candidate. The increase of sAβPPα secretion by CTS was blocked by the hydroxamate-based inhibitors GI254023X and GW280264X, and by the PKC-α inhibitor GÖ6976, suggesting involvement of the ADAM10 and PKC-α in CTS-induced α-secretase cleavage. In other experiments, CTS induced the phosphorylation of PKC-α indicating that PKC-α is involved in CTS-induced sAβPPα secretion. Furthermore, treatment of neuroblastoma cells with CTS induced the co-translocation of ADAM10 and PKC-α to the cell membrane, the site at which AβPP was cleaved, and this translocation was significantly reduced by GÖ6976. These results suggest that CTS-induced sAβPPα secretion is regulated by a PKC-α and ADAM10 cascade in neuroblastoma cells and may be involved in the lowering of Aβ production.

  4. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    PubMed Central

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  5. Investigation of the catalytic mechanism of a synthetic DNAzyme with protein-like functionality: an RNaseA mimic?

    PubMed

    Thomas, Jason M; Yoon, Jung-Ki; Perrin, David M

    2009-04-22

    The protein enzyme ribonuclease A (RNaseA) cleaves RNA with catalytic perfection, although with little sequence specificity, by a divalent metal ion (M(2+))-independent mechanism in which a pair of imidazoles provides general acid and base catalysis, while a cationic amine provides electrostatic stabilization of the transition state. Synthetic imitation of this remarkable organo-catalyst ("RNaseA mimicry") has been a longstanding goal in biomimetic chemistry. The 9(25)-11 DNAzyme contains synthetically modified nucleotides presenting both imidazole and cationic amine side chains, and catalyzes RNA cleavage with turnover in the absence of M(2+) similarly to RNaseA. Nevertheless, the catalytic roles, if any, of the "protein-like" functional groups have not been defined, and hence the question remains whether 9(25)-11 engages any of these functionalities to mimic aspects of the mechanism of RNaseA. To address this question, we report a mechanistic investigation of 9(25)-11 catalysis wherein we have employed a variety of experiments, such as DNAzyme functional group deletion, mechanism-based affinity labeling, and bridging and nonbridging phosphorothioate substitution of the scissile phosphate. Several striking parallels exist between the results presented here for 9(25)-11 and the results of analogous experiments applied previously to RNaseA. Specifically, our results implicate two particular imidazoles in general acid and base catalysis and suggest that a specific cationic amine stabilizes the transition state via diastereoselective interaction with the scissile phosphate. Overall, 9(25)-11 appears to meet the minimal criteria of an RNaseA mimic; this demonstrates how added synthetic functionality can expand the mechanistic repertoire available to a synthetic DNA-based catalyst.

  6. John Adams' essential tremor.

    PubMed

    Louis, Elan D; Kavanagh, Patricia

    2005-12-01

    John Adams (1735-1826), one of the signers of the Declaration of Independence, was the second President of the United States. Adams had tremor for many years, about which little has been written. We examined John Adams' penmanship over a 62-year period and studied his correspondence and diaries. It is not clear when Adams' tremor began, although in a diary entry dated 6 December 1760, when Adams was 25 years old, there is evidence of low-amplitude kinetic tremor. The tremor continued in his written correspondence, becoming more persistent over time. Later in life, the clarity of his written correspondence diminished, with greater decomposition of characters and a reduction in the size of individual characters. This finding raises some speculation as to whether Adams could have been developing some parkinsonism, although the evidence in favor of this is not compelling. The most likely diagnosis was essential tremor.

  7. A Disintegrin and Metalloproteinase10 (ADAM10) Regulates NOTCH Signaling during Early Retinal Development.

    PubMed

    Toonen, Joseph A; Ronchetti, Adam; Sidjanin, D J

    2016-01-01

    ADAM10 and ADAM17 are two closely related members of the ADAM (a disintegrin and metalloprotease) family of membrane-bound sheddases, which proteolytically cleave surface membrane proteins. Both ADAM10 and ADAM17 have been implicated in the proteolytic cleavage of NOTCH receptors and as such regulators of NOTCH signaling. During retinal development, NOTCH signaling facilitates retinal neurogenesis by maintaining progenitor cells in a proliferative state and by mediating retinal cell fates. However, the roles of ADAM10 and ADAM17 in the retina are not well defined. In this study, we set out to clarify the roles of ADAM10 and ADAM17 during early retinal development. The retinal phenotype of conditionally abated Adam17 retinae (Adam17 CKO) did not differ from the controls whereas conditionally ablated Adam10 retinae (Adam10 CKO) exhibited abnormal morphogenesis characterized by the formation of rosettes and a loss of retinal laminae phenotypically similar to morphological abnormalities identified in mice with retinal NOTCH signaling deficiency. Additionally, Adam10 CKO retinae exhibited abnormal neurogenesis characterized by fewer proliferating progenitor cells and greater differentiation of early photoreceptors and retinal ganglion cells. Moreover, constitutive activation of the NOTCH1-intracellular domain (N1-ICD) rescued Adam10 CKO abnormal neurogenesis, as well as abnormal retinal morphology by maintaining retinal cells in the progenitor state. Collectively these findings provide in vivo genetic evidence that ADAM10, and not ADAM17, is indispensable for proper retinal development as a regulator of NOTCH signaling.

  8. A Disintegrin and Metalloproteinase10 (ADAM10) Regulates NOTCH Signaling during Early Retinal Development

    PubMed Central

    Toonen, Joseph A.; Ronchetti, Adam; Sidjanin, D. J.

    2016-01-01

    ADAM10 and ADAM17 are two closely related members of the ADAM (a disintegrin and metalloprotease) family of membrane-bound sheddases, which proteolytically cleave surface membrane proteins. Both ADAM10 and ADAM17 have been implicated in the proteolytic cleavage of NOTCH receptors and as such regulators of NOTCH signaling. During retinal development, NOTCH signaling facilitates retinal neurogenesis by maintaining progenitor cells in a proliferative state and by mediating retinal cell fates. However, the roles of ADAM10 and ADAM17 in the retina are not well defined. In this study, we set out to clarify the roles of ADAM10 and ADAM17 during early retinal development. The retinal phenotype of conditionally abated Adam17 retinae (Adam17 CKO) did not differ from the controls whereas conditionally ablated Adam10 retinae (Adam10 CKO) exhibited abnormal morphogenesis characterized by the formation of rosettes and a loss of retinal laminae phenotypically similar to morphological abnormalities identified in mice with retinal NOTCH signaling deficiency. Additionally, Adam10 CKO retinae exhibited abnormal neurogenesis characterized by fewer proliferating progenitor cells and greater differentiation of early photoreceptors and retinal ganglion cells. Moreover, constitutive activation of the NOTCH1-intracellular domain (N1-ICD) rescued Adam10 CKO abnormal neurogenesis, as well as abnormal retinal morphology by maintaining retinal cells in the progenitor state. Collectively these findings provide in vivo genetic evidence that ADAM10, and not ADAM17, is indispensable for proper retinal development as a regulator of NOTCH signaling. PMID:27224017

  9. Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells

    PubMed Central

    Hollenhorst, Peter C.; Ferris, Mary W.; Hull, Megan A.; Chae, Heejoon; Kim, Sun; Graves, Barbara J.

    2011-01-01

    The aberrant expression of an oncogenic ETS transcription factor is implicated in the progression of the majority of prostate cancers, 40% of melanomas, and most cases of gastrointestinal stromal tumor and Ewing's sarcoma. Chromosomal rearrangements in prostate cancer result in overexpression of any one of four ETS transcription factors. How these four oncogenic ETS genes differ from the numerous other ETS genes expressed in normal prostate and contribute to tumor progression is not understood. We report that these oncogenic ETS proteins, but not other ETS factors, enhance prostate cell migration. Genome-wide binding analysis matched this specific biological function to occupancy of a unique set of genomic sites highlighted by the presence of ETS- and AP-1-binding sequences. ETS/AP-1-binding sequences are prototypical RAS-responsive elements, but oncogenic ETS proteins activated a RAS/MAPK transcriptional program in the absence of MAPK activation. Thus, overexpression of oncogenic ETS proteins can replace RAS/MAPK pathway activation in prostate cells. The genomic description of this ETS/AP-1-regulated, RAS-responsive, gene expression program provides a resource for understanding the role of these ETS factors in both an oncogenic setting and the developmental processes where these genes normally function. PMID:22012618

  10. The ADAMS interactive interpreter

    SciTech Connect

    Rietscha, E.R.

    1990-12-17

    The ADAMS (Advanced DAta Management System) project is exploring next generation database technology. Database management does not follow the usual programming paradigm. Instead, the database dictionary provides an additional name space environment that should be interactively created and tested before writing application code. This document describes the implementation and operation of the ADAMS Interpreter, an interactive interface to the ADAMS data dictionary and runtime system. The Interpreter executes individual statements of the ADAMS Interface Language, providing a fast, interactive mechanism to define and access persistent databases. 5 refs.

  11. ADAM15 expression is downregulated in melanoma metastasis compared to primary melanoma

    SciTech Connect

    Ungerer, Christopher; Doberstein, Kai; Boehm, Beate; Pfeilschifter, Josef; Mihic-Probst, Daniela; Gutwein, Paul

    2010-10-22

    Research highlights: {yields} Strong ADAM15 expression is found in normal melanocytes. {yields} ADAM15 expression is significantly downregulated in patients with melanoma metastasis. {yields} TGF-{beta} can downregulate ADAM15 expression in melanoma cells. {yields} Overexpression of ADAM15 in melanoma cells inhibits migration, proliferation and invasion of melanoma cells. {yields} Conclusion: ADAM15 represents an tumor suppressor protein in melanoma. -- Abstract: In a mouse melanoma metastasis model it has been recently shown that ADAM15 overexpression in melanoma cells significantly reduced the number of metastatic nodules on the lung. Unfortunately, the expression of ADAM15 in human melanoma tissue has not been determined so far. In our study, we characterized the expression of ADAM15 in tissue micro-arrays of patients with primary melanoma with melanoma metastasis. ADAM15 was expressed in melanocytes and endothelial cells of benign nevi and melanoma tissue. Importantly, ADAM15 was significantly downregulated in melanoma metastasis compared to primary melanoma. We further demonstrate that IFN-{gamma} and TGF-{beta} downregulate ADAM15 protein levels in melanoma cells. To investigate the role of ADAM15 in melanoma progression, we overexpressed ADAM15 in melanoma cells. Importantly, overexpression of ADAM15 in melanoma cells reduced the migration, invasion and the anchorage dependent and independent cell growth of melanoma cells. In summary, the downregulation of ADAM15 plays an important role in melanoma progression and ADAM15 act as a tumorsuppressor in melanoma.

  12. miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells.

    PubMed

    Das, Sonia G; Romagnoli, Mathilde; Mineva, Nora D; Barillé-Nion, Sophie; Jézéquel, Pascal; Campone, Mario; Sonenshein, Gail E

    2016-04-02

    ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease. Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, β1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels. We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a β1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression. We have shown for the first

  13. Targeting ADAM12 in human disease: head, body or tail?

    PubMed

    Jacobsen, J; Wewer, U M

    2009-01-01

    ADAM12/meltrin alpha is a type I transmembrane multidomain protein involved in tumor progression and other severe diseases, including osteoarthritis, and as such could be considered as a potential drug target. In addition to protease activity, ADAM12 possesses cell binding and cell signaling properties. This functional trinity is reflected in the structure of ADAM12, which can be divided into head, body, and tail. The head of the protein (consisting of the pro and catalytic domains) mediates processing of growth factors and cytokines and has been implicated in epidermal growth factor (EGF) and insulin-like growth factor receptor signaling. The body of the protein (consisting of the disintegrin, cysteine-rich, and EGF-like domains) is involved in contacts with the extracellular matrix and other cells through interactions with integrins and syndecans. Finally, the tail of the protein (consisting of the cytoplasmic domain) is engaged in interactions with intracellular signaling molecules. In many studies, ADAM12 overexpression has been correlated with disease, and ADAM12 has been shown to promote tumor growth and progression in cancer. On the other hand, protective effects of ADAM12 in disease have also been reported. Future investigations should address the precise mechanisms of ADAM12 in disease and biology in order to counterbalance the benefits from targeting ADAM12 therapeutically with possible side effects. This review describes the biology of ADAM12, its association with disease, and evaluates the possible approaches to targeting ADAM12 in human disease.

  14. Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

    PubMed Central

    Gires, O; Zimber-Strobl, U; Gonnella, R; Ueffing, M; Marschall, G; Zeidler, R; Pich, D; Hammerschmidt, W

    1997-01-01

    Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent. PMID:9359753

  15. Evolution of strategies to prepare synthetic mimics of carboxylate-bridged diiron protein active sites.

    PubMed

    Do, Loi H; Lippard, Stephen J

    2011-12-01

    We present a comprehensive review of research conducted in our laboratory in pursuit of the long-term goal of reproducing the structures and reactivity of carboxylate-bridged diiron centers used in biology to activate dioxygen for the conversion of hydrocarbons to alcohols and related products. This article describes the evolution of strategies devised to achieve these goals and illustrates the challenges in getting there. Particular emphasis is placed on controlling the geometry and coordination environment of the diiron core, preventing formation of polynuclear iron clusters, maintaining the structural integrity of model complexes during reactions with dioxygen, and tuning the ligand framework to stabilize desired oxygenated diiron species. Studies of the various model systems have improved our understanding of the electronic and physical characteristics of carboxylate-bridged diiron units and their reactivity toward molecular oxygen and organic moieties. The principles and lessons that have emerged from these investigations will guide future efforts to develop more sophisticated diiron protein model complexes.

  16. Design, Synthesis, and Dynamics of a Green Fluorescent Protein Fluorophore Mimic with an Ultrafast Switching Function.

    PubMed

    Paolino, Marco; Gueye, Moussa; Pieri, Elisa; Manathunga, Madushanka; Fusi, Stefania; Cappelli, Andrea; Latterini, Loredana; Pannacci, Danilo; Filatov, Michael; Léonard, Jérémie; Olivucci, Massimo

    2016-08-10

    While rotary molecular switches based on neutral and cationic organic π-systems have been reported, structurally homologous anionic switches providing complementary properties have not been prepared so far. Here we report the design and preparation of a molecular switch mimicking the anionic p-HBDI chromophore of the green fluorescent protein. The investigation of the mechanism and dynamics of the E/Z switching function is carried out both computationally and experimentally. The data consistently support axial rotary motion occurring on a sub-picosecond time scale. Transient spectroscopy and trajectory simulations show that the nonadiabatic decay process occurs in the vicinity of a conical intersection (CInt) between a charge transfer state and a covalent/diradical state. Comparison of our anionic p-HBDI-like switch with the previously reported cationic N-alkyl indanylidene pyrrolinium switch mimicking visual pigments reveals that these similar systems translocate, upon vertical excitation, a similar net charge in the same axial direction.

  17. Upregulation of APP, ADAM10 and ADAM17 in the denervated mouse dentate gyrus.

    PubMed

    Del Turco, Domenico; Schlaudraff, Jessica; Bonin, Michael; Deller, Thomas

    2014-01-01

    The disintegrin and metalloproteinases ADAM10 and ADAM17 are regarded as the most important α-secretases involved in the physiological processing of amyloid precursor protein (APP) in brain. Since it has been suggested that processing of APP by α-secretases could be involved in the reorganization of the brain following injury, we studied mRNA expression of the two α-secretases Adam10 and Adam17, the ß-secretase Bace1, and the App-gene family (App, Aplp1, Aplp2) in the dentate gyrus of the mouse following entorhinal denervation. Using laser microdissection, tissue was harvested from the outer molecular layer and the granule cell layer of the denervated dentate gyrus. Expression levels of candidate genes were assessed using Affymetrix GeneChip Mouse Gene 1.0 ST arrays and reverse transcription-quantitative PCR, revealing an upregulation of Adam10 mRNA and Adam17 mRNA in the denervated outer molecular layer and an upregulation of Adam10 mRNA and App mRNA in the dentate granule cell layer. Immunolabeling for ADAM10 or ADAM17 in combination with markers for astro- and microglia revealed an increased labeling of ADAM10 and ADAM17 in the denervated outer molecular layer that was associated with reactive astrocytes but not with microglia. Collectively, these data show that denervation affects the expression level of APP and its two most important α-secretases. This suggests that APP-processing could be shifted towards the non-amyloidogenic pathway in denervated areas of the brain and, thus, towards the formation of neuroprotective APP cleavage products, such as APPsα.

  18. Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein.

    PubMed

    Moriel, Danilo G; Heras, Begoña; Paxman, Jason J; Lo, Alvin W; Tan, Lendl; Sullivan, Matthew J; Dando, Samantha J; Beatson, Scott A; Ulett, Glen C; Schembri, Mark A

    2016-03-15

    Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection. Uropathogenic E. coli (UPEC) is the primary cause of urinary tract infection (UTI), a disease of major significance to human health. Globally, the incidence of UPEC-mediated UTI is strongly associated with increasing antibiotic resistance, making this extremely common infection a major public health concern. In this report, we describe

  19. Alternative mRNA splicing generates two distinct ADAM12 prodomain variants.

    PubMed

    Duhachek-Muggy, Sara; Li, Hui; Qi, Yue; Zolkiewska, Anna

    2013-01-01

    Human ADAM12, transcript variant 1 (later on referred to as Var-1b), present in publicly available databases contains the sequence 5'-GTAATTCTG-3' at the nucleotide positions 340-348 of the coding region, at the 3' end of exon 4. The translation product of this variant, ADAM12-Lb, includes the three amino acid motif (114)VIL(116) in the prodomain. This motif is not conserved in ADAM12 from different species and is not present in other human ADAMs. Currently, it is not clear whether a shorter variant, Var-1a, encoding the protein version without the (114)VIL(116) motif, ADAM12-La, is expressed in human. In this work, we have established that human mammary epithelial cells and breast cancer cells express both Var-1a and Var-1b transcripts. Importantly, the proteolytic processing and intracellular trafficking of the corresponding ADAM12-La and ADAM12-Lb proteins are different. While ADAM12-La is cleaved and trafficked to the cell surface in a manner similar to ADAM12 in other species, ADAM12-Lb is retained in the ER and is not proteolytically processed. Furthermore, the relative abundance of ADAM12-La and ADAM12-Lb proteins detected in several breast cancer cell lines varies significantly. We conclude that the canonical form of transmembrane ADAM12 is represented by Var-1a/ADAM12-La, rather than Var-1b/ADAM12-Lb currently featured in major sequence databases.

  20. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    NASA Astrophysics Data System (ADS)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  1. Mimics of Host Defense Proteins; Strategies for Translation to Therapeutic Applications.

    PubMed

    Scott, Richard W; Tew, Gregory N

    2017-01-01

    New infection treatments are urgently needed to combat the rising threat of multi-drug resistant bacteria. Despite early clinical set-backs attention has re-focused on host defense proteins (HDPs), as potential sources for new and effective antimicrobial treatments. HDPs appear to act at multiple targets and their repertoire includes disruptive membrane and intracellular activities against numerous types of pathogens as well as immune modulatory functions in the host. Importantly, these novel activities are associated with a low potential for emergence of resistance and little crossresistance with other antimicrobial agents. Based on these properties, HDPs appear to be ideal candidates for new antibiotics; however, their development has been plagued by the many therapeutic limitations associated with natural peptidic agents. This review focuses on HDP mimetic approaches aimed to improve metabolic stability, pharmacokinetics, safety and manufacturing processes. Early efforts with β-peptide or peptoid analogs focused on recreating stable facially amphiphilic structures but demonstrated that antimicrobial activity was modulated by more, complex structural properties. Several approaches have used lipidation to increase the hydrophobicity and membrane activity. One lead compound, LTX-109, has entered clinical study as a topical agent to treat impetigo and nasal decolonization. In a more significant departure from the amino acid like peptidomimetics, considerable effort has been directed at developing amphiphilic compounds that recapitulate the structural and biological properties of HDPs on small abiotic scaffolds. The lead compound from this approach, brilacidin, has completed two phase 2 studies as an intravenous agent for skin infections.

  2. ADAM12 is expressed by astrocytes during experimental demyelination.

    PubMed

    Baertling, Fabian; Kokozidou, Maria; Pufe, Thomas; Clarner, Tim; Windoffer, Reinhard; Wruck, Christoph J; Brandenburg, Lars-Ove; Beyer, Cordian; Kipp, Markus

    2010-04-22

    A disintegrin and metalloproteinase (ADAM) 12 represents a member of a large family of similarly structured multi-domain proteins. In the central nervous system (CNS), ADAM12 has been suggested to play a role in brain development, glioblastoma cell proliferation, and in experimental autoimmune encephalomyelitis. Furthermore, ADAM12 was reported to be almost exclusively expressed by oligodendrocytes and could, therefore, be considered as suitable marker for this cell type. In the present study, we investigated ADAM12 expression in the healthy and pathologically altered murine CNS. As pathological paradigm, we used the cuprizone demyelination model in which myelin loss during multiple sclerosis is imitated. Besides APC(+) oligodendrocytes, SMI311(+) neurons and GFAP(+) astrocytes express ADAM12 in the adult mouse brain. ADAM12 expression was further analyzed in vitro. After the induction of demyelination, we observed that activated astrocytes are the main source of ADAM12 in brain regions affected by oligodendrocyte loss. Exposure of astrocytes in vitro to either lipopolysaccharides (LPS), tumor necrosis factor alpha (TNFalpha), glutamate, or hydrogen peroxide revealed a highly stimulus-specific regulation of ADAM12 expression which was not seen in microglial BV2 cells. It appears that LPS- and TNFalpha-induced ADAM12 expression is mediated via the classic NFkappaB pathway. In summary, we demonstrated that ADAM12 is not a suitable marker for oligodendrocytes. Our results further suggest that ADAM12 might be implicated in the course of distinct CNS diseases such as demyelinating disorders.

  3. Molecular and Structural Characterization of a Novel Escherichia coli Interleukin Receptor Mimic Protein

    PubMed Central

    Moriel, Danilo G.; Paxman, Jason J.; Lo, Alvin W.; Tan, Lendl; Sullivan, Matthew J.; Dando, Samantha J.; Beatson, Scott A.

    2016-01-01

    ABSTRACT Urinary tract infection (UTI) is a disease of extremely high incidence in both community and nosocomial settings. UTIs cause significant morbidity and mortality, with approximately 150 million cases globally per year. Uropathogenic Escherichia coli (UPEC) is the primary cause of UTI and is generally treated empirically. However, the rapidly increasing incidence of UTIs caused by multidrug-resistant UPEC strains has led to limited available treatment options and highlights the urgent need to develop alternative treatment and prevention strategies. In this study, we performed a comprehensive analysis to define the regulation, structure, function, and immunogenicity of recently identified UPEC vaccine candidate C1275 (here referred to as IrmA). We showed that the irmA gene is highly prevalent in UPEC, is cotranscribed with the biofilm-associated antigen 43 gene, and is regulated by the global oxidative stress response OxyR protein. Localization studies identified IrmA in the UPEC culture supernatant. We determined the structure of IrmA and showed that it adopts a unique domain-swapped dimer architecture. The dimeric structure of IrmA displays similarity to those of human cytokine receptors, including the interleukin-2 receptor (IL-2R), interleukin-4 receptor (IL-4R), and interleukin-10 receptor (IL-10R) binding domains, and we showed that purified IrmA can bind to their cognate cytokines. Finally, we showed that plasma from convalescent urosepsis patients contains high IrmA antibody titers, demonstrating the strong immunogenicity of IrmA. Taken together, our results indicate that IrmA may play an important role during UPEC infection. PMID:26980835

  4. A Critical Reappraisal of Neutrophil Extracellular Traps and NETosis Mimics Based on Differential Requirements for Protein Citrullination

    PubMed Central

    Konig, Maximilian F.; Andrade, Felipe

    2016-01-01

    NETosis, an antimicrobial form of neutrophil cell death, is considered a primary source of citrullinated autoantigens in rheumatoid arthritis (RA) and immunogenic DNA in systemic lupus erythematosus (SLE). Activation of the citrullinating enzyme peptidylarginine deiminase type 4 (PAD4) is believed to be essential for neutrophil extracellular trap (NET) formation and NETosis. PAD4 is therefore viewed as a promising therapeutic target to inhibit the formation of NETs in both diseases. In this review, we examine the evidence for PAD4 activation during NETosis and provide experimental data to suggest that protein citrullination is not a universal feature of NETs. We delineate two distinct biological processes, leukotoxic hypercitrullination (LTH) and defective mitophagy, which have been erroneously classified as “NETosis.” While these NETosis mimics share morphological similarities with NETosis (i.e., extracellular DNA release), they are biologically distinct. As such, these processes can be readily classified by their stimuli, activation of distinct biochemical pathways, the presence of hypercitrullination, and antimicrobial effector function. NETosis is an antimicrobial form of cell death that is NADPH oxidase-dependent and not associated with hypercitrullination. In contrast, LTH is NADPH oxidase-independent and not bactericidal. Rather, LTH represents a bacterial strategy to achieve immune evasion. It is triggered by pore-forming pathways and equivalent signals that cumulate in calcium-dependent hyperactivation of PADs, protein hypercitrullination, and neutrophil death. The generation of citrullinated autoantigens in RA is likely driven by LTH, but not NETosis. Mitochondrial DNA (mtDNA) expulsion, the result of a constitutive defect in mitophagy, represents a second NETosis mimic. In the presence of interferon-α and immune complexes, this process can generate highly interferogenic oxidized mtDNA, which has previously been mistaken for NETosis in SLE

  5. Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

    PubMed Central

    Kleino, Iivari; Järviluoma, Annika; Hepojoki, Jussi; Huovila, Ari Pekka; Saksela, Kalle

    2015-01-01

    A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior. PMID:25825872

  6. ADAM-17 and TIMP3 protein and mRNA expression in spinal cord white matter of rats with acute experimental autoimmune encephalomyelitis.

    PubMed

    Plumb, Jonnie; Cross, Alison K; Surr, Jessica; Haddock, Gail; Smith, Terence; Bunning, Rowena A D; Woodroofe, M Nicola

    2005-07-01

    Tumour necrosis factor (TNF) is a major immunomodulatory and proinflammatory cytokine implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). ADAM-17 cleaves membrane-bound TNF into its soluble form. The distribution and level of ADAM-17 expression within spinal cords of Lewis rats with EAE was investigated. ADAM-17 was associated with endothelial cells in the naïve and pre-disease spinal cords. In peak disease astrocytic and inflammatory cells expressed ADAM-17. Upregulation of ADAM-17 mRNA expression was coupled with a decrease in mRNA levels of its inhibitor TIMP3 suggesting a role for ADAM-17 in EAE pathogenesis.

  7. Characterization of Mammalian ADAM2 and Its Absence from Human Sperm

    PubMed Central

    Choi, Heejin; Jin, Sora; Kwon, Jun Tae; Kim, Jihye; Jeong, Juri; Kim, Jaehwan; Jeon, Suyeon; Park, Zee Yong; Jung, Kang-Jin; Park, Kwangsung; Cho, Chunghee

    2016-01-01

    The members of the ADAM (a disintegrin and metalloprotease) family are membrane-anchored multi-domain proteins that play prominent roles in male reproduction. ADAM2, which was one of the first identified ADAMs, is the best studied ADAM in reproduction. In the male germ cells of mice, ADAM2 and other ADAMs form complexes that contribute to sperm-sperm adhesion, sperm-egg interactions, and the migration of sperm in the female reproductive tract. Here, we generated specific antibodies against mouse and human ADAM2, and investigated various features of ADAM2 in mice, monkeys and humans. We found that the cytoplasmic domain of ADAM2 might enable the differential association of this protein with other ADAMs in mice. Western blot analysis with the anti-human ADAM2 antibodies showed that ADAM2 is present in the testis and sperm of monkeys. Monkey ADAM2 was found to associate with chaperone proteins in testis. In humans, we identified ADAM2 as a 100-kDa protein in the testis, but failed to detect it in sperm. This is surprising given the results in mice and monkeys, but it is consistent with the failure of ADAM2 identification in the previous proteomic analyses of human sperm. These findings suggest that the reproductive functions of ADAM2 differ between humans and mice. Our protein analysis showed the presence of potential ADAM2 complexes involving yet-unknown proteins in human testis. Taken together, our results provide new information regarding the characteristics of ADAM2 in mammalian species, including humans. PMID:27341348

  8. The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.

    PubMed

    Kennaway, Christopher K; Obarska-Kosinska, Agnieszka; White, John H; Tuszynska, Irina; Cooper, Laurie P; Bujnicki, Janusz M; Trinick, John; Dryden, David T F

    2009-02-01

    Type-I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M(2)S(1) methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.

  9. The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein

    PubMed Central

    Kennaway, Christopher K.; Obarska-Kosinska, Agnieszka; White, John H.; Tuszynska, Irina; Cooper, Laurie P.; Bujnicki, Janusz M.; Trinick, John; Dryden, David T. F.

    2009-01-01

    Type-I DNA restriction–modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M2S1 methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years. PMID:19074193

  10. Self-assembled bifunctional surface mimics an enzymatic and templating protein for the synthesis of a metal oxide semiconductor

    PubMed Central

    Kisailus, David; Truong, Quyen; Amemiya, Yosuke; Weaver, James C.; Morse, Daniel E.

    2006-01-01

    The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein’s catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (γ-Ga2O3). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned. PMID:16585518

  11. The interaction of a histidine-rich protein hpn with the membrane mimics: implications for pathologic roles of Hpn in Helicobacter pylori.

    PubMed

    Zhou, Qinglu; Qi, Shuang; Sun, Xuesong; Ge, Ruiguang

    2014-04-01

    Hpn is a small histidine-rich protein in Helicobacter pylori. This protein has been shown to play roles in nickel storage and detoxification and to exhibit cytotoxicity to gastric epithelial cells. Hpn can be secreted outside of the bacterium and forms amyloid-like structures. To study the interactions between Hpn and membrane mimics, which may further our understanding of the pathologic roles of this bacterium. Various biochemical and biophysical methods, such as secondary structure determination be CD, calcein release assay with fluorescence spectrometry, and Laurdan and Prodan generalized polarization determination have been used to characterize the interaction between Hpn and membrane mimics. Membrane mimics induced the formation of α-helix in Hpn. The interaction disrupts the integrity of the membrane mimics and leads to the release of inner calcein probe. The experiments involving the Laurdan and Prodan fluorescence indicated that increasing the total protein/lipid ratio leads to a less ordered and more hydrated lipid membrane structure close to the water/lipid interface of lipid bilayers modeling the mitochondrial inner membrane. The present data indicated that Hpn may take part in the pathological roles of Helicobacter pylori through membrane interactions. © 2014 John Wiley & Sons Ltd.

  12. Cellular roles of ADAM12 in health and disease.

    PubMed

    Kveiborg, Marie; Albrechtsen, Reidar; Couchman, John R; Wewer, Ulla M

    2008-01-01

    ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.

  13. Engineered protein connectivity to actin mimics PDZ-dependent recycling of G protein-coupled receptors but not its regulation by Hrs.

    PubMed

    Lauffer, Benjamin E L; Chen, Stanford; Melero, Cristina; Kortemme, Tanja; von Zastrow, Mark; Vargas, Gabriel A

    2009-01-23

    Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the beta2-adrenergic receptor (beta2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na+/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the delta-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.

  14. ADAM10: a new player in breast cancer progression?

    PubMed Central

    Mullooly, Maeve; McGowan, Patricia M; Kennedy, Susan A; Madden, Stephen F; Crown, John; O' Donovan, Norma; Duffy, Michael J

    2015-01-01

    Background: The ADAM proteases are best known for their role in shedding the extracellular domain of transmembrane proteins. Among the transmembrane proteins shed by ADAM10 are notch, HER2, E-cadherin, CD44, L1 and the EGFR ligands, EGF and betacellulin. As cleavage of several of these proteins has been implicated in cancer formation and progression, we hypothesised that ADAM10 is also involved in these processes. Methods: ADAM10 expression was decreased by RNA interference and the effects of this on cell numbers, invasion and migration were determined. We also examined the effect of ADAM10 inhibition on breast cancer cell line invasion and migration. Results: Using the triple-negative (TN) breast cancer cell lines, BT20, MDA-MB-231 and the non-TN cell line MDA-MB-453, knockdown of ADAM10 expression significantly decreased in vitro migration (P<0.01; for each cell line). Similarly, treatment with the ADAM10-selective inhibitor GI254023X reduced migration in the three cell lines (for BT20, P<0.001; for MDA-MB-231, P=0.005; for MDA-MB-453, P=0.023). In contrast, neither knockdown of ADAM10 nor treatment with the ADAM10-selective inhibitor GI254023X significantly affected cell numbers. Using extracts of primary breast cancers, higher levels of ADAM10 were found more frequently in high-grade vs low-grade tumours (P<0.001) and in oestrogen receptor (ER)-negative compared with ER-positive tumours (P=0.005). Analysis of pooled publicly available data sets found that high levels of ADAM10 mRNA were associated with adverse outcome in patients with the basal subtype of breast cancer. Conclusions: Based on our combined cell line and breast cancer extract data, we conclude that ADAM10 is likely to be involved in breast cancer progression, especially in the basal subtype. PMID:26284334

  15. The ADAMs family of proteases: new biomarkers and therapeutic targets for cancer?

    PubMed Central

    2011-01-01

    The ADAMs are transmembrane proteins implicated in proteolysis and cell adhesion. Forty gene members of the family have been identified, of which 21 are believed to be functional in humans. As proteases, their main substrates are the ectodomains of other transmembrane proteins. These substrates include precursor forms of growth factors, cytokines, growth factor receptors, cytokine receptors and several different types of adhesion molecules. Although altered expression of specific ADAMs has been implicated in different diseases, their best-documented role is in cancer formation and progression. ADAMs shown to play a role in cancer include ADAM9, ADAM10, ADAM12, ADAM15 and ADAM17. Two of the ADAMs, i.e., ADAM10 and 17 appear to promote cancer progression by releasing HER/EGFR ligands. The released ligands activate HER/EGFR signalling that culminates in increased cell proliferation, migration and survival. Consistent with a causative role in cancer, several ADAMs are emerging as potential cancer biomarkers for aiding cancer diagnosis and predicting patient outcome. Furthermore, a number of selective ADAM inhibitors, especially against ADAM10 and ADAM17, have been shown to have anti-cancer effects. At least one of these inhibitors is now undergoing clinical trials in patients with breast cancer. PMID:21906355

  16. A doppel alpha-helix peptide fragment mimics the copper(II) interactions with the whole protein.

    PubMed

    La Mendola, Diego; Magrì, Antonio; Campagna, Tiziana; Campitiello, Maria Anna; Raiola, Luca; Isernia, Carla; Hansson, Orjan; Bonomo, Raffaele P; Rizzarelli, Enrico

    2010-06-01

    The doppel protein (Dpl) is the first homologue of the prion protein (PrP(C)) to be discovered; it is overexpressed in transgenic mice that lack the prion gene, resulting in neurotoxicity. The whole prion protein is able to inhibit Dpl neurotoxicity, and its N-terminal domain is the determinant part of the protein function. This region represents the main copper(II) binding site of PrP(C). Dpl is able to bind at least one copper ion, and the specific metal-binding site has been identified as the histidine residue at the beginning of the third helical region. However, a reliable characterization of copper(II) coordination features has not been reported. In a previous paper, we studied the copper(II) interaction with a peptide that encompasses only the loop region potentially involved in metal binding. Nevertheless, we did not find a complete match between the EPR spectroscopic parameters of the copper(II) complexes formed with the synthesized peptide and those reported for the copper(II) binding sites of the whole protein. Herein, the synthesis of the human Dpl peptide fragment hDpl(122-139) (Ac-KPDNKLHQQVLWRLVQEL-NH(2)) and its copper(II) complex species are reported. This peptide encompasses the third alpha helix and part of the loop linking the second and the third helix of human doppel protein. The single-point-mutated peptide, hDpl(122-139)D124N, in which aspartate 124 replaces an asparagine residue, was also synthesized. This peptide was used to highlight the role of the carboxylate group on both the conformation preference of the Dpl fragment and its copper(II) coordination features. NMR spectroscopic measurements show that the hDpl(122-139) peptide fragment is in the prevailing alpha-helix conformation. It is localized within the 127-137 amino acid residue region that represents a reliable conformational mimic of the related protein domain. A comparison with the single-point-mutated hDpl(122-139)D124N reveals the significant role played by the aspartic

  17. Ansel Adams: early works

    NASA Astrophysics Data System (ADS)

    Throckmorton, Jodi

    2010-02-01

    Ansel Adams (1902-1984), photographer, musician, naturalist, explorer, critic, and teacher, was a giant in the field of landscape photography. In his images of the unspoiled Western landscape, he strove to capture the sublime: the transcendentalist concept that nature can generate the experience of awe for the viewer. Many viewers are familiar with the heroic, high-contrast prints on high-gloss paper that Adams made to order beginning in the 1970s; much less well known are the intimate prints that the artist crafted earlier in his career. This exhibition focuses on these masterful small prints from the 1920s into the 1950s. During this time period, Adams's printing style changed dramatically. The painterly, soft-focus, warm-toned style of the Parmelian Prints of the High Sierras from the 1920s evolved into the sharp-focus style of the f/64 school of photography that Adams co-founded in the 1930s with Edward Weston and Imogen Cunningham. After World War II, Adams opted for a cooler, higher-contrast look for his prints. Throughout the various styles in which he chose to work, Adams explored the power of nature and succeeded in establishing landscape photography as a legitimate form of modern art.

  18. The T4 phage DNA mimic protein Arn inhibits the DNA binding activity of the bacterial histone-like protein H-NS.

    PubMed

    Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H-J

    2014-09-26

    The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties.

  19. The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS*

    PubMed Central

    Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H.-J.

    2014-01-01

    The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties. PMID:25118281

  20. Suppression of tunicamycin-induced CD44v6 ectodomain shedding and apoptosis is correlated with temporal expression patterns of active ADAM10, MMP-9 and MMP-13 proteins in Caki-2 renal carcinoma cells.

    PubMed

    Kim, Yeoun-Hee; Jung, Jae-Chang

    2012-11-01

    CD44v6 has been shown to coordinate the activation of anti-apoptotic molecules as well as resistance to apoptosis. Here, we investigated CD44v6 ectodomain shedding in Caki-2 human renal carcinoma cells as well as its underlying mechanisms. Exposure of cells to tunicamycin (TM)-induced apoptosis was accompanied by cleavage of caspase-3, PARP-1 and CD44v6 ectodomain. TM-induced apoptosis was also closely associated with endoplasmic reticulum (ER) stress, as shown by increased expression of GRP-78 and CHOP proteins. Furthermore, induction of matrix metallo-proteinase (MMP)-13, MMP-9 and ADAM10 expression was highly stimulated by tunicamycin in a time- and dose-dependent manner. TM-induced PARP-1 cleavage was significantly inhibited by treatment with GM6001 (a broad spectrum MMP inhibitor), MMP-9/-13 inhibitor and GI254023X (specific ADAM10 inhibitor). In addition, inhibition of all examined MMPs resulted in reversal of TM-induced apoptosis as well as increased cell viability. When considering the functional implications of MMP-9 and ADAM10, it is likely that active MMP-9 and ADAM10 help regulate the cellular levels of CD44v6 through cleavage of CD44v6 ectodomain during TM-induced apoptosis of Caki-2 cells. Collectively, these findings suggest that multiple TM-induced MMPs may cooperate to induce apoptosis.

  1. Optimal Hydrophobicity in Ring-Opening Metathesis Polymerization-Based Protein Mimics Required for siRNA Internalization.

    PubMed

    deRonde, Brittany M; Posey, Nicholas D; Otter, Ronja; Caffrey, Leah M; Minter, Lisa M; Tew, Gregory N

    2016-06-13

    Exploring the role of polymer structure for the internalization of biologically relevant cargo, specifically siRNA, is of critical importance to the development of improved delivery reagents. Herein, we report guanidinium-rich protein transduction domain mimics (PTDMs) based on a ring-opening metathesis polymerization scaffold containing tunable hydrophobic moieties that promote siRNA internalization. Structure-activity relationships using Jurkat T cells and HeLa cells were explored to determine how the length of the hydrophobic block and the hydrophobic side chain compositions of these PTDMs impacted siRNA internalization. To explore the hydrophobic block length, two different series of diblock copolymers were synthesized: one series with symmetric block lengths and one with asymmetric block lengths. At similar cationic block lengths, asymmetric and symmetric PTDMs promoted siRNA internalization in the same percentages of the cell population regardless of the hydrophobic block length; however, with 20 repeat units of cationic charge, the asymmetric block length had greater siRNA internalization, highlighting the nontrivial relationships between hydrophobicity and overall cationic charge. To further probe how the hydrophobic side chains impacted siRNA internalization, an additional series of asymmetric PTDMs was synthesized that featured a fixed hydrophobic block length of five repeat units that contained either dimethyl (dMe), methyl phenyl (MePh), or diphenyl (dPh) side chains and varied cationic block lengths. This series was further expanded to incorporate hydrophobic blocks consisting of diethyl (dEt), diisobutyl (diBu), and dicyclohexyl (dCy) based repeat units to better define the hydrophobic window for which our PTDMs had optimal activity. High-performance liquid chromatography retention times quantified the relative hydrophobicities of the noncationic building blocks. PTDMs containing the MePh, diBu, and dPh hydrophobic blocks were shown to have superior

  2. Clinical significance of ADAM10 expression in laryngeal carcinoma.

    PubMed

    You, Bo; Gu, Miao; Cao, Xiaolei; Li, Xingyu; Shi, Si; Shan, Ying; You, Yiwen

    2017-03-01

    Recent findings suggest that upregulated a disintegrin and metalloproteinase (ADAM)10 expression participates in the progression of multiple types of cancer. However, the expression pattern and clinicopathological significance of ADAM10, and its potential prognostic role in laryngeal carcinoma remains to be explored. The present study firstly determined the significantly elevated expression status of ADAM10 protein and messenger RNA in laryngeal carcinoma tissues compared with that in adjacent non-tumor tissues by western blotting and reverse transcription-quantitative polymerase chain reaction analysis. Next, the expression of ADAM10 and the proliferation marker Ki-67 was examined in 78 laryngeal carcinoma and 35 adjacent non-tumor specimens using immunohistochemistry. Overexpressed ADAM10 in laryngeal carcinoma was detected, which correlated with T classification (P<0.01), clinical stage (P<0.01), pathology (P=0.034) and Ki-67 expression (P<0.01). Furthermore, the expression of ADAM10 was positively correlated with the expression of Ki-67 (R(2)=0.22; P<0.01). The Kaplan-Meier method revealed that the group with overexpressed ADAM10 exhibited shorter overall survival time compared with those with low ADAM10 expression. Our findings indicated that ADAM10 serves a notable role in the progression and prognosis of laryngeal carcinoma.

  3. TMPRSS2 and ADAM17 Cleave ACE2 Differentially and Only Proteolysis by TMPRSS2 Augments Entry Driven by the Severe Acute Respiratory Syndrome Coronavirus Spike Protein

    PubMed Central

    Heurich, Adeline; Hofmann-Winkler, Heike; Gierer, Stefanie; Liepold, Thomas; Jahn, Olaf

    2014-01-01

    The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors. PMID:24227843

  4. ADAM12 and ADAM17 gene expression in laser-capture microdissected and non-microdissected breast tumors.

    PubMed

    Narita, Diana; Seclaman, Edward; Ilina, Razvan; Cireap, Natalia; Ursoniu, Sorin; Anghel, Andrei

    2011-06-01

    ADAM (a disintegrin and metalloprotease)12 and ADAM17 are multidomain transmembrane proteins involved in ectodomain shedding of cytokines, growth factors and adhesion molecules, with pivotal activities in the tumor microenvironment. The aim of this study was to confirm the up-regulation of ADAM17 and ADAM12 gene splicing variants in breast tumors and to delineate their expression between laser-capture microdissected (LCM) and non-microdissected breast tumors. The gene expression was analyzed by quantitative-reverse transcription-PCR in a total sample of 109 breast tumors paired with corresponding non-neoplastic breast tissues. ADAM12 and 17 proteins expression for corresponding tissue samples was confirmed by immunohistochemistry. ADAM12S, 12L and 17 genes were significantly up-regulated in either malign or benign LCM samples when compared to non-tumor controls. For non-LCM samples, it was obtained also an increased expression for ADAM12 and 17 genes in cancers, while in benign tumors only ADAM12 variants were significantly up-regulated compared to controls. When benign versus malignant tumors were compared, in LCM samples all investigated genes displayed a higher expression in cancers, whereas in non-LCM, ADAM12 variants were overexpressed in benign samples. The increased expression of ADAM12 protein in the tumor cells and stroma of benign breast diseases was immunohistochemically confirmed. These differences between LCM and non-LCM samples were explained by the contribution of the stroma to the expression of this marker. This study underlines the accuracy conferred by homogenous LCM samples on gene expression profiles and confers further evidence regarding the role of ADAM12 and 17 in the breast tumorigenesis and progression.

  5. ADAM -- Interface Module Reference Manual

    NASA Astrophysics Data System (ADS)

    Chipperfield, A. J.; Kelly, B. D.; Wright, S. L.

    ADAM Interface Modules provide an interface between ADAM application programs and the rest of the system. This document describes in detail the facilities available with ADAM Interface Modules and the rules for using them. It is intended as a reference manual and should shed light on some of the finer points of the ADAM parameter system. Readers requiring an introduction to Interface Modules should read SG/4.

  6. Was Adam a Real Person?

    ERIC Educational Resources Information Center

    Lamoureux, Denis O.

    2011-01-01

    Belief in the historicity of Adam has been held firmly throughout the history of the church. In the light of modern biblical criticism and the evolutionary sciences, some conservative Christians are now questioning whether or not Adam was a real person. This paper argues that the existence of Adam in the opening chapters of scripture reflects an…

  7. Was Adam a Real Person?

    ERIC Educational Resources Information Center

    Lamoureux, Denis O.

    2011-01-01

    Belief in the historicity of Adam has been held firmly throughout the history of the church. In the light of modern biblical criticism and the evolutionary sciences, some conservative Christians are now questioning whether or not Adam was a real person. This paper argues that the existence of Adam in the opening chapters of scripture reflects an…

  8. All about Adam.

    ERIC Educational Resources Information Center

    Bradley, Ann

    1992-01-01

    Rochester Teachers Association President Adam Urbanski is kingpin of a new breed of union leaders who want to be partners, not adversaries, in the school improvement crusade. Despite his good intentions, many people in his hometown are disgruntled with him. The article describes his work over the past five years. (SM)

  9. Adams v. State.

    PubMed

    1998-01-01

    The Supreme Court of Georgia, on 4 May 1998, held that a state statute permitting a crime victim who is significantly exposed to HIV request an HIV blood test on the person charged with the crime and arrested does not violate the Fourth Amendment right against unreasonable searches, nor does it violate privacy or equal protection rights. Malik Adams attacked and struggled with police officers during arrest. In the struggle, Adams's and an officer's hands, on which there were bleeding wounds, came in contact. Even though Adams did not have any outward AIDS symptoms, the State filed a motion to compel HIV testing. The Supreme Court of Georgia held that, because the statute compelling HIV testing serves the compelling state interest of preventing the public's exposure to HIV, the search, in this case the taking and sampling of blood, was reasonable. The statute also did not violate Adams's right to privacy or state or federal equal protection clauses. The judgment of the Superior court was affirmed.

  10. Adams v. State.

    PubMed

    1998-01-01

    Court Decision: 498 South Eastern Reporter, 2d Series 268; 1998 May 4 (date of decision). The Supreme Court of Georgia held that a state statute permitting a crime victim who is significantly exposed to HIV request an HIV blood test on the person charged with the crime and arrested does not violate the Fourth Amendment right against unreasonable searches, nor does it violate privacy or equal protection rights. Malik Adams attacked and struggled with police officers during arrest. In the struggle, Adams's and an officer's hands, on which there were bleeding wounds, came in contact. Even though Adams did not have any outward AIDS symptoms, the State filed a motion to compel HIV testing. The Supreme Court of Georgia held that, because the statute compelling HIV testing serves the compelling state interest of preventing the public's exposure to HIV, the search, in this case the taking and sampling of blood, was reasonable. The statute also did not violate Adams's right to privacy or state or federal equal protection clauses.

  11. The Adams Family

    ERIC Educational Resources Information Center

    Douven, Igor; Verbrugge, Sara

    2010-01-01

    According to Adams's Thesis, the acceptability of an indicative conditional sentence goes by the conditional probability of its consequent given its antecedent. We test, for the first time, whether this thesis is descriptively correct and show that it is not; in particular, we show that it yields the wrong predictions for people's judgments of the…

  12. The Adams Family

    ERIC Educational Resources Information Center

    Douven, Igor; Verbrugge, Sara

    2010-01-01

    According to Adams's Thesis, the acceptability of an indicative conditional sentence goes by the conditional probability of its consequent given its antecedent. We test, for the first time, whether this thesis is descriptively correct and show that it is not; in particular, we show that it yields the wrong predictions for people's judgments of the…

  13. High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein-Protein Interaction

    SciTech Connect

    He, Shihan; Senter, Timothy J.; Pollock, Jonathan; Han, Changho; Upadhyay, Sunil Kumar; Purohit, Trupta; Gogliotti, Rocco D.; Lindsley, Craig W.; Cierpicki, Tomasz; Stauffer, Shaun R.; Grembecka, Jolanta

    2014-10-02

    The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ~288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in MIV-6R, which inhibits the menin–MLL interaction with IC50 = 56 nM. Treatment with MIV-6 demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules.

  14. Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

    PubMed

    Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

    2017-08-15

    Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor.IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

  15. Connective tissue growth factor is a substrate of ADAM28

    SciTech Connect

    Mochizuki, Satsuki; Tanaka, Rena; Shimoda, Masayuki; Onuma, Junko; Fujii, Yutaka; Jinno, Hiromitsu; Okada, Yasunori

    2010-11-26

    Research highlights: {yields} The hyper-variable region in the cysteine-rich domain of ADAM28 binds to C-terminal domain of CTGF. {yields} ADAM28 cleaves CTGF alone and CTGF in the CTGF/VEGF{sub 165} complex. {yields} CTGF digestion by ADAM28 releases biologically active VEGF{sub 165} from the complex. {yields} ADAM28, CTGF and VEGF{sub 165} are commonly co-expressed by carcinoma cells in human breast carcinoma tissues. {yields} These suggest that ADAM28 promotes VEGF{sub 165}-induced angiogenesis in the breast carcinomas by selective CTGF digestion in the CTGF/VEGF{sub 165} complex. -- Abstract: ADAM28, a member of the ADAM (a disintegrin and metalloproteinase) gene family, is over-expressed by carcinoma cells and the expression correlates with carcinoma cell proliferation and progression in human lung and breast carcinomas. However, information about substrates of ADAM28 is limited. We screened interacting molecules of ADAM28 in human lung cDNA library by yeast two-hybrid system and identified connective tissue growth factor (CTGF). Binding of CTGF to proADAM28 was demonstrated by yeast two-hybrid assay and protein binding assay. ADAM28 cleaved CTGF in dose- and time-dependent manners at the Ala{sup 181}-Tyr{sup 182} and Asp{sup 191}-Pro{sup 192} bonds in the hinge region of the molecule. ADAM28 selectively digested CTGF in the complex of CTGF and vascular endothelial growth factor{sub 165} (VEGF{sub 165}), releasing biologically active VEGF{sub 165} from the complex. RT-PCR and immunohistochemical analyses demonstrated that ADAM28, CTGF and VEGF are commonly co-expressed in the breast carcinoma tissues. These data provide the first evidence that CTGF is a novel substrate of ADAM28 and suggest that ADAM28 may promote VEGF{sub 165}-induced angiogenesis in the breast carcinomas by the CTGF digestion in the CTGF/VEGF{sub 165} complex.

  16. Ectodomain shedding of the cell adhesion molecule Nectin-4 in ovarian cancer is mediated by ADAM10 and ADAM17.

    PubMed

    Buchanan, Petra C; Boylan, Kristin L M; Walcheck, Bruce; Heinze, Rachel; Geller, Melissa A; Argenta, Peter A; Skubitz, Amy P N

    2017-04-14

    We previously showed that the cell adhesion molecule Nectin-4 is overexpressed in ovarian cancer tumors, and its cleaved extracellular domain can be detected in the serum of ovarian cancer patients. The ADAM (adisintegrin and metalloproteinase) proteases are involved in ectodomain cleavage of transmembrane proteins, and ADAM17 is known to cleave Nectin-4 in breast cancer. However, the mechanism of Nectin-4 cleavage in ovarian cancer has not yet been determined. Analysis of ovarian cancer gene microarray data showed that higher expression of Nectin-4, ADAM10, and ADAM17 is associated with significantly decreased progression-free survival. We quantified Nectin-4 shedding from the surface of ovarian cancer cells after stimulation with lysophosphatidic acid. We report that ADAM17 and ADAM10 cleave Nectin-4 and release soluble Nectin-4 (sN4). Small molecule inhibitors and siRNA knockdown of both ADAM proteases confirmed these results. In matched samples from 11 high-grade serous ovarian cancer patients, we detected 2-20-fold more sN4 in ascites fluid than serum. Co-incubation of ovarian cancer cells with ascites fluid significantly increased sN4 shedding, which could be blocked using a dual inhibitor of ADAM10 and ADAM17. Furthermore, we detected RNA for Nectin-4, ADAM10, and ADAM17 in primary ovarian carcinoma tumors, secondary omental metastases, and ascites cells isolated from serous ovarian cancer patients. In a signaling pathway screen, lysophosphatidic acid increased phosphorylation of AKT, EGF receptor, ERK1/2, JNK1/2/3, and c-Jun. Understanding the function of Nectin-4 shedding in ovarian cancer progression is critical to facilitate its development as both a serum biomarker and a therapeutic target for ovarian cancer. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Tetraspanin15 regulates cellular trafficking and activity of the ectodomain sheddase ADAM10.

    PubMed

    Prox, Johannes; Willenbrock, Michael; Weber, Silvio; Lehmann, Tobias; Schmidt-Arras, Dirk; Schwanbeck, Ralf; Saftig, Paul; Schwake, Michael

    2012-09-01

    A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.

  18. A Substrate Mimic Allows High-Throughput Assay of the FabA Protein and Consequently the Identification of a Novel Inhibitor of Pseudomonas aeruginosa FabA

    PubMed Central

    Moynié, Lucile; Hope, Anthony G.; Finzel, Kara; Schmidberger, Jason; Leckie, Stuart M.; Schneider, Gunter; Burkart, Michael D.; Smith, Andrew D.; Gray, David W.; Naismith, James H.

    2016-01-01

    Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathways that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-Acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FASII pathway, into a high-throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of Escherichia coli acyl carrier protein and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalises affinity and suggests future design opportunities. Employing NAC fatty acid mimics to develop further high-throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials. PMID:26562505

  19. The plasma membrane: Penultimate regulator of ADAM sheddase function.

    PubMed

    Reiss, Karina; Bhakdi, Sucharit

    2017-11-01

    ADAM10 and ADAM17 are the best characterized members of the ADAM (A Disintegrin and Metalloproteinase) - family of transmembrane proteases. Both are involved diverse physiological and pathophysiological processes. ADAMs are known to be regulated by posttranslational mechanisms. However, emerging evidence indicates that the plasma membrane with its unique dynamic properties may additionally play an important role in controlling sheddase function. Membrane events that could contribute to regulation of ADAM-function are summarized. Surface expression of peptidolytic activity should be differentiated from ADAM-sheddase function since the latter additionally requires that the protease finds its substrate in the lipid bilayer. We propose that this is achieved through horizontal and vertical reorganization of membrane nanoarchitecture coordinately occurring at the sites of sheddase activation. Reshuffling of nanodomains thereby guides traffic of enzyme and substrate to each other. For ADAM17 phosphatidylserine exposure is required to then induce its shedding function. The novel concept that physicochemical properties of the lipid bilayer govern the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017. Published by Elsevier B.V.

  20. Molecular profiling of ADAM12 gene in breast cancers.

    PubMed

    Nariţa, Diana; Anghel, A; Seclaman, E; Ilina, R; Cireap, Natalia; Ursoniu, S

    2010-01-01

    ADAMs (a disintegrin and metalloproteinase) family have been associated with the process of proteolytic "shedding" of membrane-associated proteins ectodomain and hence the rapid modulation of key cell signaling pathways in tissues microenvironment. A variety of cytokines, chemokines and growth factors which are initially produced as transmembrane proforms are activated by these sheddase activities. ADAM12 is highly expressed in rapidly growing tissues such as placenta and malignant tumors and it was found as one of the Candidate Cancer Genes in a comprehensive mutational analysis of human breast cancers. Our aim was to determine the gene expression profile of ADAM12 in breast cancers in comparison with normal breast and to correlate their level of expression with the clinical and pathological characteristics of breast cancers. Gene expression of ADAM12 spliced variants (12L and 12S) was evaluated using quantitative reverse-transcription PCR in samples obtained by laser capture microdissection from 38 patients with breast cancers and compared with adjacent healthy breast tissues. Both ADAM12L and 12S expression were significantly up-regulated in breast cancers, while in the normal breast, we found a very low expression. ADAM12L expression was significantly correlated with the histopathological types and, although not statistically significant, ADAM12 both variants were up-regulated in high-grade, highly-proliferative and HER2÷neu positive tumors. From these preliminary results, we found that ADAM12 could be an interesting marker and eventually a therapeutic target for breast cancer.

  1. Reversible non-stick behaviour of a bacterial protein polymer provides a tuneable molecular mimic for cell and tissue engineering.

    PubMed

    Roque, Ana I; Soliakov, Andrei; Birch, Mark A; Philips, Sion R; Shah, Deepan S H; Lakey, Jeremy H

    2014-05-01

    Yersina pestis, the bubonic plague bacterium, is coated with a polymeric protein hydrogel for protection from host defences. The protein, which is robust and non-stick, resembles structures found in many eukaryotic extracellular-matrix proteins. Cells grown on the natural polymer cannot adhere and grow poorly; however, when cell-adhesion motifs are inserted into the protein, the cells proliferate.

  2. Cell-surface metalloprotease ADAM12 is internalized by a clathrin- and Grb2-dependent mechanism.

    PubMed

    Stautz, Dorte; Leyme, Anthony; Grandal, Michael Vibo; Albrechtsen, Reidar; van Deurs, Bo; Wewer, Ulla; Kveiborg, Marie

    2012-11-01

    ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell-adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell-surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin-dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline-rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src-homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor-bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin-dependent pathway and Grb2.

  3. Disintegrin and metalloproteinases (ADAMs) expression in gastroesophageal reflux disease and in esophageal adenocarcinoma.

    PubMed

    Kauttu, T; Mustonen, H; Vainionpää, S; Krogerus, L; Ilonen, I; Räsänen, J; Salo, J; Puolakkainen, P

    2017-01-01

    Clinically useful marker molecules for the progression of gastroesophageal reflux disease and Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) are lacking. Many adenocarcinomas and inflammatory conditions exhibit increased expression of ADAMs, 'a disintegrin and metalloproteinases'. We assessed the expression of five ADAMs (9, 10, 12, 17, 19) in three esophageal cell lines (Het-1A, OE19, OE33) by RT-PCR and Western blotting, and in human samples of normal esophagus, esophagitis, BE, Barrett's dysplasia, and EAC by RT-PCR, and in selected samples by immunohistochemistry. EAC patients showed increased mRNA expression of ADAMs 9, 12, 17 and 19, as compared to controls. At immunohistochemistry, ADAM9 and ADAM10 proteins were increased in EAC. Patient samples also showed increased mRNA expression of ADAM12 in esophagitis, of ADAM9 in BE, and of ADAMs 9, 12 and 19 in Barrett's dysplasia, as compared to controls. Two EAC cell lines showed increased ADAM9 mRNA. ADAM9 expression is increased in EAC. Its predecessors show increased ADAM9 mRNA expression. The importance of the alterations in ADAM expression for the development of EAC, and their use as marker molecules, warrant further studies.

  4. Multiple non-catalytic ADAMs are novel integrin α4 ligands.

    PubMed

    Wang, Lei; Hoggard, Jason A; Korleski, Erica D; Long, Gideon V; Ree, Brandy C; Hensley, Kenneth; Bond, Stephen R; Wolfsberg, Tyra G; Chen, JianMing; Zeczycki, Tonya N; Bridges, Lance C

    2017-09-14

    The ADAM (a disintegrin and metalloprotease) protein family uniquely exhibits both catalytic and adhesive properties. In the well-defined process of ectodomain shedding, ADAMs transform latent, cell-bound substrates into soluble, biologically active derivatives to regulate a spectrum of normal and pathological processes. In contrast, the integrin ligand properties of ADAMs are not fully understood. Emerging models posit that ADAM-integrin interactions regulate shedding activity by localizing or sequestering the ADAM sheddase. Interestingly, 8 of the 21 human ADAMs are predicted to be catalytically inactive. Unlike their catalytically active counterparts, integrin recognition of these "dead" enzymes has not been largely reported. The present study delineates the integrin ligand properties of a group of non-catalytic ADAMs. Here we report that human ADAM11, ADAM23, and ADAM29 selectively support integrin α4-dependent cell adhesion. This is the first demonstration that the disintegrin-like domains of multiple catalytically inactive ADAMs are ligands for a select subset of integrin receptors that also recognize catalytically active ADAMs.

  5. Reconstituting redox active centers of heme-containing proteins with biomineralized gold toward peroxidase mimics with strong intrinsic catalysis and electrocatalysis for H2O2 detection.

    PubMed

    Zhang, Liyan; Li, Shuai; Dong, Minmin; Jiang, Yao; Li, Ru; Zhang, Shuo; Lv, Xiaoxia; Chen, Lijun; Wang, Hua

    2017-01-15

    A facile and efficient enzymatic reconstitution methodology has been proposed for high-catalysis peroxidase mimics by remolding the redox active centers of heme-containing proteins with the in-site biomineralized gold using hemoglobin (Hb) as a model. Catalytic hemin (Hem) was extracted from the active centers of Hb for the gold biomineralization and then reconstituted into apoHb to yield the Hem-Au@apoHb nanocomposites showing dramatically improved intrinsic catalysis and electrocatalysis over natural Hb and Hem. The biomineralized gold, on the one hand, would act as "nanowires" to promote the electron transferring of the nanocomposites. On the other hand, it would create a reactivity pathway to pre-organize and accumulate more substrates towards the active sites of the peroxidase mimics. Steady-state kinetics studies indicate that Hem-Au@apoHb could present much higher substrate affinity (lower Michaelis constants) and intrinsic catalysis even than some natural peroxidases. Moreover, the application feasibility of the prepared artificial enzymes was demonstrated by colorimetric assays and direct electrocatalysis for H2O2 sensing, showing a detection limitation low as 0.45μM. Importantly, such a catalysis active-center reconstitution protocol may circumvent the substantial improvement of the intrinsic catalysis and electrocatalysis of diverse heme-containing proteins or enyzmes toward the extensive applications in the chemical, enviromental, and biomedical catalysis fields.

  6. ADAM17 mediates OSCC development in an orthotopic murine model

    PubMed Central

    2014-01-01

    Background ADAM17 is one of the main sheddases of the cells and it is responsible for the cleavage and the release of ectodomains of important signaling molecules, such as EGFR ligands. Despite the known crosstalk between ADAM17 and EGFR, which has been considered a promising targeted therapy in oral squamous cell carcinoma (OSCC), the role of ADAM17 in OSCC development is not clear. Method In this study the effect of overexpressing ADAM17 in cell migration, viability, adhesion and proliferation was comprehensively appraised in vitro. In addition, the tumor size, tumor proliferative activity, tumor collagenase activity and MS-based proteomics of tumor tissues have been evaluated by injecting tumorigenic squamous carcinoma cells (SCC-9) overexpressing ADAM17 in immunodeficient mice. Results The proteomic analysis has effectively identified a total of 2,194 proteins in control and tumor tissues. Among these, 110 proteins have been down-regulated and 90 have been up-regulated in tumor tissues. Biological network analysis has uncovered that overexpression of ADAM17 regulates Erk pathway in OSCC and further indicates proteins regulated by the overexpression of ADAM17 in the respective pathway. These results are also supported by the evidences of higher viability, migration, adhesion and proliferation in SCC-9 or A431 cells in vitro along with the increase of tumor size and proliferative activity and higher tissue collagenase activity as an outcome of ADAM17 overexpression. Conclusion These findings contribute to understand the role of ADAM17 in oral cancer development and as a potential therapeutic target in oral cancer. In addition, our study also provides the basis for the development of novel and refined OSCC-targeting approaches. PMID:24495306

  7. Altered expression of zinc finger proteins, ADAMs, and integrin-related proteins following treatment of cultured human cells with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine.

    PubMed

    Jin, Jinghua; Gao, Zhihua; Guo, Lei; Yang, Jun; Yu, Yingnian

    2003-01-01

    Proteomic analysis is an important approach to characterize the proteome and study protein functions. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be overlooked by conventional biochemical techniques. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an alkylating agent that can induce nontargeted mutagenesis in treated cells, although the mechanism remains unclear. Using an efficient proteomic method, we identified several cellular proteins that are responsive to low-concentration MNNG treatment in human FL cells. After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images then were analyzed with 2D analysis software. More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). Thirty-one proteins only detected in MNNG-treated or control cells were subjected to in-gel digestion with trypsin and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting. Eighteen of theses proteins have been identified, including several zinc finger proteins, two members of the ADAMs (a disintegrin and metalloprotease domain) family, and two integrins. Most of these proteins have unknown functions and their involvement in the cellular responses to alkylating agents have not been reported. Therefore, our findings may offer new insights into the mechanisms of low-concentration MNNG-induced nontargeted mutagenesis and these proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens.

  8. Human and Murine Interleukin 23 Receptors Are Novel Substrates for A Disintegrin and Metalloproteases ADAM10 and ADAM17*

    PubMed Central

    Franke, Manuel; Schröder, Jutta; Monhasery, Niloufar; Ackfeld, Theresa; Hummel, Thorben M.; Rabe, Björn; Garbers, Christoph; Becker-Pauly, Christoph; Floss, Doreen M.; Scheller, Jürgen

    2016-01-01

    IL-23 (interleukin 23) regulates immune responses against pathogens and plays a major role in the differentiation and maintenance of TH17 cells and the development of autoimmune diseases and cancer. The IL-23 receptor (IL-23R) complex consists of the unique IL-23R and the common IL-12 receptor β1 (IL-12Rβ1). Differential splicing generates antagonistic soluble IL-23R (sIL-23R) variants, which might limit IL-23-mediated immune responses. Here, ectodomain shedding of human and murine IL-23R was identified as an alternative pathway for the generation of sIL-23R. Importantly, proteolytically released sIL-23R has IL-23 binding activity. Shedding of IL-23R was induced by stimulation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), but not by ionomycin. PMA-induced shedding was abrogated by an ADAM (A disintegrin and metalloprotease) 10 and 17 selective inhibitor, but not by an ADAM10 selective inhibitor. ADAM17-deficient but not ADAM10-deficient HEK293 cells failed to shed IL-23R after PMA stimulation, demonstrating that ADAM17 but not ADAM10 cleaves the IL-23R. Constitutive shedding was, however, inhibited by an ADAM10 selective inhibitor. Using deletions and specific amino acid residue exchanges, we identified critical determinants of ectodomain shedding within the stalk region of the IL-23R. Finally, interaction studies identified domains 1 and 3 of the IL-23R as the main ADAM17 binding sites. In summary, we describe human and murine IL-23R as novel targets for protein ectodomain shedding by ADAM10 and ADAM17. PMID:26961870

  9. Direct measurement of the protein response to an electrostatic perturbation that mimics the catalytic cycle in ketosteroid isomerase.

    PubMed

    Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J; Boxer, Steven G

    2011-10-04

    Understanding how electric fields and their fluctuations in the active site of enzymes affect efficient catalysis represents a critical objective of biochemical research. We have directly measured the dynamics of the electric field in the active site of a highly proficient enzyme, Δ(5)-3-ketosteroid isomerase (KSI), in response to a sudden electrostatic perturbation that simulates the charge displacement that occurs along the KSI catalytic reaction coordinate. Photoexcitation of a fluorescent analog (coumarin 183) of the reaction intermediate mimics the change in charge distribution that occurs between the reactant and intermediate state in the steroid substrate of KSI. We measured the electrostatic response and angular dynamics of four probe dipoles in the enzyme active site by monitoring the time-resolved changes in the vibrational absorbance (IR) spectrum of a spectator thiocyanate moiety (a quantitative sensor of changes in electric field) placed at four different locations in and around the active site, using polarization-dependent transient vibrational Stark spectroscopy. The four different dipoles in the active site remain immobile and do not align to the changes in the substrate electric field. These results indicate that the active site of KSI is preorganized with respect to functionally relevant changes in electric fields.

  10. Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

    PubMed Central

    Bhaduri, Asani; Misra, Richa; Maji, Abhijit; Bhetaria, Preetida J.; Mishra, Sonakshi; Arora, Gunjan; Singh, Lalit Kumar; Dhasmana, Neha; Dubey, Neha; Virdi, Jugsharan Singh; Singh, Yogendra

    2014-01-01

    Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins. PMID:24505389

  11. Mycobacterium tuberculosis cyclophilin A uses novel signal sequence for secretion and mimics eukaryotic cyclophilins for interaction with host protein repertoire.

    PubMed

    Bhaduri, Asani; Misra, Richa; Maji, Abhijit; Bhetaria, Preetida J; Mishra, Sonakshi; Arora, Gunjan; Singh, Lalit Kumar; Dhasmana, Neha; Dubey, Neha; Virdi, Jugsharan Singh; Singh, Yogendra

    2014-01-01

    Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

  12. High Glucose Up-regulates ADAM17 through HIF-1α in Mesangial Cells*

    PubMed Central

    Li, Renzhong; Uttarwar, Lalita; Gao, Bo; Charbonneau, Martine; Shi, Yixuan; Chan, John S. D.; Dubois, Claire M.; Krepinsky, Joan C.

    2015-01-01

    We previously showed that ADAM17 mediates high glucose-induced matrix production by kidney mesangial cells. ADAM17 expression is increased in diabetic kidneys, suggesting that its up-regulation may augment high glucose profibrotic responses. We thus studied the effects of high glucose on ADAM17 gene regulation. Primary rat mesangial cells were treated with high glucose (30 mm) or mannitol as osmotic control. High glucose dose-dependently increased ADAM17 promoter activity, transcript, and protein levels. This correlated with augmented ADAM17 activity after 24 h versus 1 h of high glucose. We tested involvement of transcription factors shown in other settings to regulate ADAM17 transcription. Promoter activation was not affected by NF-κB or Sp1 inhibitors, but was blocked by hypoxia-inducible factor-1α (HIF-1α) inhibition or down-regulation. This also prevented ADAM17 transcript and protein increases. HIF-1α activation by high glucose was shown by its increased nuclear translocation and activation of the HIF-responsive hypoxia-response element (HRE)-luciferase reporter construct. Assessment of ADAM17 promoter deletion constructs coupled with mutation analysis and ChIP studies identified HIF-1α binding to its consensus element at −607 as critical for the high glucose response. Finally, inhibitors of epidermal growth factor receptor (EGFR) and downstream PI3K/Akt, or ADAM17 itself, prevented high glucose-induced HIF-1α activation and ADAM17 up-regulation. Thus, high glucose induces ADAM17 transcriptional up-regulation in mesangial cells, which is associated with augmentation of its activity. This is mediated by HIF-1α and requires EGFR/ADAM17 signaling, demonstrating the potentiation by ADAM17 of its own up-regulation. ADAM17 inhibition thus provides a potential novel therapeutic strategy for the treatment of diabetic nephropathy. PMID:26175156

  13. DNA mimics a self-protein that may be a target for some anti-DNA antibodies in systemic lupus erythematosus.

    PubMed

    Zack, D J; Yamamoto, K; Wong, A L; Stempniak, M; French, C; Weisbart, R H

    1995-02-15

    Recent studies suggest that some anti-DNA Abs in systemic lupus erythematosus may actually be Abs to specific proteins and that binding to dsDNA is a nonspecific cross-reactive event. To identify such proteins that bind to anti-DNA Abs, a cDNA expression library from human placenta was screened with mAb 3E10, a pathogenic anti-dsDNA Ab. MAb 3E10 was shown to bind to a 44-amino-acid fragment of HP8, a newly identified protein with amino acid sequence homology to the family of SPARC extracellular matrix proteins. To determine if Ab binding to both dsDNA and HP8 protein occurs through a common binding site, and therefore represents molecular mimicry, the Ab binding domains for protein and DNA were mapped. Chain recombinations between mAb 3E10 and a non-anti-DNA mAb showed that both the heavy and the light chains of mAb 3E10 were essential for anti-dsDNA and anti-HP8 reactivity. Mutagenesis experiments demonstrated that dsDNA and HP8 shared several critical binding residues located in all three complementarity-determining regions of mAb 3E10 VH. Moreover, Abs to HP8 were demonstrated in the sera of a subset of lupus patients. These results indicate that DNA mimics the HP8 protein in binding a lupus Ab, and that this protein may be a target for a subpopulation of anti-dsDNA Abs in systemic lupus erythematosus.

  14. New Mathematical Dimensions: Adam's Story

    ERIC Educational Resources Information Center

    Manizade, Agida

    2009-01-01

    Adam, an 11th grader, was identified as gifted and accepted into a two week summer enrichment program. He signed up for "Geometry with Flash Programming." He had no prior programming experience but had a strong and healthy self-image as mathematics student. Although Adam had a positive attitude toward mathematics and saw himself as a successful…

  15. Changing Perceptions in "Adam Bede."

    ERIC Educational Resources Information Center

    Loges, Max L.

    From the very beginning of "Adam Bede," the idea of sight or perception is emphasized. Indeed by reference to a quotation from Wordsworth, George Eliot announces the purpose of the novel: to reveal clearly, to remove from the shade. While most of the characters in "Adam Bede" do not perceive events clearly and must have their…

  16. New Mathematical Dimensions: Adam's Story

    ERIC Educational Resources Information Center

    Manizade, Agida

    2009-01-01

    Adam, an 11th grader, was identified as gifted and accepted into a two week summer enrichment program. He signed up for "Geometry with Flash Programming." He had no prior programming experience but had a strong and healthy self-image as mathematics student. Although Adam had a positive attitude toward mathematics and saw himself as a successful…

  17. Toward Functional Carboxylate-Bridged Diiron Protein Mimics: Achieving Structural Stability and Conformational Flexibility Using a Macrocylic Ligand Framework

    PubMed Central

    Do, Loi H.; Lippard, Stephen J.

    2011-01-01

    A dinucleating macrocycle, H2PIM, containing phenoxylimine metal-binding units has been prepared. Reaction of H2PIM with [Fe2(Mes)4] (Mes = 2,4,6-trimethylphenyl) and sterically hindered carboxylic acids, Ph3CCO2H or ArTolCO2H (2,6-bis(p-tolyl)benzoic acid), afforded complexes [Fe2(PIM)(Ph3CCO2)2] (1) and [Fe2(PIM)(ArTolCO2)2] (2), respectively. X-ray diffraction studies revealed that these diiron(II) complexes closely mimic the active site structures of the hydroxylase components of bacterial multi-component monooxygenases (BMMs), particularly the syn disposition of the nitrogen donor atoms and the bridging μ-η1η2 and μ-η1η1 modes of the carboxylate ligands at the diiron(II) centers. Cyclic voltammograms of 1 and 2 displayed quasi-reversible redox couples at +16 and +108 mV vs. ferrocene/ferrocenium, respectively. Treatment of 2 with silver perchlorate afforded a silver(I)/iron(III) heterodimetallic complex, [Fe2(μ-OH)2(ClO4)2(PIM)(ArTolCO2)Ag] (3), which was structurally and spectroscopically characterized. Complexes 1 and 2 both react rapidly with dioxygen. Oxygenation of 1 afforded a (μ-hydroxo)diiron(III) complex [Fe2(μ-OH)(PIM)(Ph3CCO2)3] (4), a hexa(μ-hydroxo)tetrairon(III) complex [Fe4(μ-OH)6(PIM)2(Ph3CCO2)2] (5), and an unidentified iron(III) species. Oxygenation of 2 exclusively formed di(carboxylato)diiron(III) compounds, a testimony to the role of the macrocylic ligand in preserving the dinuclear iron center under oxidizing conditions. X-ray crystallographic and 57Fe Mössbauer spectroscopic investigations indicated that 2 reacts with dioxygen to give a mixture of (μ-oxo)diiron(III) [Fe2(μ-O)(PIM)(ArTolCO2)2] (6) and di(μ-hydroxo)diiron(III) [Fe2(μ-OH)2(PIM)(ArTolCO2)2] (7) units in the same crystal lattice. Compounds 6 and 7 spontaneously convert to a tetrairon(III) complex, [Fe4(μ-OH)6(PIM)2(ArTolCO2)2] (8), when treated with excess H2O. PMID:21682286

  18. Peptide–oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    PubMed Central

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte S.; Thomsen, Rasmus P.; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W.; Wengel, Jesper; Jensen, Knud J.

    2016-01-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide–oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design. PMID:27464951

  19. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic.

    PubMed

    Lou, Chenguang; Martos-Maldonado, Manuel C; Madsen, Charlotte S; Thomsen, Rasmus P; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W; Wengel, Jesper; Jensen, Knud J

    2016-07-28

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

  20. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    NASA Astrophysics Data System (ADS)

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte S.; Thomsen, Rasmus P.; Midtgaard, Søren Roi; Christensen, Niels Johan; Kjems, Jørgen; Thulstrup, Peter W.; Wengel, Jesper; Jensen, Knud J.

    2016-07-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain formation have never been realized for de novo protein design. Here, we show the applicability of peptide-oligonucleotide conjugates for self-assembly of higher-ordered protein-like structures. The resulting nano-assemblies were characterized by ultraviolet-melting, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering and transmission electron microscopy. These studies revealed the formation of the desired triple helix and coiled coil domains at low concentrations, while a dimer of trimers was dominating at high concentration. CD spectroscopy showed an extraordinarily high degree of α-helicity for the peptide moieties in the assemblies. The results validate the use of orthogonal self-assembly principles as a paradigm for de novo protein design.

  1. Myxoma Virus Immunomodulatory Protein M156R is a Structural Mimic of Eukaryotic Translation Initiation Factor eIF2 alpha

    SciTech Connect

    Ramelot, Theresa A.; Cort, John R.; Yee, Adelinda; Liu, Furong; Goshe, Michael B.; Edwards, Aled M.; Smith, Richard D.; Arrowsmith, Cheryl H.; Dever, Thomas E.; Kennedy, Michael A.

    2002-10-04

    M156R, the product of the myxoma virus M156R open reading frame, is a protein of unknown function. However, several homologs of M156R from other viruses are immunomodulatory proteins that bind to interferon-induced protein kinase PKR and inhibit phosphorylation of the eukaryotic translation initiation factor eIF2a. In this study, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel b-barrel with two of the strands connected by a long loop and a short a-helix. The similarity between M156R and the predicted S1 motif structure of eIF2a suggests that the viral homologs are pseudosubstrate inhibitors of PKR that mimic eIF2a in order to compete for binding to PKR. A homology modeled structure of the well studied vaccinia virus K3L was generated based on alignment with M156R. Residues important for binding to PKR are conserved residues on the surface of the b-barrel and in the mobile loop, identifying the putative PKR recognition motif.

  2. Metalloproteinases ADAM10 and ADAM17 Mediate Migration and Differentiation in Glioblastoma Sphere-Forming Cells.

    PubMed

    Siney, Elodie J; Holden, Alexander; Casselden, Elizabeth; Bulstrode, Harry; Thomas, Gareth J; Willaime-Morawek, Sandrine

    2017-07-01

    Glioblastoma is the most common form of primary malignant brain tumour. These tumours are highly proliferative and infiltrative resulting in a median patient survival of only 14 months from diagnosis. The current treatment regimens are ineffective against the small population of cancer stem cells residing in the tumourigenic niche; however, a new therapeutic approach could involve the removal of these cells from the microenvironment that maintains the cancer stem cell phenotype. We have isolated multipotent sphere-forming cells from human high grade glioma (glioma sphere-forming cells (GSCs)) to investigate the adhesive and migratory properties of these cells in vitro. We have focused on the role of two closely related metalloproteinases ADAM10 and ADAM17 due to their high expression in glioblastoma and GSCs and their ability to activate cytokines and growth factors. Here, we report that ADAM10 and ADAM17 inhibition selectively increases GSC, but not neural stem cell, migration and that the migrated GSCs exhibit a differentiated phenotype. We also observed a correlation between nestin, a stem/progenitor marker, and fibronectin, an extracellular matrix protein, expression in high grade glioma tissues. GSCs adherence on fibronectin is mediated by α5β1 integrin, where fibronectin further promotes GSC migration and is an effective candidate for in vivo cancer stem cell migration out of the tumourigenic niche. Our results suggest that therapies against ADAM10 and ADAM17 may promote cancer stem cell migration away from the tumourigenic niche resulting in a differentiated phenotype that is more susceptible to treatment.

  3. Essential role of eIF5-mimic protein in animal development is linked to control of ATF4 expression

    USDA-ARS?s Scientific Manuscript database

    Translational control of ATF4 through upstream ORFs (uORFs) plays an important role in eukaryotic gene regulation. While ATF4 translation is typically induced by inhibitory phosphorylation of eIF2, ATF4 translation can be also induced by expression of a new translational inhibitor protein, eIF5-mimi...

  4. The alpha secretase ADAM10: A metalloprotease with multiple functions in the brain.

    PubMed

    Saftig, Paul; Lichtenthaler, Stefan F

    2015-12-01

    Proteins belonging to the 'A Disintegrin And Metalloproteinase' (ADAM) family are membrane-anchored proteases that are able to cleave the extracellular domains of several membrane-bound proteins in a process known as 'ectodomain shedding'. In the central nervous system, ADAM10 has attracted the most attention, since it was described as the amyloid precursor protein α-secretase over ten years ago. Despite the excitement over the potential of ADAM10 as a novel drug target in Alzheimer disease, the physiological functions of ADAM10 in the brain are not yet well understood. This is largely because of the embryonic lethality of ADAM10-deficient mice, which results from the loss of cleavage and signaling of the Notch receptor, another ADAM10 substrate. However, the recent generation of conditional ADAM10-deficient mice and the identification of further ADAM10 substrates in the brain has revealed surprisingly numerous and fundamental functions of ADAM10 in the development of the embryonic brain and also in the homeostasis of adult neuronal networks. Mechanistically, ADAM10 controls these functions by utilizing unique postsynaptic substrates in the central nervous system, in particular synaptic cell adhesion molecules, such as neuroligin-1, N-cadherin, NCAM, Ephrin A2 and A5. Consequently, a dysregulation of ADAM10 activity is linked to psychiatric and neurological diseases, such as epilepsy, fragile X syndrome and Huntington disease. This review highlights the recent progress in understanding the substrates and function as well as the regulation and cell biology of ADAM10 in the central nervous system and discusses the value of ADAM10 as a drug target in brain diseases.

  5. Functional analysis of a breast cancer-associated mutation in the intracellular domain of the metalloprotease ADAM12.

    PubMed

    Stautz, Dorte; Wewer, Ulla M; Kveiborg, Marie

    2012-01-01

    A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer.

  6. Involvement of ADAM10 in axonal outgrowth and myelination of the peripheral nerve.

    PubMed

    Jangouk, Parastoo; Dehmel, Thomas; Meyer Zu Hörste, Gerd; Ludwig, Andreas; Lehmann, Helmar C; Kieseier, Bernd C

    2009-12-01

    The disintegrin and metalloproteinase 10 (ADAM10) is a membrane-anchored metalloproteinase with both proteolytic and disintegrin characteristics. Here, we investigate the expression, regulation, and functional role of ADAM10 in axonal outgrowth and myelination of the peripheral nerve. Expression pattern analysis of 11 ADAM family members in co-cultures of rat dorsal root ganglia (DRG) neurons and Schwann cells (SCs) demonstrated the most pronounced mRNA expression for ADAM10. In further studies, ADAM10 was found to be consistently upregulated in DRG-SC co-cultures before the induction of myelination. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons before the induction of myelination. Inhibition of ADAM10 activity by the hydroxamate-based inhibitors GI254023X and GW280264X resulted in a significant decrease in the mean axonal length. These data suggest that ADAM10 represents a prerequisite for myelination, although its activity is not required during the process of myelination itself as demonstrated by expression analysis of myelin protein zero (P0) and Sudan black staining. Hence, during the process of myelin formation, ADAM10 is highly upregulated and appears to be critically involved in axonal outgrowth that is a requirement for myelination in the peripheral nerve.

  7. Nonpolar substitution at C-terminus of the prion protein, a mimic of GPI anchor, partially impairs amyloid fibrils formation

    PubMed Central

    Breydo, Leonid; Sun, Ying; Makarava, Natallia; Lee, Cheng-I; Novitskaia, Vera; Bocharova, Olga; Kao, Joseph P.Y.; Baskakov, Ilia V.

    2008-01-01

    In contrast to most amyloidogenic proteins or peptides that do not contain any significant post-translational modifications, the prion protein (PrP) is modified with either one or two polysaccharides and a GPI anchor which attaches PrP to the plasma membrane. Like other amyloidogenic proteins, however, PrP adopts a fibrillar shape when converted to a disease-specific conformation. Therefore, PrP polymerization offers a unique opportunity to examine the effects of biologically relevant non-peptidic modifications on conversion to the amyloid conformation. To test the extent to which a long hydrophobic chain at the C-terminus affects the intrinsic amyloidogenic propensity of PrP, we modified recombinant PrP with a N-myristoylamido-maleimidyl group, which can serve as a membrane anchor. We show that while this modification increases the affinity of PrP for the cell membrane, it does not alter the structure of the protein. Myristoylation of PrP affected amyloid formation in two ways: (i) it substantially decreased the extent of fibrillation, presumably due to off-pathway aggregation, and (ii) it prohibited assembly of filaments into higher-order fibrils by preventing their lateral association. The negative effect on lateral association was abolished if the myristoylated moiety at the C-terminus was replaced by a polar group of similar size or by a hydrophobic group of smaller size. When preformed PrP fibrils were provided as seeds, myristoylated PrP supported fibril elongation and formation of higher-order fibrils composed of several filaments. Our studies illustrate that, despite a bulky hydrophobic moiety at C-terminus, myristoylated PrP can still incorporate into fibrillar structure, and that the C-terminal hydrophobic substitution does not affect the size of the proteinase K resistant core, but controls the mode of lateral assembly of filaments into higher-order fibrils. PMID:17223707

  8. Mechanistic insight into interaction of Sodium Dodecyl Sulphate to asialylated form of glycoprotein: A mimic of membrane protein-lipid system.

    PubMed

    Zaidi, Nida; Khan, Rizwan Hasan

    2017-10-01

    The SDS-glycoprotein system is mimic of membrane protein-lipid system. Fate of glycoprotein, conformation and the interactive forces involved in membrane milieu are expected to be decided by the net charge on glycoprotein that may change during acidic environment in a range of pathological states, including cancer, stroke, and ischemia. Asialofetuin (ASF; asialylated form of glycoprotein) and SDS interaction is studied when glycoprotein bears varying range of net charge (i.e. at different pH's) by steady state and time-resolved spectroscopic, calorimetric and microscopic approaches. SDS interacts differently with ASF when protein is in cationic (at pH 2, 3 and 4) and in anionic states (pH 7.4). ASF undergo aggregation at pH 2, 3 and 4 whereas have enhancement in α-helical structure at pH 7.4 at sub-micellar concentrations of SDS. At pH 2, 3 and 4, the positively charged ASF interacts electrostatically with negatively charged head groups of SDS, leaving its hydrophobic tail free to interact with other protein-SDS complex and consequently lead to amyloid formation. However, at pH 7.4, the ASF interacts hydrophobically with SDS and an increase in α-helical content occurs that constrains the environment of Trp51 and consequently decreases movement of Trp conformers. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Phosphorylated, cellulose-based substrates as potential adsorbents for bone morphogenetic proteins in biomedical applications: a protein adsorption screening study using cytochrome C as a bone morphogenetic protein mimic.

    PubMed

    Mucalo, Michael R; Kato, Katsuya; Yokogawa, Yoshiyuki

    2009-06-01

    Screening studies aimed at identifying useful biomedical materials that (when combined with implants) can attract bone morphogenetic proteins to their surfaces have been conducted. In this paper, the screening process has involved carrying out protein adsorption studies using cytochrome C, as a BMP protein mimic on phosphorylated cellulose-based substrates. These studies have shown that phosphorylation of cellulose produces materials that are capable of attracting the adsorption of cytochrome C to their surface. In contrast, negligible cytochrome C adsorption was observed on the unphosphorylated cellulose-based materials. The selective uptake of the positively charged cytochrome C (from solutions at pH 9.51) by the negatively charged phosphorylated cotton and microcrystalline cellulose substrates was primarily due to this protein's high isoelectric point (i.e.p) of 9.8 which gives it a positive charge at pHprotein adsorption behaviour, this property with respect to phosphorylated materials and its potential use for selective BMP adsorption onto biomedical materials, have not been reported directly in the literature. The work thus shows that the phosphorylated cellulose-based substrates should be seriously considered as carrier materials that could be used (with preloaded BMPs) as part of an implant system to assist in implant healing.

  10. The Adams family.

    PubMed

    Douven, Igor; Verbrugge, Sara

    2010-12-01

    According to Adams's Thesis, the acceptability of an indicative conditional sentence goes by the conditional probability of its consequent given its antecedent. We test, for the first time, whether this thesis is descriptively correct and show that it is not; in particular, we show that it yields the wrong predictions for people's judgments of the acceptability of important subclasses of the class of inferential conditionals. Experimental results are presented that reveal an interaction effect between, on the one hand, the type of inferential connection between a conditional's antecedent and its consequent and, on the other, the judged acceptability of the conditional in relation to the conditional probability of its consequent given its antecedent. Specifically, these results suggest a family of theses, each pertaining to a different type of conditional, about how conditionals relate to the relevant conditional probabilities.

  11. High ADAM8 expression is associated with poor prognosis in patients with hepatocellular carcinoma.

    PubMed

    Zhang, Yun; Tan, Yong-Fei; Jiang, Chao; Zhang, Kai; Zha, Tian-Zhou; Zhang, Miao

    2013-01-01

    In this study,we investigated the ADAM8 expression in hepatocellular carcinoma (HCC) and its correlation with clinicopathologic features,including the survival of patients with HCC. Furthermore,we examined the biological processes regulated by ADAM8 during the development of using HepG2 cell line as a model system. We used immunohistochemistry to compare ADAM8 protein expression in HCC and normal liver tissues and further analyze the ADAM8 protein expression in clinicopathologically characterized 105 HCC cases.We stably knocked down the endogenous expression level of ADAM8 in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells,we examined in vitro cell growth by MTT assay,anchorage-independent growth by soft-agar colony formation assay and cell migration/invasion by transwell and boyden chamber assay. And in addition,we also investigated the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice. Protein expression level of ADAM8 was markedly higher in HCC tissues than that in the normal liver tissues (P = 0.0058).In addition,high expression of ADAM8 protein was positively correlated with serum AFP elevation,tumor size,histological differentiation,tumor recurrence,tumor metastasis,and tumor stage. Patients with higher ADAM8 expression showed a significantly shorter overall survival time than patients with low ADAM8 expression. Multivariate analysis suggested that ADAM8 expression might be an independent prognostic indicator (p = 0.016) for the survival of patients with HCC. ADAM8-specific shRNA (shADAM8) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells,the shADAM8 cells exhibited significantly reduced in vitro cell growth,anchorage-independent growth,cell migration and invasion (p < 0.05).In vivo,the xenograft transplants from shADAM8 cells gave rise to much smaller

  12. A new gene for asthma: would you ADAM and Eve it?

    PubMed

    Cookson, William

    2003-04-01

    Recently, a novel gene was reported to underlie asthma. Linkage to the short arm of chromosome 20 in a genome screen was followed by positive tests of association that centre on the gene for a membrane-anchored zinc-dependent metalloproteinase known as ADAM33. The domain structure of the ADAM33 protein gives capabilities of proteolysis, adhesion, cell fusion and intracellular signalling. Although its function is at present unknown, these potential actions of ADAM33 provide many possibilities for further research.

  13. Systematic substrate identification indicates a central role for the metalloprotease ADAM10 in axon targeting and synapse function

    PubMed Central

    Kuhn, Peer-Hendrik; Colombo, Alessio Vittorio; Schusser, Benjamin; Dreymueller, Daniela; Wetzel, Sebastian; Schepers, Ute; Herber, Julia; Ludwig, Andreas; Kremmer, Elisabeth; Montag, Dirk; Müller, Ulrike; Schweizer, Michaela; Saftig, Paul; Bräse, Stefan; Lichtenthaler, Stefan F

    2016-01-01

    Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain. DOI: http://dx.doi.org/10.7554/eLife.12748.001 PMID:26802628

  14. The role of CDCP1 (CUB domain-containing protein 1) and ADAM12 (a disintegrin and metalloproteinase 12) in ovarian cancer.

    PubMed

    Vlad, Catalin; Kubelac, Paul; Onisim, Andrea; Irimie, Alexandru; Achimas-Cadariu, Patriciu

    2015-01-01

    Ovarian cancer (OC) is the second most common gynecological cancer and the deadliest in industrialized countries, with poor outcomes that indicate an urgent need to provide a greater insight into the molecular mechanisms underlying OC. Insights into the complex tumor microenviroment show that besides tumor islets, OC biomarkers can derive from newly formed blood vessels that have endothelial cells with a different molecular signature in comparison with their normal counterparts. In this view, recent research has been able to highlight promising candidates such as CDCP1 and ADAM12. Our present review summarises their implications in cancer progression with a focus on OC.

  15. Medial prefrontal cortical injections of c-fos antisense oligonucleotides transiently lower c-Fos protein and mimic amphetamine withdrawal behaviours.

    PubMed

    Persico, A M; Schindler, C W; Davis, S C; Ambrosio, E; Uhl, G R

    1998-02-01

    Prefrontal cerebral cortical areas display decreased expression of several transcription factor/immediate-early genes, including c-fos, during amphetamine withdrawal. Antisense strategies can help to test possible roles for this prefrontal c-fos down-regulation in the behavioural correlates of amphetamine withdrawal. Medial prefrontal cortical injections delivering 1.7 nmoles of anti c-fos oligonucleotides revealed an approximately 3 h half-life for phosphothioate and a 15 min half-life for phosphodiester oligonucleotides. Antisense phosphothioates complementary to the c-fos translational start site reduced levels of c-Fos protein, while exerting modest and variable effects on c-fos messenger RNA levels. Neither missense phosphorothioate nor antisense phosphodiester oligonucleotides significantly reduced levels of either c-fos messenger RNA or protein. Animals injected with anti c-fos phosphothioate oligonucleotides into the medial prefrontal cortex displayed marked reductions in linear locomotor activity and repetitive movements measured in a novel environment, effects not seen when missense oligonucleotides were used or when animals were accustomed to the activity monitor prior to antisense oligonucleotide injection. Behavioural changes produced by prefrontal cortical injections of c-fos antisense oligonucleotides closely mimic alterations recorded during amphetamine withdrawal. Prefrontal c-fos could thus conceivably play roles in the neurobiological underpinnings of psychostimulant withdrawal and of responses to stressors such as exposure to novel environments.

  16. Neuronal ADAM10 Promotes Outgrowth of Small-Caliber Myelinated Axons in the Peripheral Nervous System.

    PubMed

    Meyer zu Horste, Gerd; Derksen, Angelika; Stassart, Ruth; Szepanowski, Fabian; Thanos, Melissa; Stettner, Mark; Boettcher, Christina; Lehmann, Helmar C; Hartung, Hans-Peter; Kieseier, Bernd C

    2015-11-01

    The regulation of myelination and axonal outgrowth in the peripheral nervous system is controlled by a complex signaling network involving various signaling pathways. Members of the A Disintegrin And Metalloproteinase (ADAM) family are membrane-anchored proteinases with both proteolytic and disintegrin characteristics that modulate the function of signaling molecules. One family member, ADAM17, is known to influence myelination by cleaving and thus regulating one of the key signals, neuregulin-1, which controls peripheral nervous system myelination. A similar function for ADAM10 had been suggested by previous in vitro studies. Here, we assessed whether ADAM10 exerts a similar function in vivo and deleted ADAM10 in a cell type-specific manner in either neurons or Schwann cells. We found that ADAM10 is not required in either Schwann cells or neurons for normal myelination during development or for remyelination after injury. Instead, ADAM10 is required specifically in neurons for the outgrowth of myelinated small-fiber axons in vitro and after injury in vivo. Thus, we report for the first time a neuron-intrinsic function of ADAM10 in axonal regeneration that is distinct from that of the related protein family member ADAM17 and that may have implications for targeting ADAM function in nervous system diseases.

  17. Control of ADAM17 activity by regulation of its cellular localisation

    PubMed Central

    Lorenzen, Inken; Lokau, Juliane; Korpys, Yvonne; Oldefest, Mirja; Flynn, Charlotte M.; Künzel, Ulrike; Garbers, Christoph; Freeman, Matthew; Grötzinger, Joachim; Düsterhöft, Stefan

    2016-01-01

    An important, irreversible step in many signalling pathways is the shedding of membrane-anchored proteins. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. This central role in signalling implies that ADAM17 activity has to be tightly regulated, including at the level of localisation. Most mature ADAM17 is localised intracellularly, with only a small amount at the cell surface. We found that ADAM17 is constitutively internalised by clathrin-coated pits and that physiological stimulators such as GPCR ligands induce ADAM17-mediated shedding, but do not alter the cell-surface abundance of the protease. In contrast, the PKC-activating phorbol ester PMA, often used as a strong inducer of ADAM17, causes not only proteolysis by ADAM17 but also a rapid increase of the mature protease at the cell surface. This is followed by internalisation and subsequent degradation of the protease. Eventually, this leads to a substantial downregulation of mature ADAM17. Our results therefore imply that physiological activation of ADAM17 does not rely on its relocalisation, but that PMA-induced PKC activity drastically dysregulates the localisation of ADAM17. PMID:27731361

  18. Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

    PubMed

    Calle, Luis P; Echeverria, Begoña; Franconetti, Antonio; Serna, Sonia; Fernández-Alonso, M Carmen; Diercks, Tammo; Cañada, F Javier; Ardá, Ana; Reichardt, Niels-Christian; Jiménez-Barbero, Jesús

    2015-08-03

    Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. A unique capsular polysaccharide structure from the psychrophilic marine bacterium Colwellia psychrerythraea 34H that mimics antifreeze (glyco)proteins.

    PubMed

    Carillo, Sara; Casillo, Angela; Pieretti, Giuseppina; Parrilli, Ermenegilda; Sannino, Filomena; Bayer-Giraldi, Maddalena; Cosconati, Sandro; Novellino, Ettore; Ewert, Marcela; Deming, Jody W; Lanzetta, Rosa; Marino, Gennaro; Parrilli, Michelangelo; Randazzo, Antonio; Tutino, Maria L; Corsaro, M Michela

    2015-01-14

    The low temperatures of polar regions and high-altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a γ-proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one- and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanics and dynamics calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

  20. Parkinson’s Protein α-Synuclein Binds Efficiently and with a Novel Conformation to Two Natural Membrane Mimics

    PubMed Central

    Kumar, Pravin; Segers-Nolten, Ine M. J.; Schilderink, Nathalie; Subramaniam, Vinod; Huber, Martina

    2015-01-01

    Binding of human α-Synuclein, a protein associated with Parkinson’s disease, to natural membranes is thought to be crucial in relation to its pathological and physiological function. Here the binding of αS to small unilamellar vesicles mimicking the inner mitochondrial and the neuronal plasma membrane is studied in situ by continuous wave and pulsed electron paramagnetic resonance. Local binding information of αS spin labeled by MTSL at positions 56 and 69 respectively shows that also helix 2 (residues 50–100) binds firmly to both membranes. By double electron-electron resonance (DEER) on the mutant spin labeled at positions 27 and 56 (αS 27/56) a new conformation on the membrane is found with a distance of 3.6 nm/ 3.7 nm between residues 27 and 56. In view of the low negative charge density of these membranes, the strong interaction is surprising, emphasizing that function and pathology of αS could involve synaptic vesicles and mitochondria. PMID:26588454

  1. Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein

    SciTech Connect

    Davidson, Amy; Leeper, Thomas C.; Athanassiou, Zafiria; Patora-Komisarska, Krystyna; Karn, Jonathan; Robinson, John A.; Varani, Gabriele

    2009-07-21

    The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide–RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.

  2. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic

    PubMed Central

    Mendez, Aaron S; Alfaro, Jennifer; Morales-Soto, Marisol A; Dar, Arvin C; McCullagh, Emma; Gotthardt, Katja; Li, Han; Acosta-Alvear, Diego; Sidrauski, Carmela; Korennykh, Alexei V; Bernales, Sebastian; Shokat, Kevan M; Walter, Peter

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.05434.001 PMID:25986605

  3. ADAM10 as a target for anti-cancer therapy.

    PubMed

    Moss, Marcia L; Stoeck, Alexander; Yan, Wenbo; Dempsey, Peter J

    2008-02-01

    There is a great unmet medical need in the area of cancer treatment. A potential therapeutic target for intervention in cancer is ADAM10. ADAM10 is a disintegrin-metalloproteinase that processes membrane bound proteins from the cell surface to yield soluble forms. Pharmaceutical companies are actively seeking out inhibitors of ADAM10 for treatments in cancer as the enzyme is known to release the ErbB receptor, HER2/ErbB2 from the cell membrane, an event that is necessary for HER2 positive tumor cells to proliferate. ADAM10 is also capable of processing betacellulin indicating that an inhibitor could be used against EGFR/ErbB1 and/or HER4/ErbB4 receptor positive tumor cells that are betacellulin-dependent. ADAM10 is the principle sheddase for several other molecules associated with cancer proliferation, differentiation, adhesion and migration such as Notch, E-cadherin, CD44 and L1 adhesion molecule indicating that targeting ADAM10 with specific inhibitors could be beneficial.

  4. ω-Turn: a novel β-turn mimic in globular proteins stabilized by main-chain to side-chain C−H···O interaction.

    PubMed

    Dhar, Jesmita; Chakrabarti, Pinak; Saini, Harpreet; Raghava, Gajendra Pal Singh; Kishore, Raghuvansh

    2015-02-01

    Mimicry of structural motifs is a common feature in proteins. The 10-membered hydrogen-bonded ring involving the main-chain C − O in a β-turn can be formed using a side-chain carbonyl group leading to Asx-turn. We show that the N − H component of hydrogen bond can be replaced by a C(γ) -H group in the side chain, culminating in a nonconventional C − H···O interaction. Because of its shape this β-turn mimic is designated as ω-turn, which is found to occur ∼ three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C − H···O interaction occurring between the terminal residues, constraining the torsion angles ϕi + 1, ψi + 1, ϕi + 2 and χ'1(i + 2) (using the interacting C(γ) atom). Based on these angles there are two types of ω-turns, each of which can be further divided into two groups. C(β) -branched side-chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal-binding sites. N-linked glycosylation occurs at the consensus pattern Asn-Xaa-Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω-turn, which may be the recognition site for protein modification. Location between two β-strands is the most common occurrence in protein tertiary structure, and being generally exposed ω-turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. © 2014 Wiley Periodicals, Inc.

  5. N-Glycosylation Regulates ADAM8 Processing and Activation*

    PubMed Central

    Srinivasan, Srimathi; Romagnoli, Mathilde; Bohm, Andrew; Sonenshein, Gail E.

    2014-01-01

    The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)α-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ERα-negative breast cancer cells but not on ERα-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8. PMID:25336660

  6. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution.

    PubMed

    Filonov, Grigory S; Moon, Jared D; Svensen, Nina; Jaffrey, Samie R

    2014-11-19

    Genetically encoded fluorescent ribonucleic acids (RNAs) have diverse applications, including imaging RNA trafficking and as a component of RNA-based sensors that exhibit fluorescence upon binding small molecules in live cells. These RNAs include the Spinach and Spinach2 aptamers, which bind and activate the fluorescence of fluorophores similar to that found in green fluorescent protein. Although additional highly fluorescent RNA-fluorophore complexes would extend the utility of this technology, the identification of novel RNA-fluorophore complexes is difficult. Current approaches select aptamers on the basis of their ability to bind fluorophores, even though fluorophore binding alone is not sufficient to activate fluorescence. Additionally, aptamers require extensive mutagenesis to efficiently fold and exhibit fluorescence in living cells. Here we describe a platform for rapid generation of highly fluorescent RNA-fluorophore complexes that are optimized for function in cells. This procedure involves selection of aptamers on the basis of their binding to fluorophores, coupled with fluorescence-activated cell sorting (FACS) of millions of aptamers expressed in Escherichia coli. Promising aptamers are then further optimized using a FACS-based directed evolution approach. Using this approach, we identified several novel aptamers, including a 49-nt aptamer, Broccoli. Broccoli binds and activates the fluorescence of (Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one. Broccoli shows robust folding and green fluorescence in cells, and increased fluorescence relative to Spinach2. This reflects, in part, improved folding in the presence of low cytosolic magnesium concentrations. Thus, this novel fluorescence-based selection approach simplifies the generation of aptamers that are optimized for expression and performance in living cells.

  7. Evolution of Vertebrate Adam Genes; Duplication of Testicular Adams from Ancient Adam9/9-like Loci

    PubMed Central

    Wei, Shuo

    2015-01-01

    Members of the disintegrin metalloproteinase (ADAM) family have important functions in regulating cell-cell and cell-matrix interactions as well as cell signaling. There are two major types of ADAMs: the somatic ADAMs (sADAMs) that have a significant presence in somatic tissues, and the testicular ADAMs (tADAMs) that are expressed predominantly in the testis. Genes encoding tADAMs can be further divided into two groups: group I (intronless) and group II (intron-containing). To date, tAdams have only been reported in placental mammals, and their evolutionary origin and relationship to sAdams remain largely unknown. Using phylogenetic and syntenic tools, we analyzed the Adam genes in various vertebrates ranging from fishes to placental mammals. Our analyses reveal duplication and loss of some sAdams in certain vertebrate species. In particular, there exists an Adam9-like gene in non-mammalian vertebrates but not mammals. We also identified putative group I and group II tAdams in all amniote species that have been examined. These tAdam homologues are more closely related to Adams 9 and 9-like than to other sAdams. In all amniote species examined, group II tAdams lie in close vicinity to Adam9 and hence likely arose from tandem duplication, whereas group I tAdams likely originated through retroposition because of their lack of introns. Clusters of multiple group I tAdams are also common, suggesting tandem duplication after retroposition. Therefore, Adam9/9-like and some of the derived tAdam loci are likely preferred targets for tandem duplication and/or retroposition. Consistent with this hypothesis, we identified a young retroposed gene that duplicated recently from Adam9 in the opossum. As a result of gene duplication, some tAdams were pseudogenized in certain species, whereas others acquired new expression patterns and functions. The rapid duplication of Adam genes has a major contribution to the diversity of ADAMs in various vertebrate species. PMID:26308360

  8. Evolution of Vertebrate Adam Genes; Duplication of Testicular Adams from Ancient Adam9/9-like Loci.

    PubMed

    Bahudhanapati, Harinath; Bhattacharya, Shashwati; Wei, Shuo

    2015-01-01

    Members of the disintegrin metalloproteinase (ADAM) family have important functions in regulating cell-cell and cell-matrix interactions as well as cell signaling. There are two major types of ADAMs: the somatic ADAMs (sADAMs) that have a significant presence in somatic tissues, and the testicular ADAMs (tADAMs) that are expressed predominantly in the testis. Genes encoding tADAMs can be further divided into two groups: group I (intronless) and group II (intron-containing). To date, tAdams have only been reported in placental mammals, and their evolutionary origin and relationship to sAdams remain largely unknown. Using phylogenetic and syntenic tools, we analyzed the Adam genes in various vertebrates ranging from fishes to placental mammals. Our analyses reveal duplication and loss of some sAdams in certain vertebrate species. In particular, there exists an Adam9-like gene in non-mammalian vertebrates but not mammals. We also identified putative group I and group II tAdams in all amniote species that have been examined. These tAdam homologues are more closely related to Adams 9 and 9-like than to other sAdams. In all amniote species examined, group II tAdams lie in close vicinity to Adam9 and hence likely arose from tandem duplication, whereas group I tAdams likely originated through retroposition because of their lack of introns. Clusters of multiple group I tAdams are also common, suggesting tandem duplication after retroposition. Therefore, Adam9/9-like and some of the derived tAdam loci are likely preferred targets for tandem duplication and/or retroposition. Consistent with this hypothesis, we identified a young retroposed gene that duplicated recently from Adam9 in the opossum. As a result of gene duplication, some tAdams were pseudogenized in certain species, whereas others acquired new expression patterns and functions. The rapid duplication of Adam genes has a major contribution to the diversity of ADAMs in various vertebrate species.

  9. John Adams - an outstanding career.

    PubMed

    Lewin, David

    2016-12-07

    A distinguished nurse, teacher, researcher and historian, John Adams was educated at Aylesbury Grammar School and graduated from Selwyn College, Cambridge, with a degree in theological and religious studies.

  10. iRhoms 1 and 2 are essential upstream regulators of ADAM17-dependent EGFR signaling

    PubMed Central

    Li, Xue; Maretzky, Thorsten; Weskamp, Gisela; Monette, Sébastien; Qing, Xiaoping; Issuree, Priya Darshinee A.; Crawford, Howard C.; McIlwain, David R.; Mak, Tak W.; Salmon, Jane E.; Blobel, Carl P.

    2015-01-01

    The metalloproteinase ADAM17 (a disintegrin and metalloprotease 17) controls EGF receptor (EGFR) signaling by liberating EGFR ligands from their membrane anchor. Consequently, a patient lacking ADAM17 has skin and intestinal barrier defects that are likely caused by lack of EGFR signaling, and Adam17−/− mice die perinatally with open eyes, like Egfr−/− mice. A hallmark feature of ADAM17-dependent EGFR ligand shedding is that it can be rapidly and posttranslationally activated in a manner that requires its transmembrane domain but not its cytoplasmic domain. This suggests that ADAM17 is regulated by other integral membrane proteins, although much remains to be learned about the underlying mechanism. Recently, inactive Rhomboid 2 (iRhom2), which has seven transmembrane domains, emerged as a molecule that controls the maturation and function of ADAM17 in myeloid cells. However, iRhom2−/− mice appear normal, raising questions about how ADAM17 is regulated in other tissues. Here we report that iRhom1/2−/− double knockout mice resemble Adam17−/− and Egfr−/− mice in that they die perinatally with open eyes, misshapen heart valves, and growth plate defects. Mechanistically, we show lack of mature ADAM17 and strongly reduced EGFR phosphorylation in iRhom1/2−/− tissues. Finally, we demonstrate that iRhom1 is not essential for mouse development but regulates ADAM17 maturation in the brain, except in microglia, where ADAM17 is controlled by iRhom2. These results provide genetic, cell biological, and biochemical evidence that a principal function of iRhoms1/2 during mouse development is to regulate ADAM17-dependent EGFR signaling, suggesting that iRhoms1/2 could emerge as novel targets for treatment of ADAM17/EGFR-dependent pathologies. PMID:25918388

  11. Modulation of integrin α4β1 by ADAM28 promotes lymphocyte adhesion and transendothelial migration.

    PubMed

    McGinn, Owen J; English, William R; Roberts, Stephanie; Ager, Ann; Newham, Peter; Murphy, Gillian

    2011-10-01

    ADAMs (a disintegrin and metalloproteinase) are a family of type I transmembrane glycoproteins related to snake venom metalloproteases and disintegrins. They are regulatory proteins that modulate intercellular adhesion and the bioavailability of growth factors, and have been implicated in many disease states, including cancer, immunity and inflammation. One member of the ADAM family, ADAM28, has been reported to bind to the integrin α4β1 in humans; however, the distribution of ADAM28 and the biological consequences of ADAM28-α4β1 interactions are yet to be fully elucidated. The expression of ADAM28 in human and murine tissues was examined by multiple Affymetrix microarray analyses, real-time RT-PCR (reverse transcription-PCR) and immunohistochemical staining. We found that ADAM28 has a relatively restricted expression pattern in mouse and human and is highly expressed in the B-lymphocyte lineage, including chronic lymphocytic leukaemic B-cells. The murine B-lymphoma line L1-2 and recombinant soluble murine ADAM28 were used to investigate ADAM28-α4β1 interactions. Our data reveal that ADAM28 binding to α4β1 is typical of integrin-ligand interactions, since it is attenuated by anti-functional integrin antibodies, and is enhanced by Mn2+ and the integrin mAb (monoclonal antibody) 9EG7. However, a key finding was that soluble ADAM28 unexpectedly enhanced α4β1-dependent cell adhesion to VCAM-1 (vascular cell adhesion molecule-1). In so doing ADAM28 was able to influence lymphocyte adhesion to, and migration through, endothelial monolayers, suggesting a physiological role for ADAM28 in regulating the specific spatial and temporal transendothelial migration of lymphocytes.

  12. Molecular Mechanisms of TNFR-associated Factor 6 (TRAF6) Utilization by the Oncogenic Viral Mimic of CD40, Latent Membrane Protein 1 (LMP1)*

    PubMed Central

    Arcipowski, Kelly M.; Stunz, Laura L.; Graham, John P.; Kraus, Zachary J.; Bush, Tony J. Vanden; Bishop, Gail A.

    2011-01-01

    Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1. PMID:21262968

  13. Adam Smith and dependency.

    PubMed

    Ozler, Sule

    2012-06-01

    The focus of this paper is the works and life of Adam Smith, who is widely recognized as the father and founder of contemporary economics. Latent content analysis is applied to his seminal text in economics, An Inquiry into the Nature and Causes of the Wealth of Nations (1776). The results reveal that Smith considers dependence on others a problem and sees the solution to this problem in impersonalized interdependence. In addition, his views on social dependency and personal dependency, reflected in his Lectures on Jurisprudence (1963) and The Theory of Moral Sentiments (1759), are analyzed. This analysis suggests a central tension between dependence and independence in Smith's writings. The personal dependency patterns he exhibited in his life, which also suggest a tension between dependence and independence, are identified through a reading of his biographies. Based on insights from psychoanalytic literature, this paper proposes that developing the ideas in the Wealth of Nations was part of Smith's creative solution to this tension. In particular, his solution to one individual's dependence on another was through a system of impersonalized interdependence. In other words, Smith defended against his personal dependence through his economic theorizing.

  14. Adam Smith on population.

    PubMed

    Spengler, J J

    1970-11-01

    Abstract Adam Smith dealt with questions of population mainly in his Wealth of Nations. His discussion falls roughly under five heads and reflects in considerable measure his image of the English economy. (1) A country's population capacity, given the average level of consumption, was conditioned by the stock of land, the skill with which it was cultivated, and the degree to which division of labour could be increased and thereby augment output for domestic use and sale in external markets. (2) Growth of population was essentially in response to growth of the demand for labour and served to increase division of labour. (3) The social mechanisms underlying elevation of the scale of living are touched upon, and in an optimistic spirit. (4) The distribution of a country's population responded to its progress in opulence, with the rate of this progress conditioned by the degree to which inappropriate (e.g. mercantilist) policies were avoided. (5) Smith dealt briefly with such matters as colonies, education, size of economy, environmental influences, and public policy, all of which he recognized as significant for the quantity and quality of a country's numbers.

  15. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development

    PubMed Central

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development. PMID:27043020

  16. ADAM10 Is Involved in Cell Junction Assembly in Early Porcine Embryo Development.

    PubMed

    Kwon, Jeongwoo; Jeong, Sung-min; Choi, Inchul; Kim, Nam-Hyung

    2016-01-01

    ADAM10 (A Disintegrin and Metalloprotease domain-containing protein 10) is a cell surface protein with a unique structure possessing both potential adhesion and protease domains. However, the role of ADAM10 in preimplantation stage embryos is not clear. In this study, we examined the expression patterns and functional roles of ADAM10 in porcine parthenotes during preimplantation development. The transcription level of ADAM10 dramatically increased from the morula stage onward. Immunostaining revealed that ADAM10 was present in both the nucleus and cytoplasm in early cleavage stage embryos, and localized to the apical region of the outer cells in morula and blastocyst embryos. Knockdown (KD) of ADAM10 using double strand RNA did not alter preimplantation embryo development until morula stage, but resulted in significantly reduced development to blastocyst stage. Moreover, the KD blastocyst showed a decrease in gene expression of adherens and tight junction (AJ/TJ), and an increase in trophectoderm TJ permeability by disrupting TJ assembly. Treatment with an ADAM10 specific chemical inhibitor, GI254023X, at the morula stage also inhibited blastocyst development and led to disruption of TJ assembly. An in situ proximity ligation assay demonstrated direct interaction of ADAM10 with coxsackie virus and adenovirus receptor (CXADR), supporting the involvement of ADAM10 in TJ assembly. In conclusion, our findings strongly suggest that ADADM10 is important for blastocyst formation rather than compaction, particularly for TJ assembly and stabilization in preimplantation porcine parthenogenetic development.

  17. Secretion-Positive LGI1 Mutations Linked to Lateral Temporal Epilepsy Impair Binding to ADAM22 and ADAM23 Receptors

    PubMed Central

    Dazzo, Emanuela; Belluzzi, Elisa; Malacrida, Sandro; Vitiello, Libero; Greggio, Elisa; Tosatto, Silvio C. E.

    2016-01-01

    Autosomal dominant lateral temporal epilepsy (ADTLE) is a focal epilepsy syndrome caused by mutations in the LGI1 gene, which encodes a secreted protein. Most ADLTE-causing mutations inhibit LGI1 protein secretion, and only a few secretion-positive missense mutations have been reported. Here we describe the effects of four disease-causing nonsynonymous LGI1 mutations, T380A, R407C, S473L, and R474Q, on protein secretion and extracellular interactions. Expression of LGI1 mutant proteins in cultured cells shows that these mutations do not inhibit protein secretion. This finding likely results from the lack of effects of these mutations on LGI1 protein folding, as suggested by 3D protein modelling. In addition, immunofluorescence and co-immunoprecipitation experiments reveal that all four mutations significantly impair interaction of LGI1 with the ADAM22 and ADAM23 receptors on the cell surface. These results support the existence of a second mechanism, alternative to inhibition of protein secretion, by which ADLTE-causing LGI1 mutations exert their loss-of-function effect extracellularly, and suggest that interactions of LGI1 with both ADAM22 and ADAM23 play an important role in the molecular mechanisms leading to ADLTE. PMID:27760137

  18. Metalloproteinases ADAM12 and MMP-14 are associated with cavernous sinus invasion in pituitary adenomas.

    PubMed

    Wang, Junwen; Voellger, Benjamin; Benzel, Julia; Schlomann, Uwe; Nimsky, Christopher; Bartsch, Jörg W; Carl, Barbara

    2016-09-15

    Invasion of tumor cells critically depends on cell-cell or cell-extracellular matrix interactions. Enzymes capable of modulating these interactions belong to the proteinase families of ADAM (a disintegrin and metalloprotease) and MMP (matrix metalloprotease) proteins. Our objective is to examine their expression levels and evaluate the relationship between expression levels and cavernous sinus invasion in pituitary adenomas. Tissue samples from 35 patients with pituitary adenomas were analyzed. Quantitative real-time polymerase chain reaction (qPCR) was employed to assess mRNA expression levels for ADAM and MMP genes. Protein levels were examined using immunohistochemistry and Western Blot. Correlation analyses between expression levels and clinical parameters were performed. By silencing ADAM12 and MMP-14 with siRNA in a mouse pituitary adenoma cell line (TtT/GF), their cellular effects were investigated. In our study, nine women and 26 men were included, with a mean age of 53.1 years (range 15-84 years) at the time of surgery. There were 19 cases with cavernous sinus invasion. The proteins ADAM12 and MMP-14 were significantly up-regulated in invasive adenomas compared to noninvasive adenomas. Both human isoforms of ADAM12 (ADAM12L and ADAM12s) were involved in tumor invasion; moreover, ADAM12L was found to correlate positively with Ki-67 proliferation index in pituitary adenomas. In TtT/GF pituitary adenoma cells, silencing of ADAM12 and MMP-14 significantly inhibited cell invasion and migration, respectively, whereas only silencing of ADAM12 suppressed cell proliferation. We conclude that ADAM12 and MMP-14 are associated with cavernous sinus invasion in pituitary adenomas, which qualifies these proteins in diagnosis and therapy.

  19. ADAM8 is a negative regulator of retinal neovascularization and of the growth of heterotopically injected tumor cells in mice

    PubMed Central

    Guaiquil, Victor H.; Swendeman, Steven; Zhou, Wenhui; Guaiquil, Patricio; Weskamp, Gisela; Bartsch, Jörg W.

    2010-01-01

    ADAM8 is a member of the “a disintegrin and metalloproteinase” (ADAM) family of membrane-anchored metalloproteinases. ADAM8-deficient mice have no evident spontaneous developmental or pathological defects, and little is currently known about the role of ADAM8 in disease. Here, we investigated the contribution of ADAM8 to pathological neovascularization in mice using an oxygen-induced retinopathy (OIR) model and heterotopical injection of tumor cells. We found an increase in retinal re-vascularization but fewer neovascular tufts in the OIR model and increased growth of heterotopically injected tumor cells in Adam8−/− mice compared with wild-type controls. These results suggest that ADAM8 functions to limit both of these processes in wild-type mice. In cell-based assays, overexpression of ADAM8 increased the ectodomain shedding of several co-expressed membrane proteins with roles in angiogenesis (CD31, Tie-2, Flk-1, Flt-1, EphrinB2, EphB4, VE-cadherin, KL-1, E-selectin, and neuregulin-1β2). Thus, dysregulated expression of ADAM8 in endothelial cells in vivo could potentially increase the processing of these and other substrate proteins. Taken together, our findings suggest that inhibiting ADAM8 could be useful for promoting re-vascularization and thereby preventing formation of neovascular tufts in proliferative retinopathies. On the other hand, blocking ADAM8 could be detrimental in the context of rapidly growing tumors. PMID:20119708

  20. Binding Characteristics of Small Molecules that Mimic Nucleocapsid Protein-induced Maturation of Stem-loop-1 of HIV-1 RNA†

    PubMed Central

    Chung, Janet; Ulyanov, Nikolai B.; Guilbert, Christophe; Mujeeb, Anwer; James, Thomas L.

    2010-01-01

    As a retrovirus, the human immunodeficiency virus (HIV-1) packages two copies of the RNA genome as a dimer in the infectious virion. Dimerization is initiated at the dimer initiation site (DIS) which encompasses stem-loop 1 (SL1) in the 5’-UTR of the genome. Study of genomic dimerization has been facilitated by the discovery that short RNA fragments containing SL1 can dimerize spontaneously without any protein factors. Based on the palindromic nature of SL1, a kissing loop model has been proposed. First, a metastable kissing dimer is formed via standard Watson-Crick base pairs and then converted into a more stable extended dimer by the viral nucleocapsid protein (NCp7). This dimer maturation in vitro is believed to mimic initial steps in the RNA maturation in vivo, which is correlated with viral infectivity. We previously discovered a small molecule activator, Lys-Ala-7-amido-4-methylcoumarin (KA-AMC), which facilitates dimer maturation in vitro, and determined aspects of its structure-activity relationship. In this report, we present measurements of the binding affinity of the activators and characterization of their interactions with the SL1 RNA. Guanidinium groups and increasing positive charge on the side chain enhance affinity and activity, but features in the aromatic ring at least partially decouple affinity from activity. Although KA-AMC can bind to multiple structural motifs, NMR study showed KA-AMC preferentially binds to unique structural motifs, such as the palindromic loop and the G-rich internal loop in the SL1 RNA. NCp7 binds to SL1 only an order of magnitude tighter than the best small molecule ligand tested. The study presented here provides guidelines for design of superior small molecule binders to the SL1 RNA that have the potential of being developed as an antiviral by either interfering with SL1-NCp7 interaction at the packaging and/or maturation stages. PMID:20565056

  1. Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation

    PubMed Central

    Kozel, Caitlin; Thompson, Brytteny; Hustak, Samantha; Moore, Chelsea; Nakashima, Akio; Singh, Chingakham Ranjit; Reid, Megan; Cox, Christian; Papadopoulos, Evangelos; Luna, Rafael E.; Anderson, Abbey; Tagami, Hideaki; Hiraishi, Hiroyuki; Slone, Emily Archer; Yoshino, Ken-ichi; Asano, Masayo; Gillaspie, Sarah; Nietfeld, Jerome; Perchellet, Jean-Pierre; Rothenburg, Stefan; Masai, Hisao; Wagner, Gerhard; Beeser, Alexander; Kikkawa, Ushio; Fleming, Sherry D.; Asano, Katsura

    2016-01-01

    ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila. The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions. PMID:27325740

  2. The Cytosolic Domain of Protein-tyrosine Kinase 7 (PTK7), Generated from Sequential Cleavage by a Disintegrin and Metalloprotease 17 (ADAM17) and γ-Secretase, Enhances Cell Proliferation and Migration in Colon Cancer Cells*

    PubMed Central

    Na, Hye-Won; Shin, Won-Sik; Ludwig, Andreas; Lee, Seung-Taek

    2012-01-01

    Protein-tyrosine kinase 7 (PTK7) is a member of the defective receptor protein-tyrosine kinases and is known to function as a regulator of planar cell polarity during development. Its expression is up-regulated in some cancers including colon carcinomas. A 100-kDa fragment of PTK7 was detected in the culture media from colon cancer cells and HEK293 cells. The shed fragment was named sPTK7-Ig1–7 because its molecular mass was very similar to that of the entire extracellular domain of PTK7 that contains immunoglobulin-like loops 1 to 7 (Ig1–7). The shedding of sPTK7-Ig1–7 was enhanced by treatment with phorbol 12-myristate 13-acetate. In addition to the sPTK7-Ig1–7 found in the culture medium, two C-terminal fragments of PTK7 were detected in the cell lysates: PTK7-CTF1, which includes a transmembrane segment and a cytoplasmic domain, and PTK7-CTF2, which lacks most of the transmembrane segment from PTK7-CTF1. Analysis of PTK7 processing in the presence of various protease inhibitors or after knockdown of potential proteases suggests that shedding of PTK7 into sPTK7-Ig1–7 and PTK7-CTF1 is catalyzed by ADAM17, and further cleavage of PTK7-CTF1 into PTK7-CTF2 is mediated by the γ-secretase complex. PTK7-CTF2 localizes to the nucleus and enhances proliferation, migration, and anchorage-independent colony formation. Our findings demonstrate a novel role for PTK7 in the tumorigenesis via generation of PTK7-CTF2 by sequential cleavage of ADAM17 and γ-secretase. PMID:22665490

  3. ADAM12 induces estrogen-independence in breast cancer cells.

    PubMed

    Roy, Roopali; Moses, Marsha A

    2012-02-01

    Antiestrogen therapy has been used successfully to prolong disease-free and overall survival of ER positive breast cancer patients. However, 50% of patients with ER+ tumors fail to respond to such therapy or eventually acquire resistance to endocrine therapy, resulting in tumor progression and mortality. It is imperative, therefore, to understand the mechanisms that lead to hormone refractory breast cancer in order to develop therapeutics that can modulate the resistance to antiestrogen therapy. The protease, ADAM12, can be detected in the urine of breast cancer patients and its levels correlate with disease status, stage, and cancer risk. Within the context of this study, the authors have investigated the role of the two distinct isoforms of ADAM12 in breast tumor cell proliferation and as potential mediators of endocrine resistance. Using stable clones of ADAM12-overexpressing MCF-7 cells, the authors analyzed proliferation rates of these ER+ breast tumor cells both in estrogen-depleted medium and in the presence of the antiestrogens, tamoxifen, and ICI 182,780. Acquired estrogen resistance in these cells was analyzed using phospho-RTK analysis. Upregulation and phosphorylation of proteins were detected via immunoprecipitation and immunoblotting. EGFR and MAPK inhibitors were used to explore the mechanism of acquired estrogen resistance in breast tumor cells. It was observed that overexpression of the two isoforms, transmembrane ADAM12-L, and secreted ADAM12-S, in breast tumor cells promoted estrogen-independent proliferation. In ADAM12-L-expressing cells, estrogen-independence was a direct result of increased EGFR expression and MAPK activation, whereas, the mechanism in ADAM12-S-expressing cells may be enhanced IGF-1R signaling. The importance of the EGFR signaling pathway in the estrogen-independent growth of ADAM12-L expressing cells was highlighted by the effect of EGFR inhibitors AG1478 and PD15035 or MAPK inhibitor U0126, each of which abolished the

  4. The DNA-mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription.

    PubMed

    Melkina, Olga E; Goryanin, Ignatiy I; Zavilgelsky, Gennadii B

    2016-11-01

    The antirestriction proteins ArdA ColIb-P9, Arn T4 and Ocr T7 specifically inhibit type I and type IV restriction enzymes and belong to the family of DNA-mimic proteins because their three-dimensional structure is similar to the double-helical B-form DNA. It is proposed that the DNA-mimic proteins are able to bind nucleoid protein H-NS and alleviate H-NS-silencing of the transcription of bacterial genes. Escherichia coli lux biosensors were constructed by inserting H-NS-dependent promoters into a vector, thereby placing each fragment upstream of the promoterless Photorhabdus luminescens luxCDABE operon. It was demonstrated that the DNA-mimic proteins ArdA, Arn and Ocr activate the transcription of H-NS-dependent promoters of the lux operon of marine luminescent bacteria (mesophilic Aliivibrio fischeri and psychrophilic Aliivibrio logei), and the dps gene from E. coli. It was also demonstrated that the ArdA antirestriction protein, the genes of which are located on transmissive plasmids ColIb-P9, R64, PK101, decreases levels of H-NS silencing of the PluxC promoter during conjugation in the recipient bacteria.

  5. Characterization of the catalytic activity of the membrane-anchored metalloproteinase ADAM15 in cell-based assays.

    PubMed

    Maretzky, Thorsten; Yang, Guangli; Ouerfelli, Ouathek; Overall, Christopher M; Worpenberg, Susanne; Hassiepen, Ulrich; Eder, Joerg; Blobel, Carl P

    2009-04-28

    ADAM15 (a disintegrin and metalloproteinase 15) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM15. Here, we purified soluble recombinant ADAM15 to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM15. Overexpression of full-length membrane-anchored ADAM15 or the catalytically inactive ADAM15E-->A together with various membrane proteins resulted in increased release of the extracellular domain of the fibroblast growth factor receptor 2iiib (FGFR2iiib) by ADAM15, but not ADAM15E-->A. This provided a robust assay for a characterization of the catalytic properties of ADAM15 in intact cells. We found that increased expression of ADAM15 resulted in increased FGFR2iiib shedding, but that ADAM15 was not stimulated by phorbol esters or calcium ionophores, two commonly used activators of ectodomain shedding. Moreover, ADAM15-dependent processing of FGFR2iiib was inhibited by the hydroxamate-based metalloproteinase inhibitors marimastat, TAPI-2 and GM6001, and by 50 nM TIMP-3 (tissue inhibitor of metalloproteinases 3), but not by 100 nM TIMP-1, and only weakly by 100 nM TIMP-2. These results define key catalytic properties of ADAM15 in cells and its response to stimulators and inhibitors of ectodomain shedding. A cell-based assay for the catalytic activity of ADAM15 could aid in identifying compounds, which could be used to block the function of ADAM15 in pathological neovascularization and cancer.

  6. ADAM12: a potential target for the treatment of chronic wounds.

    PubMed

    Harsha, Asheesh; Stojadinovic, Olivera; Brem, Harold; Sehara-Fujisawa, Atsuko; Wewer, Ulla; Loomis, Cynthia A; Blobel, Carl P; Tomic-Canic, Marjana

    2008-08-01

    Wound healing is a complex process involving multiple cellular events, including cell proliferation, migration, and tissue remodeling. A disintegrin and metalloprotease 12 (ADAM12) is a membrane-anchored metalloprotease, which has been implicated in activation-inactivation of growth factors that play an important role in wound healing, including heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and insulin growth factor (IGF) binding proteins. Here, we report that expression of ADAM12 is fivefold upregulated in the nonhealing edge of chronic ulcers compared to healthy skin, based on microarrays of biopsies taken from five patients and from healthy controls (p = 0.013). The increase in ADAM12 expression in chronic ulcers was confirmed by quantitative real-time polymerase chain reaction (RT-PCR). Moreover, immunohistochemical analysis demonstrated a pronounced increase in the membranous and intracellular signal for ADAM12 in the epidermis of chronic wounds compared to healthy skin. These findings, coupled with our previous observations that lack of keratinocyte migration contributes to the pathogenesis of chronic ulcers, prompted us to evaluate how the absence of ADAM12 affects the migration of mouse keratinocytes. Skin explants from newborn ADAM12-/- or wild-type (WT) mice were used to quantify keratinocyte migration out of the explants over a period of 7 days. We found a statistically significant increase in the migration of ADAM12-/- keratinocytes compared to WT control (p = 0.0014) samples. Taken together, the upregulation of ADAM12 in chronic wounds and the increased migration of keratinocytes in the absence of ADAM12 suggest that ADAM12 is an important mediator of wound healing. We hypothesize that increased expression of ADAM12 in chronic wounds impairs wound healing through the inhibition of keratinocyte migration and that topical ADAM12 inhibitors may therefore prove useful for the treatment of chronic wounds.

  7. A Disintegrin and Metalloproteinase-12 (ADAM12): Function, Roles in Disease Progression, and Clinical Implications

    PubMed Central

    Nyren-Erickson, Erin K.; Jones, Justin M.; Srivastava, D. K.

    2013-01-01

    Background A disintegrin and metalloproteinase-12 (ADAM12) is a member of the greater ADAM family of enzymes: these are multifunctional, generally membrane-bound, zinc proteases for which there are forty genes known (21 of these appearing in humans). ADAM12 has been implicated in the pathogenesis of various cancers, liver fibrogenesis, hypertension, and asthma, and its elevation or decrease in human serum has been linked to these and other physiological/pathological conditions. Scope In this review, we begin with a brief overview of the ADAM family of enzymes and protein structure. We then discuss the role of ADAM12 in the progression and/or diagnosis of various disease conditions, and we will conclude with an exploration of currently known natural and synthetic inhibitors. Major Conclusions ADAM12 has potential to emerge as a successful drug target, although targeting the metalloproteinase domain with any specificity will be difficult to achieve due to structural similarity between the members of the ADAM and MMP family of enzymes. Overall, more research is required to establish ADAM12 being as a highly desirable biomarker and drug target of different diseases, and their selective inhibitors as potential therapeutic agents. General Significance Given the appearance of elevated levels of ADAM12 in various diseases, particularly breast cancer, our understanding of this enzyme both as a biomarker and a potential drug target could help make significant inroads into both early diagnosis and treatment of disease. PMID:23680494

  8. ADAM12-deficient zebrafish exhibit retardation in body growth at the juvenile stage without developmental defects.

    PubMed

    Tokumasu, Yudai; Iida, Atsuo; Wang, Zi; Ansai, Satoshi; Kinoshita, Masato; Sehara-Fujisawa, Atsuko

    2016-05-01

    ADAM (a disintegrin and metalloprotease) constitutes a family of multi-domain proteins that are involved in development, homeostasis, and disease. ADAM12 plays important roles in myogenesis and adipogenesis in mice; however, the precise physiological mechanisms are not known, and the function of this gene in other vertebrates has not been examined. In this study, we used a simple model vertebrate, the zebrafish, to investigate the functions of ADAM12 during development. Zebrafish adam12 is conserved with those of mammals in the synteny and the amino-acid sequence. We examined adam12 expression in zebrafish embryos by whole mount in situ hybridization and the promoter activity of the adam12 upstream sequence. We found that adam12 is strongly expressed in the cardiovascular system, erythroid progenitors, brain, and jaw cartilage during zebrafish development, and adam12-knockout zebrafish exhibited reduced body size in the juvenile stage without apparent morphological defects. Taken together, these results suggest that adam12 plays a significant role in the regulation of body growth during juvenile stage in zebrafish, although the precise molecular mechanisms await further study.

  9. Metalloprotease-disintegrin ADAM12 expression is regulated by Notch signaling via microRNA-29.

    PubMed

    Li, Hui; Solomon, Emilia; Duhachek Muggy, Sara; Sun, Danqiong; Zolkiewska, Anna

    2011-06-17

    Metalloprotease-disintegrin ADAM12 is overexpressed and frequently mutated in breast cancer. We report here that ADAM12 expression in cultured mammalian cells is up-regulated by Notch signals. Expression of a constitutively active form of Notch1 in murine fibroblasts, myoblasts, or mammary epithelial cells or activation of the endogenous Notch signaling by co-culture with ligand-expressing cells increases ADAM12 protein and mRNA levels. Up-regulation of ADAM12 expression by Notch requires new transcription, is activated in a CSL-dependent manner, and is abolished upon inhibition of IκB kinase. Expression of a constitutively active Notch1 in NIH3T3 cells increases the stability of Adam12 mRNA. We further show that the microRNA-29 family, which has a predicted conserved site in the 3'-untranslated region of mouse Adam12, plays a critical role in mediating the stimulatory effect of Notch on ADAM12 expression. In human cells, Notch up-regulates the expression of the long form, but not the short form, of ADAM12 containing a divergent 3'-untranslated mRNA region. These studies uncover a novel paradigm in Notch signaling and establish Adam12 as a Notch-related gene.

  10. Role of MicroRNA-103a Targeting ADAM10 in Abdominal Aortic Aneurysm.

    PubMed

    Jiao, Tong; Yao, Ye; Zhang, Bo; Hao, Da-Cheng; Sun, Qing-Feng; Li, Jing-Bo; Yuan, Chao; Jing, Bao; Wang, Yun-Peng; Wang, Hai-Yang

    2017-01-01

    MicroRNAs (miRNAs) are deregulated in various vascular ailments including abdominal aortic aneurysm (AAA). MiR-103 is involved in vascular, metabolic, and malignant diseases, but whether it participates in the pathogenesis of AAA remains elusive. ADAM10 plays a vital role in the formation of aneurysm, but whether miRs modulate its activity during AAA formation is totally unknown. In this study, we detected the significantly increased protein expression of ADAM10 in angiotensin II induced murine AAA specimens, while the mRNA expression of ADAM10 was similar between AAA and control, suggesting the posttranscriptional regulation. The ADAM10 specific inhibitor GI254023X dramatically reduced the macrophage infiltration of murine abdominal aorta. Bioinformatic predictions suggest that ADAM10 is the target of miR-103a/107 but the binding site is exclusive. At the cellular level, miR-103a-1 suppressed the protein expression of ADAM10, while antisense miR-103a-1 increased its expression. Particularly, with the progression of murine AAA, the mRNA expression of miR-103a/107 substantially decreased and the protein expression of ADAM10 greatly increased. Together, our data afford the new insight that miR-103a inhibited AAA growth via targeting ADAM10, which might be a promising therapeutic strategy to alleviate AAA.

  11. Role of MicroRNA-103a Targeting ADAM10 in Abdominal Aortic Aneurysm

    PubMed Central

    Jiao, Tong; Yao, Ye; Zhang, Bo; Sun, Qing-Feng; Li, Jing-Bo; Yuan, Chao; Jing, Bao; Wang, Yun-Peng

    2017-01-01

    MicroRNAs (miRNAs) are deregulated in various vascular ailments including abdominal aortic aneurysm (AAA). MiR-103 is involved in vascular, metabolic, and malignant diseases, but whether it participates in the pathogenesis of AAA remains elusive. ADAM10 plays a vital role in the formation of aneurysm, but whether miRs modulate its activity during AAA formation is totally unknown. In this study, we detected the significantly increased protein expression of ADAM10 in angiotensin II induced murine AAA specimens, while the mRNA expression of ADAM10 was similar between AAA and control, suggesting the posttranscriptional regulation. The ADAM10 specific inhibitor GI254023X dramatically reduced the macrophage infiltration of murine abdominal aorta. Bioinformatic predictions suggest that ADAM10 is the target of miR-103a/107 but the binding site is exclusive. At the cellular level, miR-103a-1 suppressed the protein expression of ADAM10, while antisense miR-103a-1 increased its expression. Particularly, with the progression of murine AAA, the mRNA expression of miR-103a/107 substantially decreased and the protein expression of ADAM10 greatly increased. Together, our data afford the new insight that miR-103a inhibited AAA growth via targeting ADAM10, which might be a promising therapeutic strategy to alleviate AAA. PMID:28357407

  12. Apparent reduction of ADAM10 in scrapie-infected cultured cells and in the brains of scrapie-infected rodents.

    PubMed

    Chen, Cao; Lv, Yan; Zhang, Bao-Yun; Zhang, Jin; Shi, Qi; Wang, Jing; Tian, Chan; Gao, Chen; Xiao, Kang; Ren, Ke; Zhou, Wei; Dong, Xiao-Ping

    2014-12-01

    It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP(C)) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP(C). Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP(C). Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.

  13. The Role of SnoN in Transforming Growth Factor β1-induced Expression of Metalloprotease-Disintegrin ADAM12*

    PubMed Central

    Solomon, Emilia; Li, Hui; Duhachek Muggy, Sara; Syta, Emilia; Zolkiewska, Anna

    2010-01-01

    Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor β1 (TGFβ1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFβ1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFβ signaling pathway, is a master regulator of ADAM12 expression in response to TGFβ1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFβ1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFβ1-induced expression of ADAM12. In a panel of TGFβ1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFβ1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFβ1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies. PMID:20457602

  14. The role of SnoN in transforming growth factor beta1-induced expression of metalloprotease-disintegrin ADAM12.

    PubMed

    Solomon, Emilia; Li, Hui; Duhachek Muggy, Sara; Syta, Emilia; Zolkiewska, Anna

    2010-07-16

    Increased expression of metalloprotease-disintegrin ADAM12 is a hallmark of several pathological conditions, including cancer, cardiovascular disease, and certain inflammatory diseases of the central nervous system or the muscoskeletal system. We show that transforming growth factor beta1 (TGFbeta1) is a potent inducer of ADAM12 mRNA and protein in mouse fibroblasts and in mouse and human mammary epithelial cells. Induction of ADAM12 is detected within 2 h of treatment with TGFbeta1, is Smad2/Smad3-dependent, and is a result of derepression of the Adam12 gene. SnoN, a negative regulator of the TGFbeta signaling pathway, is a master regulator of ADAM12 expression in response to TGFbeta1 stimulation. Overexpression of SnoN in NIH3T3 cells reduces the magnitude of ADAM12 induction by TGFbeta1 treatment. Down-regulation of SnoN expression by short hairpin RNA enhances TGFbeta1-induced expression of ADAM12. In a panel of TGFbeta1-responsive cancer cell lines with high expression of SnoN, induction of ADAM12 by TGFbeta1 is significantly impaired, suggesting that the endogenous SnoN plays a role in regulating ADAM12 expression in response to TGFbeta1. Identification of SnoN as a repressor of the ADAM12 gene should contribute to advances in the studies on the role of ADAM12 in tumor progression and in the development of other pathologies.

  15. Extravillous trophoblast-associated ADAM12 exerts pro-invasive properties, including induction of integrin beta 1-mediated cellular spreading.

    PubMed

    Biadasiewicz, Katarzyna; Fock, Valerie; Dekan, Sabine; Proestling, Katharina; Velicky, Philipp; Haider, Sandra; Knöfler, Martin; Fröhlich, Camilla; Pollheimer, Jürgen

    2014-05-01

    ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

  16. Thiazolidinediones mimic glucose starvation in facilitating Sp1 degradation through the up-regulation of beta-transducin repeat-containing protein.

    PubMed

    Wei, Shuo; Chuang, Hsiao-Ching; Tsai, Wan-Chi; Yang, Hsiao-Ching; Ho, Shiuh-Rong; Paterson, Andrew J; Kulp, Samuel K; Chen, Ching-Shih

    2009-07-01

    This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.

  17. The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents.

    PubMed

    Krute, Christina N; Bell-Temin, Harris; Miller, Halie K; Rivera, Frances E; Weiss, Andy; Stevens, Stanley M; Shaw, Lindsey N

    2015-05-01

    Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

  18. ADAMS executive and operating system

    NASA Technical Reports Server (NTRS)

    Pittman, W. D.

    1981-01-01

    The ADAMS Executive and Operating System, a multitasking environment under which a variety of data reduction, display and utility programs are executed, a system which provides a high level of isolation between programs allowing them to be developed and modified independently, is described. The Airborne Data Analysis/Monitor System (ADAMS) was developed to provide a real time data monitoring and analysis capability onboard Boeing commercial airplanes during flight testing. It inputs sensor data from an airplane performance data by applying transforms to the collected sensor data, and presents this data to test personnel via various display media. Current utilization and future development are addressed.

  19. CJD mimics and chameleons.

    PubMed

    Mead, Simon; Rudge, Peter

    2017-04-01

    Rapidly progressive dementia mimicking Creutzfeldt-Jakob disease (CJD) is a relatively rare presentation but a rewarding one to become familiar with, as the potential diagnoses range from the universally fatal to the completely reversible. Patients require urgent decisions about assessment and investigation and have quickly evolving needs for treatments and support, through symptom management and end-of-life care in most cases. We have based this pragmatic review on the experiences of a specialist prion referral centre in the UK, which, unsurprisingly, is strongly biased towards seeing patients with CJD. Cases eventually proven not to have prion disease might be described as 'CJD-mimics'; being referred from UK neurologists, these are the most challenging cases. CJD in its classical presentation is very rarely mimicked; however, it is highly heterogeneous, and atypical forms can mimic virtually all common neurodegenerative syndromes. Warning features of a mimic include generalised seizures, hyponatraemia, fever, a facial movement disorder, a normal neurological examination and a modestly rapid presentation. Contrast-enhancing lesions or MRI signal hyperintensity outside the striatum, thalamus or cortex and a cerebrospinal fluid pleocytosis are key investigation pointers to a CJD mimic.

  20. Identification of novel interaction between ADAM17 (a disintegrin and metalloprotease 17) and thioredoxin-1.

    PubMed

    Aragão, Annelize Z B; Nogueira, Maria Luiza C; Granato, Daniela C; Simabuco, Fernando M; Honorato, Rodrigo V; Hoffman, Zaira; Yokoo, Sami; Laurindo, Francisco R M; Squina, Fabio M; Zeri, Ana Carolina M; Oliveira, Paulo S L; Sherman, Nicholas E; Paes Leme, Adriana F

    2012-12-14

    ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

  1. Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1*

    PubMed Central

    Aragão, Annelize Z. B.; Nogueira, Maria Luiza C.; Granato, Daniela C.; Simabuco, Fernando M.; Honorato, Rodrigo V.; Hoffman, Zaira; Yokoo, Sami; Laurindo, Francisco R. M.; Squina, Fabio M.; Zeri, Ana Carolina M.; Oliveira, Paulo S. L.; Sherman, Nicholas E.; Paes Leme, Adriana F.

    2012-01-01

    ADAM17, which is also known as TNFα-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation. PMID:23105116

  2. MT5-MMP, ADAM-10, and N-Cadherin Act in Concert To Facilitate Synapse Reorganization after Traumatic Brain Injury

    PubMed Central

    Warren, Kelly M.; Reeves, Thomas M.

    2012-01-01

    Abstract Matrix metalloproteinases (MMPs) influence synaptic recovery following traumatic brain injury (TBI). Membrane type 5-matrix metalloproteinase (MT5-MMP) and a distintegrin and metalloproteinase-10 (ADAM-10) are membrane-bound MMPs that cleave N-cadherin, a protein critical to synapse stabilization. This study examined protein and mRNA expression of MT5-MMP, ADAM-10, and N-cadherin after TBI, contrasting adaptive and maladaptive synaptogenesis. The effect of MMP inhibition on MT5-MMP, ADAM-10, and N-cadherin was assessed during maladaptive plasticity and correlated with synaptic function. Rats were subjected to adaptive unilateral entorhinal cortical lesion (UEC) or maladaptive fluid percussion TBI+bilateral entorhinal cortical lesion (TBI+BEC). Hippocampal MT5-MMP and ADAM-10 protein was significantly elevated 2 and 7 days post-injury. At 15 days after UEC, each MMP returned to control level, while TBI+BEC ADAM-10 remained elevated. At 2 and 7 days, N-cadherin protein was below control. By the 15-day synapse stabilization phase, UEC N-cadherin rose above control, a shift not seen for TBI+BEC. At 7 days, increased TBI+BEC ADAM-10 transcript correlated with protein elevation. UEC ADAM-10 mRNA did not change, and no differences in MT5-MMP or N-cadherin mRNA were detected. Confocal imaging showed MT5-MMP, ADAM-10, and N-cadherin localization within reactive astrocytes. MMP inhibition attenuated ADAM-10 protein 15 days after TBI+BEC and increased N-cadherin. This inhibition partially restored long-term potentiation induction, but did not affect paired-pulse facilitation. Our results confirm time- and injury-dependent expression of MT5-MMP, ADAM-10, and N-cadherin during reactive synaptogenesis. Persistent ADAM-10 expression was correlated with attenuated N-cadherin level and reduced functional recovery. MMP inhibition shifted ADAM-10 and N-cadherin toward adaptive expression and improved synaptic function. PMID:22489706

  3. What's an Adam's Apple? (For Kids)

    MedlinePlus

    ... Happens in the Operating Room? What's an Adam's Apple? KidsHealth > For Kids > What's an Adam's Apple? A A A You're at the high ... the throat. This is what's called an Adam's apple. Everyone's larynx grows during puberty, but a girl's ...

  4. What's an Adam's Apple? (For Kids)

    MedlinePlus

    ... Too Short All About Puberty What's an Adam's Apple? KidsHealth > For Kids > What's an Adam's Apple? Print A A A You're at the ... the throat. This is what's called an Adam's apple. Everyone's larynx grows during puberty, but a girl's ...

  5. The shedding activity of ADAM17 is sequestered in lipid rafts

    SciTech Connect

    Tellier, Edwige; Canault, Matthias; Rebsomen, Laure; Bonardo, Bernadette; Juhan-Vague, Irene; Nalbone, Gilles; Peiretti, Franck . E-mail: Franck.Peiretti@medecine.univ-mrs.fr

    2006-12-10

    The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-disintegrin responsible for the cleavage of several biologically active transmembrane proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding activity are still poorly understood. Here, we report that during its transport through the Golgi apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts) where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of these substrates demonstrating the importance of lipid rafts in the control of this process. Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting that the entry of each of these substrates in these particular membrane microdomains is specifically regulated. Our data support the idea that one of the mechanisms regulating ADAM17 substrate cleavage involves protein partitioning in lipid rafts.

  6. TspanC8 Tetraspanins and A Disintegrin and Metalloprotease 10 (ADAM10) Interact via Their Extracellular Regions

    PubMed Central

    Noy, Peter J.; Yang, Jing; Reyat, Jasmeet S.; Matthews, Alexandra L.; Charlton, Alice E.; Furmston, Joanna; Rogers, David A.; Rainger, G. Ed; Tomlinson, Michael G.

    2016-01-01

    A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitously expressed transmembrane metalloprotease that cleaves the extracellular regions from its transmembrane substrates. ADAM10 is essential for embryonic development and is implicated in cancer, Alzheimer, and inflammatory diseases. The tetraspanins are a superfamily of 33 four-transmembrane proteins in mammals, of which the TspanC8 subgroup (Tspan5, 10, 14, 15, 17, and 33) promote ADAM10 intracellular trafficking and enzymatic maturation. However, the interaction between TspanC8s and ADAM10 has only been demonstrated in overexpression systems and the interaction mechanism remains undefined. To address these issues, an antibody was developed to Tspan14, which was used to show co-immunoprecipitation of Tspan14 with ADAM10 in primary human cells. Chimeric Tspan14 constructs demonstrated that the large extracellular loop of Tspan14 mediated its co-immunoprecipitation with ADAM10, and promoted ADAM10 maturation and trafficking to the cell surface. Chimeric ADAM10 constructs showed that membrane-proximal stalk, cysteine-rich, and disintegrin domains of ADAM10 mediated its co-immunoprecipitation with Tspan14 and other TspanC8s. This TspanC8-interacting region was required for ADAM10 exit from the endoplasmic reticulum. Truncated ADAM10 constructs revealed differential TspanC8 binding requirements for the stalk, cysteine-rich, and disintegrin domains. Moreover, Tspan15was the only TspanC8 to promote cleavage of the ADAM10 substrate N-cadherin, whereas Tspan14 was unique in reducing cleavage of the platelet collagen receptor GPVI. These findings suggest that ADAM10 may adopt distinct conformations in complex with different TspanC8s, which could impact on substrate selectivity. Furthermore, this study identifies regions of TspanC8s and ADAM10 for potential interaction-disrupting therapeutic targeting. PMID:26668317

  7. Host proteins that bind to or mimic SV40 large T antigen: using antibodies to look at protein interactions and their significance

    PubMed Central

    Mole, S. E.; Gannon, J. V.; Anton, I. A.; Ford, M. J.; Lane, D. P.

    1989-01-01

    The papovavirus SV40 is able to induce tumours in susceptible hosts and will transform cells in vitro. Its major early protein, large T antigen, is required for viral DNA synthesis, both in vivo and in vitro, and is also responsible for the oncogenic action of the virus. We have made use of an extensive library of anti-T monoclonal antibodies to investigate the cellular effects of T. Large T shares an antigenic determinant with a growth-regulated host protein, p68, which is a member of an expanding super-family of helicases with particular homology to the translation initiation factor elF-4A. We have also studied the binding and interaction of large T with two particular host components: the replicative enzyme DNA polymerase α and the proto-oncogene p53. These two proteins bind to similar regions of T and exert similar effects on its antigenic structure. We found that p53 can block the binding of DNA polymerase α to T as well as co-existing with DNA polymerase α in a trimeric complex with T. This suggests that these interactions may be important in the oncogenic and replicative action of large T.

  8. NASA Dryden test pilot Michael J. Adams

    NASA Image and Video Library

    1967-03-22

    Air Force test pilot Maj. Michael J. Adams stands beside X-15 ship number one. Adams was selected for the X-15 program in 1966 and made his first flight on Oct. 6, 1966. On Nov. 15, 1967, Adams made his seventh and final X-15 flight. The X-15 launched from the B-52, but during the ascent an electrical problem affected the X-15's control system. The aircraft crashed northwest of Cuddeback Lake, California, causing the death of Adams. He was posthumously awarded Air Force astronaut wings because his final flight exceeded 50 miles in altitude. Adams was the only pilot lost in the 199-flight X-15 program.

  9. ADAM33 and ADAM12 genetic polymorphisms and their expression in Egyptian children with asthma.

    PubMed

    Shalaby, Sally M; Abdul-Maksoud, Rehab S; Abdelsalam, Sanaa M; Abdelrahman, Hadeel M; Abdelaziz Almalky, Mohamed A

    2016-01-01

    The ADAM family is involved in some pathologic processes, such as inflammation and asthma. To assess the association between ADAM33 and ADAM12 single-nucleotide polymorphisms (SNPs) with asthma risk and severity and to investigate the effect of ADAM33 and ADAM12 polymorphisms on expression of these proteases in sputum. Two SNPs of the ADAM33 gene, F+1 (rs511898) G/A and ST+4 (rs44707) A/C, and 2 SNPs of the ADAM12 gene, rs3740199 and rs1871054, were analyzed in 400 asthma cases and 200 controls aged 3 to 14 years using the polymerase chain reaction-restriction fragment length polymorphism method. Messenger RNA expression profile of ADAM33 and ADAM12 proteases in sputum from studied groups was determined by reverse transcription polymerase chain reaction. ADAM33 F+1 homozygous mutant genotype (AA) and ST+4 heterozygous and homozygous mutant genotype (AC and CC) and mutant alleles of both polymorphisms were significantly associated with asthma risk and severity in moderate and severe subgroups. Patients with the ADAM12 (rs3740199) CC genotype were at increased risk for moderate and severe asthma. Messenger RNA levels of ADAM12 were significantly increased in asthmatic children compared with controls, whereas we were not able to detect the expression of ADAM33 in the sputum of the groups studied. The ADAM12 expression was significantly higher in homozygous CC (variant type) compared with homozygous GG (wild type) of both ADAM12 rs3740199 and rs1871054 in the asthmatic group. Our analysis suggests a likely role for ADAM33 and ADAM12 in the development of asthma in Egyptian children. Furthermore, ADAM12 polymorphisms may affect ADAM12 expression in asthma. Copyright © 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  10. ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis.

    PubMed

    Romagnoli, Mathilde; Mineva, Nora D; Polmear, Michael; Conrad, Catharina; Srinivasan, Srimathi; Loussouarn, Delphine; Barillé-Nion, Sophie; Georgakoudi, Irene; Dagg, Áine; McDermott, Enda W; Duffy, Michael J; McGowan, Patricia M; Schlomann, Uwe; Parsons, Maddy; Bartsch, Jörg W; Sonenshein, Gail E

    2014-02-01

    The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

  11. ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

    PubMed Central

    Romagnoli, Mathilde; Mineva, Nora D; Polmear, Michael; Conrad, Catharina; Srinivasan, Srimathi; Loussouarn, Delphine; Barillé-Nion, Sophie; Georgakoudi, Irene; Dagg, Áine; McDermott, Enda W; Duffy, Michael J; McGowan, Patricia M; Schlomann, Uwe; Parsons, Maddy; Bartsch, Jörg W; Sonenshein, Gail E

    2014-01-01

    The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via β1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination. Subject Category Cancer PMID:24375628

  12. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding.

    PubMed

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-12-09

    Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha. Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

  13. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

    PubMed Central

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-01-01

    Background Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Results Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα. Conclusion Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP. PMID:18067682

  14. Aberrant ADAM10 expression correlates with osteosarcoma progression

    PubMed Central

    2014-01-01

    Background Osteosarcoma is the most common type of bone cancer and is notorious for its rapid progression. The Notch signaling pathway has recently been shown to be involved in osteosarcoma. As a major sheddase of Notch receptors, ADAM10 has been implicated in many types of cancers, but its role in osteosarcoma has not been investigated. Previous studies have shown that the expression of CD31 was significantly elevated in metastatic osteosarcoma; however, its expression in nonmetastatic groups is not known. In addition, the mysterious multinucleated giant cell in giant cell-rich osteosarcoma was previously regarded as an osteoclast-like cell, but its exact identity is unclear. Method Tissue chip samples from 40 cases of nonmetastatic osteosarcoma were stained for cytoplasmic ADAM10, activated Notch1 and CD31. Osteoclasts in tumor sections were also stained for tartrate-resistant acid phosphatase (TRAP). Results Immunofluorescence staining revealed that ADAM10 expression significantly increased with the progression of osteosarcoma as well as in osteoblastic osteosarcoma, whereas the expression of the Notch intracellular domain (NICD) and CD31 was not significantly altered between different pathological stages. In addition, multinucleated giant cells in giant cell-rich osteosarcoma were also found to coexpress CD31, ADAM10 and NICD, but were negative for TRAP staining. Conclusions Our results highlight the importance of ADAM10 in the progression of osteosarcoma and suggest that the protein might be a potential therapeutic target in osteosarcoma treatment. This study also demonstrates that the multinucleated giant cell is an angiogenic tumor cell, rather than an osteoclast, and involves ADAM10/Notch1 signaling activation. PMID:24548763

  15. Expression of ADAMs and their inhibitors in sputum from patients with asthma.

    PubMed

    Paulissen, Geneviève; Rocks, Natacha; Quesada-Calvo, Florence; Gosset, Philippe; Foidart, Jean-Michel; Noel, Agnès; Louis, Renaud; Cataldo, Didier D

    2006-01-01

    ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV(1)) (r = -0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV(1) (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma.

  16. Expression of ADAMs and Their Inhibitors in Sputum from Patients with Asthma

    PubMed Central

    Paulissen, Geneviève; Rocks, Natacha; Quesada-Calvo, Florence; Gosset, Philippe; Foidart, Jean-Michel; Noel, Agnès; Louis, Renaud; Cataldo, Didier D

    2006-01-01

    ADAMs (a disintegrin and metalloprotease) constitute a family of cell surface proteins containing disintegrin and metalloprotease domains which associate features of adhesion molecules and proteases. ADAMTSs (a disintegrin and metalloprotease with thrombospondin motifs) bear thrombospondin type I motifs in C-terminal extremity, and most of them are secreted proteins. Because genetic studies have shown that ADAM-33 gene polymorphisms are associated with asthma, we designed this study to assess mRNA expression profile of several ADAM and ADAMTS proteases in sputum from patients with asthma and to investigate the relationship between expression of these proteases and asthma-associated inflammation and airway obstruction. mRNA expression profile of selected ADAM and ADAMTS proteinases (ADAM-8, -9, -10, -12, -15, -17, and -33; ADAMTS-1, -2, -15, -16, -17, -18, and -19), their physiological inhibitors TIMP-1 and TIMP-3, and RECK, a membrane-anchored MMP activity regulator, was obtained by RT-PCR analysis performed on cells collected by sputum induction from 21 patients with mild to moderate asthma and 17 healthy individuals. mRNA levels of ADAM-8, ADAM-9, ADAM-12, TIMP-1, and TIMP-3 were significantly increased, whereas mRNA levels coding for ADAMTS-1, ADAMTS-15, and RECK were significantly decreased in patients with asthma compared with control patients. ADAM-8 expression was negatively correlated with the forced expiratory volume at the first second (FEV1) (r = −0.57, P < 0.01), whereas ADAMTS-1 and RECK expressions were positively correlated to FEV1 (r = 0.45, P < 0.05, and r = 0.55, P = 0.01, respectively). We conclude that expression of ADAMs and ADAMTSs and their inhibitors is modulated in airways from patients with asthma and that these molecules may play a role in the pathogenesis of asthma. PMID:17088949

  17. SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

    PubMed Central

    Saraceno, C; Marcello, E; Di Marino, D; Borroni, B; Claeysen, S; Perroy, J; Padovani, A; Tramontano, A; Gardoni, F; Di Luca, M

    2014-01-01

    A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP. PMID:25429624

  18. ADAM12 is a prognostic factor associated with an aggressive molecular subtype of high-grade serous ovarian carcinoma.

    PubMed

    Cheon, Dong-Joo; Li, Andrew J; Beach, Jessica A; Walts, Ann E; Tran, Hang; Lester, Jenny; Karlan, Beth Y; Orsulic, Sandra

    2015-07-01

    ADAM metallopeptidase domain 12 (ADAM12) is a promising biomarker because of its low expression in normal tissues and high expression in a variety of human cancers. However, ADAM12 levels in ovarian cancer have not been well characterized. We previously identified ADAM12 as one of the signature genes associated with poor survival in high-grade serous ovarian carcinoma (HGSOC). Here, we sought to determine if high levels of the ADAM12 protein and/or messenger RNA (mRNA) are associated with clinical variables in HGSOC. We show that high protein levels of ADAM12 in banked preoperative sera are associated with shorter progression-free and overall survival. Tumor levels of ADAM12 mRNA were also associated with shorter progression-free and overall survival as well as with lymphatic and vascular invasion, and residual tumor volume following cytoreductive surgery. The majority of genes co-expressed with ADAM12 in HGSOC were transforming growth factor (TGF)β signaling targets that function in collagen remodeling and cell-matrix adhesion. In tumor sections, the ADAM12 protein and mRNA were expressed in epithelial cancer cells and surrounding stromal cells. In vitro data showed that ADAM12 mRNA levels can be increased by TGFβ signaling and direct contact between epithelial and stromal cells. High tumor levels of ADAM12 mRNA were characteristic of the mesenchymal/desmoplastic molecular subtype of HGSOC, which is known to have the poorest prognosis. Thus, ADAM12 may be a useful biomarker of aggressive ovarian cancer for which standard treatment is not effective.

  19. Parasomnias and their mimics.

    PubMed

    Malhotra, Raman K; Avidan, Alon Y

    2012-11-01

    Parasomnias are classified into nonrapid eye movement and rapid eye movement sleep parasomnias. It is important for the clinician to consider other parasomnia mimics that present to the sleep disorders clinic. These conditions can be differentiated by taking a detailed sleep history and conducting nocturnal polysomnograms to evaluate for potential comorbidities and better characterize the disorder. Mainstays of treatment include treating any underlying primary sleep disorders that induce sleep fragmentation and could trigger the parasomnia, as well as implementing safety precautions to protect patients and their household. Pharmacologic treatment of many parasomnias is also available if needed and clinically indicated.

  20. ADAM 12: a putative marker of oligodendrogliomas?

    PubMed

    Kanakis, Dimitrios; Lendeckel, Uwe; Theodosiou, Paraskevi; Dobrowolny, Henrik; Mawrin, Christian; Keilhoff, Gerburg; Bukowska, Alicia; Dietzmann, Knut; Bogerts, Bernhard; Bernstein, Hans-Gert

    2013-01-01

    ADAM 12 (meltrin alpha) belongs to a large family of molecules, consisting of members with both disintegrin and metalloproteinase properties. ADAMs have been implicated in several cell physiological processes including cell adhesion, cell fusion, proteolysis and signalling. ADAM 12 is widely expressed, including skeletal muscle, testis, bone, intestine, heart and kidney. In addition, a variety of tumours show elevated expression of ADAM12; among them being breast-, colon-, gastric- and lung-carcinoma. As to the brain, ADAM 12 has been shown previously to be expressed in rat and human oligodendrocytes. However, little is known about the expression of this protease in brain tumours. This study demonstrates the presence of ADAM 12 in non-neoplastic oligodendroglial cells of normal human brain as well as in neoplastic oligodendroglia and minigemistocytes arising from four pure oligodendrogliomas and three mixed oligoastrocytomas. Double stainings revealed a notable preference of ADAM 12 for the oligodendroglial over astroglial components. The results of immunohistochemistry are in accordance with the results obtained from the RT-PCR, which further demonstrated a mild difference concerning the mRNA concentration of ADAM 12 between similar grades of eight astrocytomas and eight oligodendrogliomas (namely four astrocytomas grade II versus four oligodendrogliomas grade II and four astrocytomas grade III versus four oligodendrogliomas grade III). Both cellular immunostaining for ADAM 12 and ADAM 12 mRNA content decrease with higher histologic grade of the tumour. Surprisingly, the latter parameter (ADAM12 mRNA) showed a significant opposite correlation to the degree of histologic tumour malignancy. From our data showing that ADAM 12 is highly expressed in, but not restricted to, oligodendrogliomas, we conclude that ADAM 12 immunohistochemistry may be a helpful tool in the diagnosis of brain tumours.

  1. ADAM 12: A Putative Marker of Oligodendrogliomas?

    PubMed Central

    Kanakis, Dimitrios; Lendeckel, Uwe; Theodosiou, Paraskevi; Dobrowolny, Henrik; Mawrin, Christian; Keilhoff, Gerburg; Bukowska, Alicia; Dietzmann, Knut; Bogerts, Bernhard; Bernstein, Hans-Gert

    2013-01-01

    ADAM 12 (meltrin alpha) belongs to a large family of molecules, consisting of members with both disintegrin and metalloproteinase properties. ADAMs have been implicated in several cell physiological processes including cell adhesion, cell fusion, proteolysis and signalling. ADAM 12 is widely expressed, including skeletal muscle, testis, bone, intestine, heart and kidney. In addition, a variety of tumours show elevated expression of ADAM12; among them being breast-, colon-, gastric- and lung-carcinoma. As to the brain, ADAM 12 has been shown previously to be expressed in rat and human oligodendrocytes. However, little is known about the expression of this protease in brain tumours. This study demonstrates the presence of ADAM 12 in non-neoplastic oligodendroglial cells of normal human brain as well as in neoplastic oligodendroglia and minigemistocytes arising from four pure oligodendrogliomas and three mixed oligoastrocytomas. Double stainings revealed a notable preference of ADAM 12 for the oligodendroglial over astroglial components. The results of immunohistochemistry are in accordance with the results obtained from the RT-PCR, which further demonstrated a mild difference concerning the mRNA concentration of ADAM 12 between similar grades of eight astrocytomas and eight oligodendrogliomas (namely four astrocytomas grade II versus four oligodendrogliomas grade II and four astrocytomas grade III versus four oligodendrogliomas grade III). Both cellular immunostaining for ADAM 12 and ADAM 12 mRNA content decrease with higher histologic grade of the tumour. Surprisingly, the latter parameter (ADAM12 mRNA) showed a significant opposite correlation to the degree of histologic tumour malignancy. From our data showing that ADAM 12 is highly expressed in, but not restricted to, oligodendrogliomas, we conclude that ADAM 12 immunohistochemistry may be a helpful tool in the diagnosis of brain tumours. PMID:23324579

  2. Regulation of mature ADAM17 by redox agents for L-selectin shedding1

    PubMed Central

    Wang, Yue; Herrera, Amy H.; Li, Ying; Belani, Kiran K.; Walcheck, Bruce

    2008-01-01

    L-selectin is constitutively expressed by neutrophils and plays a key role in directing these cells to sites of inflammation. Upon neutrophil activation, L-selectin is rapidly and efficiently down-regulated from the cell surface by ectodomain shedding. We have directly shown that ADAM17 is a primary and non-redundant sheddase of L-selection by activated neutrophils in vivo. Following cell activation, intracellular signals lead to the induction of ADAM17's enzymatic activity; however, the target of this inducer mechanism remains unclear. Our study provides evidence of an activation mechanism that involves the extracellular region of the mature form of cell surface ADAM17 and not its intracellular region. We demonstrate that the catalytic activity of purified ADAM17 lacking a pro-domain and its intracellular region is diminished under mild reducing conditions by DTT and enhanced by H2O2 oxidation. Moreover, H2O2 reversed ADAM17 inhibition by DTT. The treatment of neutrophils with H2O2 also induced L-selectin shedding in an ADAM17-dependent manner. These findings suggest that thioldisulfide conversion occurring in the extracellular region of ADAM17 may be involved in its activation. An analysis of ADAM17 revealed that within its disintegrin/cysteine-rich region are two highly conserved, vicinal cysteine sulfhydryl motifs (cysteine-X-X-cysteine, CXXC), which are well characterized targets for thiol-disulfide exchange in various other proteins. Using a cell-based ADAM17 reconstitution assay, we demonstrate that the CXXC motifs are critical for L-selectin cleavage. Taken together, our findings suggest that redox modifications of cysteinyl sulfhydryl groups in mature ADAM17 may serve as a mechanism for regulating the shedding of L-selectin following neutrophil stimulation. PMID:19201900

  3. Xenopus ADAM 13 is a metalloprotease required for cranial neural crest-cell migration.

    PubMed

    Alfandari, D; Cousin, H; Gaultier, A; Smith, K; White, J M; Darribère, T; DeSimone, D W

    2001-06-26

    Cranial neural-crest (CNC) cells originate from the lateral edge of the anterior neuroepithelium and migrate to form parts of the peripheral nervous system, muscles, cartilage, and bones of the face. Neural crest-cell migration involves the loss of adhesion from the surrounding neuroepithelium and a corresponding increase in cell adhesion to the extracellular matrix (ECM) present in migratory pathways. While proteolytic activity is likely to contribute to the regulation of neural crest-cell adhesion and migration, the role of a neural crest-specific protease in these processes has yet to be demonstrated. We previously showed that CNC cells express ADAM 13, a cell surface metalloprotease/disintegrin. Proteins of this family are known to act in cell-cell adhesion and as sheddases. ADAMs have also been proposed to degrade the ECM, but this has not yet been shown in a physiological context. Using a tissue transplantation technique, we show that Xenopus CNC cells overexpressing wild-type ADAM 13 migrate along the same hyoid, branchial, and mandibular pathways used by normal CNC cells. In contrast, CNC cell grafts that express protease-defective ADAM 13 fail to migrate along the hyoid and branchial pathways. In addition, ectopic expression of wild-type ADAM 13 results in a gain-of-function phenotype in embryos, namely the abnormal positioning of trunk neural-crest cells. We further show that explanted embryonic tissues expressing wild-type, but not protease-defective, ADAM 13 display decreased cell-matrix adhesion. Purified ADAM 13 can cleave fibronectin, and tissue culture cells that express wild-type, but not protease-defective, ADAM 13 can remodel a fibronectin substrate. Our findings support the hypothesis that the protease activity of ADAM 13 plays a critical role in neural crest-cell migration along defined pathways. We propose that the ADAM 13-dependent modification of ECM and/or other guidance molecules is a key step in the directed migration of the CNC.

  4. Down regulation of ADAM33 as a Predictive Biomarker of Aggressive Breast Cancer

    PubMed Central

    Manica, Graciele C. M.; Ribeiro, Caroline F.; Oliveira, Marco A. S. de; Pereira, Isabela T.; Chequin, Andressa; Ramos, Edneia A. S.; Klassen, Liliane M. B.; Sebastião, Ana Paula M.; Alvarenga, Larissa M.; Zanata, Silvio M.; Noronha, Lucia De; Rabinovich, Iris; Costa, Fabricio F.; Souza, Emanuel M.; Klassen, Giseli

    2017-01-01

    Breast cancer is a heterogeneous disease with differences in its clinical, molecular and biological features. Traditionally, immunohistochemical markers together with clinicopathologic parameters are used to classify breast cancer and to predict disease outcome. Triple-negative breast cancer (TNBC) is a particular type of breast cancer that is defined by a lack of expression of hormonal receptors and the HER2 gene. Most cases of TNBC also have a basal-like phenotype (BLBC) with expression of cytokeratin 5/6 and/or EGFR. A basal marker alone is insufficient for a better understanding of the tumor biology of TNBC. In that regard, the ADAM33 gene is silenced by DNA hypermethylation in breast cancer, which suggests that ADAM33 might be useful as a molecular marker. In the present study, we have produced monoclonal antibodies against the ADAM33 protein and have investigated the role of ADAM33 protein in breast cancer. We used 212 breast tumor samples and lower levels of ADAM33 were correlated with TNBC and basal-like markers. A lower level of ADAM33 was also correlated with shorter overall survival and metastasis-free survival and was considered an independent prognostic factor suggesting that ADAM33 is a novel molecular biomarker of TNBC and BLBC that might be useful as a prognostic factor. PMID:28294120

  5. The Distinct Role of ADAM17 in APP Proteolysis and Microglial Activation Related to Alzheimer's Disease.

    PubMed

    Qian, Meng; Shen, Xiaoqiang; Wang, Huanhuan

    2016-05-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease with the symptom of cognitive impairment. The deposition of amyloid β (Aβ) peptide is believed to be the primary cause to neuronal dystrophy and eventually dementia. Aβ is the proteolytic product from its precursor amyloid precursor protein (APP) by β- and γ- secretase. An optional cleavage by α-secretase happens inside the Aβ domain. ADAM17 is supposed to be the regulated α-secretase of APP. Enhanced activity of ADAM17 leads to the increasing secretion of neuroprotective soluble APP α fragment and reduction of Aβ generation, which may be benefit to the disease. ADAM17 is then considered the potential therapeutic target for AD. Microglia activation and neuroinflammation is another important event in AD pathogenesis. Interestingly, ADAM17 also participates in the cleavage of many other membrane-bound proteins, especially some inflammatory factors related to microglia activation. The facilitating role of ADAM17 in inflammation and further neuronal damage has also been illustrated. In results, the activation of ADAM17 as the solution to AD may be a tricky task. The comprehensive consideration and evaluation has to be carried out carefully before the final treatment. In the present review, the distinct role of ADAM17 in AD-related APP shedding and neuroinflammatory microglial activation will be carefully discussed.

  6. Down regulation of ADAM33 as a Predictive Biomarker of Aggressive Breast Cancer.

    PubMed

    Manica, Graciele C M; Ribeiro, Caroline F; Oliveira, Marco A S de; Pereira, Isabela T; Chequin, Andressa; Ramos, Edneia A S; Klassen, Liliane M B; Sebastião, Ana Paula M; Alvarenga, Larissa M; Zanata, Silvio M; Noronha, Lucia De; Rabinovich, Iris; Costa, Fabricio F; Souza, Emanuel M; Klassen, Giseli

    2017-03-15

    Breast cancer is a heterogeneous disease with differences in its clinical, molecular and biological features. Traditionally, immunohistochemical markers together with clinicopathologic parameters are used to classify breast cancer and to predict disease outcome. Triple-negative breast cancer (TNBC) is a particular type of breast cancer that is defined by a lack of expression of hormonal receptors and the HER2 gene. Most cases of TNBC also have a basal-like phenotype (BLBC) with expression of cytokeratin 5/6 and/or EGFR. A basal marker alone is insufficient for a better understanding of the tumor biology of TNBC. In that regard, the ADAM33 gene is silenced by DNA hypermethylation in breast cancer, which suggests that ADAM33 might be useful as a molecular marker. In the present study, we have produced monoclonal antibodies against the ADAM33 protein and have investigated the role of ADAM33 protein in breast cancer. We used 212 breast tumor samples and lower levels of ADAM33 were correlated with TNBC and basal-like markers. A lower level of ADAM33 was also correlated with shorter overall survival and metastasis-free survival and was considered an independent prognostic factor suggesting that ADAM33 is a novel molecular biomarker of TNBC and BLBC that might be useful as a prognostic factor.

  7. ADAM10 modulates calcitriol-regulated RAGE in cardiomyocytes.

    PubMed

    Lee, Ting-Wei; Kao, Yu-Hsun; Lee, Ting-I; Chen, Yi-Jen

    2017-09-01

    Receptor for advanced glycation end products (RAGE) signalling plays a critical role in the pathogenesis of cardiovascular disease. Calcitriol modulates cardiac RAGE expression. This study explored the mechanisms underlying the effect of calcitriol on RAGE and soluble RAGE (sRAGE) expression in cardiomyocytes. Western blot, ELISA, fluorometric assay and PCR analyses were used to evaluate the RAGE, sRAGE, endogenous secretory RAGE (esRAGE), Jun N-terminal kinase (JNK), and a disintegrin and metalloprotease 10 (ADAM10) expression and enzyme activity in HL-1 atrial myocytes without and with calcitriol (10 and 100 nM), nuclear factor-κB (NF-κB) inhibitor (50 μg/mL), or ADAM10 inhibitor (5 μM) incubation for 48 h. Calcitriol (10 nM) significantly reduced RAGE protein expression and increased sRAGE concentrations in HL-1 cardiomyocytes compared with control cells. These changes were associated with increased protein expression and enzyme activity of ADAM10 and higher mRNA expression of esRAGE. In the presence of ADAM10 inhibitor, however, the suppressive effect of calcitriol on RAGE was diminished. Methylglyoxal (500 μM for 10 min)-mediated JNK phosphorylation was attenuated in the presence of calcitriol (10 nM). Moreover, control and NF-κB inhibitor-treated HL-1 cells had similar RAGE and sRAGE expression, suggesting that calcitriol-mediated RAGE modulation was independent of NF-κB signalling. We showed that RAGE downregulation and increased sRAGE production by calcitriol were mediated through ADAM10 activation in cardiomyocytes. The results suggest that calcitriol has therapeutic potential in treating RAGE-mediated cardiovascular complications. © 2017 Stichting European Society for Clinical Investigation Journal Foundation.

  8. ADAMS: AIRLAB data management system user's guide

    NASA Technical Reports Server (NTRS)

    Conrad, C. L.; Ingogly, W. F.; Lauterbach, L. A.

    1986-01-01

    The AIRLAB Data Management System (ADAMS) is an online environment that supports research at NASA's AIRLAB. ADAMS provides an easy to use interactive interface that eases the task of documenting and managing information about experiments and improves communication among project members. Data managed by ADAMS includes information about experiments, data sets produced, software and hardware available in AIRLAB as well as that used in a particular experiment, and an on-line engineer's notebook. The User's Guide provides an overview of the ADAMS system as well as details of the operations available within ADAMS. A tutorial section takes the user step-by-step through a typical ADAMS session. ADAMS runs under the VAX/VMS operating system and uses the ORACLE database management system and DEC/FMS (the Forms Management System). ADAMS can be run from any VAX connected via DECnet to the ORACLE host VAX. The ADAMS system is designed for simplicity, so interactions within the underlying data management system and communications network are hidden from the user.

  9. Erbb2 up-regulation of ADAM12 expression accelerates skin cancer progression.

    PubMed

    Rao, Velidi H; Vogel, Kristen; Yanagida, Jodi K; Marwaha, Nitin; Kandel, Amrit; Trempus, Carol; Repertinger, Susan K; Hansen, Laura A

    2015-10-01

    Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness. © 2014 Wiley Periodicals, Inc.

  10. Phenotypic diversity of breast cancer-related mutations in metalloproteinase-disintegrin ADAM12.

    PubMed

    Qi, Yue; Duhachek-Muggy, Sara; Li, Hui; Zolkiewska, Anna

    2014-01-01

    Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity--R612Q and L792F--occur in triple-negative breast cancers, while loss-of-function mutations--D301H, G479E, T596A, and G668A--are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.

  11. Effect of a dominant-negative form of ADAM10 in a mouse model of Alzheimer's disease.

    PubMed

    Schroeder, Anja; Fahrenholz, Falk; Schmitt, Ulrich

    2009-01-01

    The alpha-secretase cleaves in the non-amyloidogenic pathway the amyloid-beta protein precursor (AbetaPP) within the region of the amyloid-beta peptides to prevent their formation and aggregation in the brain. Members of the ADAM family (a disintegrin and metalloprotease) are the main candidates for physiologically relevant alpha-secretases. We recently demonstrated that overexpression of ADAM10 in mice transgenic for human AbetaPP (ADAM10 x APP[V717I]) alleviated functional deficits related to Alzheimer's disease. To further demonstrate that this is due to the specific activity of alpha-secretase, we characterized mice overexpressing an inactive form of ADAM10 (ADAM10[E384A]; ADAM10-dn). Three lines of mice (controls (C57Bl/6 x FVB), APP[V717I[ transgenics and ADAM10-dn x APP[V717I] double-transgenics) were investigated with respect to learning and memory in the Morris water maze. Double-transgenic mice overexpressing ADAM10-dn behaved similar to APP[V717I] overexpressing mice. This provides further evidence that ADAM10 in vivo by its enzymatic activity is able to counteract cognitive deficits. Stimulation of alpha-secretase activity might thus be a suitable approach to study treatment strategies of Alzheimer's disease.

  12. Comparative localization of ADAMs 10 and 15 in human cerebral cortex normal aging, Alzheimer disease and Down syndrome.

    PubMed

    Bernstein, Hans-Gert; Bukowska, Alicja; Krell, Dieter; Bogerts, Bernhard; Ansorge, Siegfried; Lendeckel, Uwe

    2003-02-01

    Using immunohistochemical techniques we studied the light microscopic localization of ADAMs (A Disintegrin And Metalloprotease) 10 and 15 in different neocortical areas of the human brain during normal aging, and also in patients with Alzheimer disease (AD) and Down syndrome (DS). ADAM 10, a putative alpha-secretase involved in Notch signaling, was found in neurons of the perinatal cortex. During aging there is an increase in intraneuronal staining intensity and in the number of cortical nerve cells that contain the enzyme. Furthermore, in AD and DS brains ADAM 10 immunoreactivity was associated with diffuse and neuritic plaques. ADAM 15 was detected in perinatal cortical pyramidal cells; during aging there was also an increase in intracellular staining and the number of stained cells per volume (cell density). In AD brains ADAM 15 was seen in a few diffuse plaques. Morphometric analysis revealed a significant reduction of ADAM 10 but not ADAM 15 immunoreactive neurons in AD brains in comparison to controls. Our findings support the idea that ADAM 10 is involved in the pathophysiology of AD and DS. ADAM 15 might be linked to AD via interaction with integrin and/or src protein tyrosine kinases.

  13. ADAM17 controls endochondral ossification by regulating terminal differentiation of chondrocytes.

    PubMed

    Hall, Katherine C; Hill, Daniel; Otero, Miguel; Plumb, Darren A; Froemel, Dara; Dragomir, Cecilia L; Maretzky, Thorsten; Boskey, Adele; Crawford, Howard C; Selleri, Licia; Goldring, Mary B; Blobel, Carl P

    2013-08-01

    Endochondral ossification is a highly regulated process that relies on properly orchestrated cell-cell interactions in the developing growth plate. This study is focused on understanding the role of a crucial regulator of cell-cell interactions, the membrane-anchored metalloproteinase ADAM17, in endochondral ossification. ADAM17 releases growth factors, cytokines, and other membrane proteins from cells and is essential for epidermal growth factor receptor (EGFR) signaling and for processing tumor necrosis factor alpha. Here, we report that mice lacking ADAM17 in chondrocytes (A17ΔCh) have a significantly expanded zone of hypertrophic chondrocytes in the growth plate and retarded growth of long bones. This abnormality is caused by an accumulation of the most terminally differentiated type of chondrocytes that produces a calcified matrix. Inactivation of ADAM17 in osteoclasts or endothelial cells does not affect the zone of hypertrophic chondrocytes, suggesting that the main role of ADAM17 in the growth plate is in chondrocytes. This notion is further supported by in vitro experiments showing enhanced hypertrophic differentiation of primary chondrocytes lacking Adam17. The enlarged zone of hypertrophic chondrocytes in A17ΔCh mice resembles that described in mice with mutant EGFR signaling or lack of its ligand transforming growth factor α (TGFα), suggesting that ADAM17 regulates terminal differentiation of chondrocytes during endochondral ossification by activating the TGFα/EGFR signaling axis.

  14. Activation of peroxisome proliferator-activated receptor α stimulates ADAM10-mediated proteolysis of APP.

    PubMed

    Corbett, Grant T; Gonzalez, Frank J; Pahan, Kalipada

    2015-07-07

    Amyloid precursor protein (APP) derivative β-amyloid (Aβ) plays an important role in the pathogenesis of Alzheimer's disease (AD). Sequential proteolysis of APP by β-secretase and γ-secretase generates Aβ. Conversely, the α-secretase "a disintegrin and metalloproteinase" 10 (ADAM10) cleaves APP within the eventual Aβ sequence and precludes Aβ generation. Therefore, up-regulation of ADAM10 represents a plausible therapeutic strategy to combat overproduction of neurotoxic Aβ. Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that regulates genes involved in fatty acid metabolism. Here, we determined that the Adam10 promoter harbors PPAR response elements; that knockdown of PPARα, but not PPARβ or PPARγ, decreases the expression of Adam10; and that lentiviral overexpression of PPARα restored ADAM10 expression in Ppara(-/-) neurons. Gemfibrozil, an agonist of PPARα, induced the recruitment of PPARα:retinoid x receptor α, but not PPARγ coactivator 1α (PGC1α), to the Adam10 promoter in wild-type mouse hippocampal neurons and shifted APP processing toward the α-secretase, as determined by augmented soluble APPα and decreased Aβ production. Accordingly, Ppara(-/-) mice displayed elevated SDS-stable, endogenous Aβ and Aβ1-42 relative to wild-type littermates, whereas 5XFAD mice null for PPARα (5X/α(-/-)) exhibited greater cerebral Aβ load relative to 5XFAD littermates. These results identify PPARα as an important factor regulating neuronal ADAM10 expression and, thus, α-secretase proteolysis of APP.

  15. Validation of ADAM10 metalloprotease as a Bacillus thuringiensis Cry3Aa toxin functional receptor in Colorado potato beetle (Leptinotarsa decemlineata).

    PubMed

    Ruiz-Arroyo, V M; García-Robles, I; Ochoa-Campuzano, C; Goig, G A; Zaitseva, E; Baaken, G; Martínez-Ramírez, A C; Rausell, C; Real, M D

    2017-04-01

    Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species. © 2016 The Royal Entomological Society.

  16. Upregulation of ADAM12 contributes to accelerated cell proliferation and cell adhesion-mediated drug resistance (CAM-DR) in Non-Hodgkin's Lymphoma.

    PubMed

    Yin, Haibing; Zhong, Fei; Ouyang, Yu; Wang, Qiru; Ding, Linlin; He, Song

    2017-10-01

    ADAM12 is a member of a disintegrin and metalloproteinase family and has been reported to participate in the development of variety of tumors. However, the role of ADAM12 in Non-Hodgkin Lymphoma (NHL) has not been investigated. The present study was undertaken to determine the expression and biologic function of ADAM12 in human NHL. First, we constructed a model of cell adhesion in NHL, the mRNA, and protein level of ADAM12 in suspension and the adhesion model was analyzed by RT-PCR and western blot. Then, flow cytometry assay and western blot were used to investigate the mechanism of ADAM12 in the proliferation of NHL cells. In vitro, after using siRNA interfering ADAM12 expression, we performed adhesion assay and cell viability assay to determine the effect of ADAM12 on adhesive rate and drug sensitivity. ADAM12 was lowly expressed in suspended cells and highly expressed in adherent NHL cells. In addition, ADAM12 was positively correlated with the proliferation and apoptosis of NHL cells by regulating the expression of p-AKT and p-GSK-3β. Furthermore, ADAM12 promoted cell adhesion-mediated drug resistance (CAM-DR) in DLBCL via AKT signaling pathway. Our data support a role for ADAM12 in NHL cell proliferation, adhesion, and drug resistance, and it may pave the way for a novel therapeutic approach for CAM-DR in NHL.

  17. A positive feedback loop between HER2 and ADAM12 in human head and neck cancer cells increases migration and invasion.

    PubMed

    Rao, V H; Kandel, A; Lynch, D; Pena, Z; Marwaha, N; Deng, C; Watson, P; Hansen, L A

    2012-06-07

    Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.

  18. ADAM12 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity.

    PubMed

    Stautz, Dorte; Sanjay, Archana; Hansen, Matilde Thye; Albrechtsen, Reidar; Wewer, Ulla M; Kveiborg, Marie

    2010-01-01

    ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.

  19. The Historical World of Henry Adams

    ERIC Educational Resources Information Center

    Blaser, Kent

    1976-01-01

    This paper examines Henry Adams' writings on history by considering four topics which comprise the basis of his thinking: politics, religion, sex, and science. Adam's main goal was to make history a means of exploring the most significant dimensions of human being. (Author/RM)

  20. ADAM12 redistributes and activates MMP-14, resulting in gelatin degradation, reduced apoptosis and increased tumor growth.

    PubMed

    Albrechtsen, Reidar; Kveiborg, Marie; Stautz, Dorte; Vikeså, Jonas; Noer, Julie B; Kotzsh, Alexander; Nielsen, Finn Cilius; Wewer, Ulla M; Fröhlich, Camilla

    2013-10-15

    Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVβ3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVβ3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.

  1. Asteroid shape modelling with ADAM

    NASA Astrophysics Data System (ADS)

    Viikinkoski, Matti; Kaasalainen, Mikko; Durech, Josef

    2015-08-01

    Technological advancements have made it possible to obtain highly detailed images of asteroids, yet 3-D shape reconstruction remains a challenge. Shape inversion is an ill-posed inverse problem as systematic errors, shadowing effects due to non-convex features, and the limitations of the imaging systems render the direct inversion impossible. Moreover, the coverage of one observation session alone is seldom sufficient for 3-D reconstruction, necessitating a method for the integration of widely different, complementary data sources into a coherent shape solution.We present a new 3-D shape reconstruction method for asteroid models. ADAM, an acronym for all-data asteroid modelling, is a general procedure for combining disk-resolved observational data into a shape model. ADAM handles all disk-resolved data in a uniform manner via 2-D Fourier Transform. Almost all disk-resolved data sources are supported: adaptive optics and other images, range-Doppler radar data, and thermal infrared interferometry.As case studies, we examine the shape of (41) Daphne using the adaptive optics images and photometry, and create a model of the asteroid 2000 ET70 from the range-Doppler radar images. Finally, we combine ALMA science verification data, adaptive optics images, occultations, and lightcurve data to study the shape of the large main-belt asteroid (3) Juno.

  2. Recombinant disintegrin domain of ADAM15 inhibits the proliferation and migration of Bel-7402 cells

    SciTech Connect

    Hou, Y.; Chu, M.; Du, F.F.; Lei, J.Y.; Chen, Y.; Zhu, R.Y.; Gong, X.H.; Ma, X.; Jin, J.

    2013-06-14

    Highlights: •rhddADAM15 inhibited the proliferation and migration of Bel-7402 cells. •rhddADAM15 inhibited growth and metastasis of Bel-7402 cells in zebrafish xenograft. •rhddADAM15 induced apoptosis in Bel-7402 cells and somatic cells of zebrafish. •Cell-cycle in Bel-7402 cells showed a partial G{sub 2}/S arrest. •Activity of caspases 8, 9 and 3 was increased in rhddADAM15-treated Bel-7402 cells. -- Abstract: ADAM15 (A Disintegrin And Metalloproteinase 15), a transmembrane protein containing seven domains, interacts with some integrins via its disintegrin domain and overexpresses in many solid tumors. In this study, the effect of the recombinant human disintegrin domain (rhddADAM15) on the proliferation and migration of Bel-7402 cells was evaluated in vitro and in vivo in zebrafish xenografts. rhddADAM15 (4 μM) severely inhibited the proliferation and migration of Bel-7402 cells, inducing a partial G{sub 2}/S arrest and morphological nucleus changes of apoptosis. Moreover, the activity of caspases 8, 9 and 3 in Bel-7402 cells was increased. In addition, the zebrafish was used as a model for apoptosis-induction and tumor-xenograft. rhddADAM15 (1 pM) inhibited the growth and metastasis of Bel-7402 cell xenografts in zebrafish and a lower concentration (0.1 pM) induced severe apoptosis in the somatic cells of zebrafish. In conclusion, our data identified rhddADAM15 as a potent inhibitor of tumor growth and metastasis, making it a promising tool for use in anticancer treatment.

  3. ADAM12-directed ectodomain shedding of E-cadherin potentiates trophoblast fusion.

    PubMed

    Aghababaei, M; Hogg, K; Perdu, S; Robinson, W P; Beristain, A G

    2015-12-01

    Trophoblasts, placental cells of epithelial lineage, undergo extensive differentiation to form the cellular components of the placenta. Trophoblast progenitor cell differentiation into the multinucleated syncytiotrophoblast is a key developmental process required for placental function, where defects in syncytiotrophoblast formation and turnover associate with placental pathologies and link to poor pregnancy outcomes. The cellular and molecular processes governing syncytiotrophoblast formation are poorly understood, but require the activation of pathways that direct cell fusion. The protease, A Disintegrin and Metalloproteinase 12 (ADAM12), controls cell fusion in myoblasts and is highly expressed in the placenta localizing to multiple trophoblast populations. However, the importance of ADAM12 in regulating trophoblast fusion is unknown. Here, we describe a function for ADAM12 in regulating trophoblast fusion. Using two distinct trophoblast models of cell fusion, we show that ADAM12 is dynamically upregulated and is under the transcriptional control of protein kinase A. siRNA-directed loss of ADAM12 impedes spontaneous fusion of primary cytotrophoblasts, whereas overexpression of the secreted variant, ADAM12S, potentiates cell fusion in the Bewo trophoblast cell line. Mechanistically, both ectopic and endogenous levels of ADAM12 were shown to control trophoblast fusion through E-cadherin ectodomain shedding and remodeling of intercellular boundaries. This study describes a novel role for ADAM12 in placental development, specifically highlighting its importance in controlling the differentiation of villous cytotrophoblasts into multinucleated cellular structures. Moreover, this work identifies E-cadherin as a novel ADAM12 substrate, and highlights the significance that cell adhesion molecule ectodomain shedding has in normal development.

  4. Amyotrophic lateral sclerosis mimic syndromes

    PubMed Central

    Ghasemi, Majid

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) misdiagnosis has many broad implications for the patient and the neurologist. Potentially curative treatments exist for certain ALS mimic syndromes, but delay in starting these therapies may have an unfavorable effect on outcome. Hence, it is important to exclude similar conditions. In this review, we discuss some of the important mimics of ALS. PMID:27326363

  5. A disintegrin and metalloproteinase 12 (ADAM12) localizes to invasive trophoblast, promotes cell invasion and directs column outgrowth in early placental development.

    PubMed

    Aghababaei, M; Perdu, S; Irvine, K; Beristain, A G

    2014-03-01

    During pregnancy, stromal- and vascular-remodeling trophoblasts serve critical roles in directing placental development acquiring pro-invasive characteristics. The A Disintegrin and Metalloproteinase (ADAM) family of multifunctional proteins direct cellular processes across multiple organ systems via their intrinsic catalytic, cell adhesive and intracellular signaling properties. ADAM12, existing as two distinct splice variants (ADAM12L and ADAM12S), is highly expressed in the human placenta and promotes cell migration and invasion in several tumor cell lines; however, its role in trophoblast biology is unknown. In this study, ADAM12 was localized to anchoring trophoblast columns in first trimester placentas and to highly invasive extracellular matrix-degrading trophoblasts in placental villous explants. The importance of ADAM12 in directing trophoblast invasion was tested using loss-of and gain-of-function strategies, where siRNA-directed knockdown of ADAM12 inhibited trophoblast cell invasion while over-expression promoted migration and invasion in two trophoblastic cell models. In placental villous explant cultures, siRNA-directed loss of ADAM12 significantly dampened trophoblast column outgrowth. Additionally, we provide functional evidence for the ADAM12S variant in promoting trophoblast invasion and column outgrowth through a mechanism requiring its catalytic activity. This is the first study to assign a function for ADAM12 in trophoblast biology, where ADAM12 may play a central role regulating the behavior of invasive trophoblast subsets in early pregnancy. This study also underlines the importance of ADAM12L and ADAM12S in directing cell motility in normal developmental processes outside of cancer, specifically highlighting a potentially important function of ADAM12S in directing early placental development.

  6. Insulin treatment attenuates renal ADAM17 and ACE2 shedding in diabetic Akita mice.

    PubMed

    Salem, Esam S B; Grobe, Nadja; Elased, Khalid M

    2014-03-15

    Angiotensin-converting enzyme 2 (ACE2) is located in several tissues and is highly expressed in renal proximal tubules, where it degrades the vasoconstrictor angiotensin II (ANG II) to ANG-(1-7). Accumulating evidence supports protective roles of ACE2 in several disease states, including diabetic nephropathy. A disintegrin and metalloprotease (ADAM) 17 is involved in the shedding of several transmembrane proteins, including ACE2. Our previous studies showed increased renal ACE2, ADAM17 expression, and urinary ACE2 in type 2 diabetic mice (Chodavarapu H, Grobe N, Somineni HK, Salem ES, Madhu M, Elased KM. PLoS One 8: e62833, 2013). The aim of the present study was to determine the effect of insulin on ACE2 shedding and ADAM17 in type 1 diabetic Akita mice. Results demonstrate increased renal ACE2 and ADAM17 expression and increased urinary ACE2 fragments (≈70 kDa) and albumin excretion in diabetic Akita mice. Immunostaining revealed colocalization of ACE2 with ADAM17 in renal tubules. Renal proximal tubular cells treated with ADAM17 inhibitor showed reduced ACE2 shedding into the media, confirming ADAM17-mediated shedding of ACE2. Treatment of Akita mice with insulin implants for 20 wk normalized hyperglycemia and decreased urinary ACE2 and albumin excretion. Insulin also normalized renal ACE2 and ADAM17 but had no effect on tissue inhibitor of metalloproteinase 3 (TIMP3) protein expression. There was a positive linear correlation between urinary ACE2 and albuminuria, blood glucose, plasma creatinine, glucagon, and triglycerides. This is the first report showing an association between hyperglycemia, cardiovascular risk factors, and increased shedding of urinary ACE2 in diabetic Akita mice. Urinary ACE2 could be used as a biomarker for diabetic nephropathy and as an index of intrarenal ACE2 status.

  7. Hypoxia-induced ADAM 17 expression is mediated by RSK1-dependent C/EBPβ activation in human lung fibroblasts.

    PubMed

    Chen, Jing-Yun; Lin, Chien-Huang; Chen, Bing-Chang

    2017-08-01

    Hypoxia was identified as a mediator of lung fibrosis in patients with chronic obstructive asthma (COA). Overexpression of a disintegrin and metalloproteinase 17 (ADAM 17) and connective tissue growth factor (CTGF) leads to development of tissue fibrosis. However, the signaling pathway in hypoxia-induced ADAM 17 expression remains poorly defined. In this study, we investigated the roles that ribosomal S-6 kinase 1 (RSK1)/CCAAT/enhancer-binding protein β (C/EBPβ)-dependent ADAM 17 expression plays in hypoxia-induced CTGF expression in human lung fibroblasts. We observed that hypoxia caused increases in ADAM 17 expression and ADAM 17-luciferase activity in WI-38 cells. Hypoxia-induced CTGF-luciferase activity and CTGF expression were reduced in cells transfected with small interfering (si)RNA of ADAM 17 in WI-38 cells. Moreover, hypoxia-induced ADAM 17 expression was reduced by RSK1 siRNA and C/EBPβ siRNA. Hypoxia caused time-dependent increases in RSK1 phosphorylation at Thr359/Ser363. Exposure of cells to hypoxia resulted in increased C/EBPβ phosphorylation at Thr266 and C/EBPβ-luciferase activity in time-dependent manners, and these effects were suppressed by RSK1 siRNA. Hypoxia induced recruitment of C/EBPβ to the ADAM 17 promoter. Furthermore, CTGF-luciferase activity induced by hypoxia was attenuated by RSK1 siRNA and C/EBPβ siRNA. These results suggest that hypoxia instigates the RSK1-dependent C/EBPβ signaling pathway, which in turn initiates binding of C/EBPβ to the ADAM 17 promoter and ultimately induces ADAM 17 expression in human lung fibroblasts. Moreover, RSK1/C/EBPβ-dependent ADAM 17 expression is involved in hypoxia-induced CTGF expression. Our results suggest possible therapeutic approaches for treating hypoxia-mediated lung fibrosis in COA. Copyright © 2017. Published by Elsevier Ltd.

  8. The Disintegrin and Metalloprotease ADAM12 Is Associated with TGF-β-Induced Epithelial to Mesenchymal Transition.

    PubMed

    Ruff, Michaël; Leyme, Anthony; Le Cann, Fabienne; Bonnier, Dominique; Le Seyec, Jacques; Chesnel, Franck; Fattet, Laurent; Rimokh, Ruth; Baffet, Georges; Théret, Nathalie

    2015-01-01

    The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-β and promotes TGF-β-dependent signaling through interaction with the type II receptor of TGF-β. Here we explore the implication of ADAM12 in TGF-β-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-β-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-β receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-β-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.

  9. Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays.

    PubMed

    Maretzky, Thorsten; Swendeman, Steven; Mogollon, Elin; Weskamp, Gisela; Sahin, Umut; Reiss, Karina; Blobel, Carl P

    2017-04-13

    ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Castleman disease: the great mimic.

    PubMed

    Bonekamp, David; Horton, Karen M; Hruban, Ralph H; Fishman, Elliot K

    2011-10-01

    Castleman disease is a nonclonal lymphoproliferative disorder and one of the more common causes of nonneoplastic lymphadenopathy. Because of its diverse manifestations and ability to affect any body region, Castleman disease is a great mimic of both benign and malignant abnormalities in the neck, chest, abdomen, and pelvis. Castleman disease most commonly manifests as unicentric disease (unicentric Castleman disease) with a hyperenhancing lymph nodal mass and should be considered in the differential diagnosis of lymphoma, metastatic adenopathy, and infectious and/or inflammatory diseases that result in adenopathy. Castleman disease includes a spectrum of pathologic variants, including the classic hyaline vascular type, the less common plasma cell variant of Castleman disease, and the more recently described multicentric Castleman disease and Castleman disease associated with human herpesvirus 8. Castleman disease has been associated with the human immunodeficiency virus, lymphoma, POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome, paraneoplastic pemphigus, and plasma cell dyscrasias. Aggressive forms of Castleman disease with systemic manifestations may occur. Unicentric hyaline vascular Castleman disease is often curable with surgery; however, multicentric Castleman disease may require steroid treatment, chemotherapy, antiviral medication, or the use of antiproliferative regimens because a surgical procedure cannot be curative in this setting. Supplemental material available at http://radiographics.rsna.org/lookup/suppl/doi:10.1148/rg.316115502/-/DC1. © RSNA, 2011.

  11. ADAM8 is selectively upregulated in endothelial cells and is associated with angiogenesis after spinal cord injury in adult mice

    PubMed Central

    Mahoney, Edward T.; Benton, Richard L.; Maddie, Melissa A.; Whittemore, Scott R.; Hagg, Theo

    2009-01-01

    Endothelial cell (EC) loss and subsequent angiogenesis occurs over the first week after spinal cord injury (SCI). To identify molecular mechanisms that could be targeted with intravenous (i.v.) treatments we determined whether transmembrane A Disintegrin And Metalloprotease (ADAM) proteins are expressed in ECs of the injured spinal cord. ADAMs bind to integrins which are important for EC survival and angiogenesis. Female adult C57Bl/6 mice with a spinal cord contusion had progressively more ADAM8 (CD156) immunostaining in blood vessels and individual ECs between 1 and 28 days following injury. Uninjured spinal cords had little ADAM8 staining. The increase in ADAM8 mRNA and protein was confirmed in spinal cord lysates, and ADAM8 mRNA was present in FACS-enriched ECs. ADAM8 co-localized extensively and exclusively with the EC marker PECAM and also with i.v. injected lectins. I.v. injected isolectin B4 (IB4) labels a subpopulation of blood vessels at and within the injury epicenter 3-7 days after injury, coincident with angiogenesis. Both ADAM8 and the proliferation marker Ki-67 were present in IB4-positive microvessels. ADAM8-positive proliferating cells were seen at the leading end of IB4-positive blood vessels. Angiogenesis was confirmed by BrdU incorporation, binding of i.v. injected nucleolin antibodies, and MT1-MMP immunostaining in a subset of blood vessels. These data suggest that ADAM8 is vascular-selective and plays a role in proliferation and/or migration of ECs during angiogenesis following SCI. PMID:19003792

  12. Recombinant Sendai viruses expressing fusion proteins with two furin cleavage sites mimic the syncytial and receptor-independent infection properties of respiratory syncytial virus.

    PubMed

    Rawling, Joanna; Cano, Olga; Garcin, Dominique; Kolakofsky, Daniel; Melero, José A

    2011-03-01

    Cell entry by paramyxoviruses requires fusion between viral and cellular membranes. Paramyxovirus infection also gives rise to the formation of multinuclear, fused cells (syncytia). Both types of fusion are mediated by the viral fusion (F) protein, which requires proteolytic processing at a basic cleavage site in order to be active for fusion. In common with most paramyxoviruses, fusion mediated by Sendai virus F protein (F(SeV)) requires coexpression of the homologous attachment (hemagglutinin-neuraminidase [HN]) protein, which binds to cell surface sialic acid receptors. In contrast, respiratory syncytial virus fusion protein (F(RSV)) is capable of fusing membranes in the absence of the viral attachment (G) protein. Moreover, F(RSV) is unique among paramyxovirus fusion proteins since F(RSV) possesses two multibasic cleavage sites, which are separated by an intervening region of 27 amino acids. We have previously shown that insertion of both F(RSV) cleavage sites in F(SeV) decreases dependency on the HN attachment protein for syncytium formation in transfected cells. We now describe recombinant Sendai viruses (rSeV) that express mutant F proteins containing one or both F(RSV) cleavage sites. All cleavage-site mutant viruses displayed reduced thermostability, with double-cleavage-site mutants exhibiting a hyperfusogenic phenotype in infected cells. Furthermore, insertion of both F(RSV) cleavage sites in F(SeV) reduced dependency on the interaction of HN with sialic acid for infection, thus mimicking the unique ability of RSV to fuse and infect cells in the absence of a separate attachment protein.

  13. ADAM13 function is required in the 3 dimensional context of the embryo during cranial neural crest cell migration in Xenopus laevis

    PubMed Central

    Cousin, Hélène; Abbruzzese, Genevieve; McCusker, Catherine; Alfandari, Dominique

    2012-01-01

    The cranial neural crest (CNC) is a population of cells that arises from the lateral part of the developing brain, migrates ventrally and coordinates the entire craniofacial development of vertebrates. Many molecules are involved in CNC migration including the transmembrane metalloproteases ADAM13 and 19. We have previously shown that these ADAMs cleave a number of extracellular proteins and modify the transcription of a number of genes, and that both of these activities are important for cell migration. Here we show that the knock down of ADAM13 inhibits CNC migration in vivo but not in vitro, indicating that ADAM13 function is required in the 3-dimentional context of the embryo. We further show that the migration of CNC that do not express ADAM13 and ADAM19 can be rescued in vivo by co-grafting wild type CNC. Furthermore, the migration of CNC lacking ADAM13 can be rescued by mechanically separating the CNC from the surrounding ectoderm and mesoderm. Finally, we show that ADAM13 function is autonomous to CNC tissue, as the migration of morphant CNC can only be rescued by ADAM13 expression in the CNC and not the surrounding tissues. Together our results suggest that ADAM13 changes CNC interaction with the extracellular environment and that this change is necessary for their migration in vivo. PMID:22683825

  14. Integrin alpha5beta1 and ADAM-17 interact in vitro and co-localize in migrating HeLa cells.

    PubMed

    Bax, Daniel V; Messent, Anthea J; Tart, Jonathan; van Hoang, Mien; Kott, Jane; Maciewicz, Rose A; Humphries, Martin J

    2004-05-21

    Tumor necrosis factor (TNF) alpha-converting enzyme (TACE/ADAM-17) has diverse roles in the proteolytic processing of cell surface molecules and, due to its ability to process TNFalpha, is a validated therapeutic target for anti-inflammatory therapies. Unlike a number of other ADAM proteins, which interact with integrin receptors via their disintegrin domains, there is currently no evidence for an ADAM-17-integrin association. By analyzing the adhesion of a series of cell lines with recombinant fragments of the extracellular domain of ADAM-17, we now demonstrate a functional interaction between ADAM-17 and alpha(5)beta(1) integrin in a trans orientation. Because ADAM-17-mediated adhesion was sensitive to RGD peptides and EDTA, and the integrin-binding site within ADAM-17 was narrowed down to the disintegrin/cysteine-rich region, the two molecules appear to have a ligand-receptor relationship mediated by the alpha(5)beta(1) ligand binding pocket. Intriguingly, ADAM-17 and alpha(5)beta(1) were found to co-localize in both membrane ruffles and focal adhesions in HeLa cells. When confluent HeLa cell monolayers were wounded, ADAM-17 and alpha(5)beta(1) redistributed to the leading edge and co-localized, which is suggestive of a cis orientation. We postulate that the interaction of ADAM-17 with alpha(5)beta(1) may target or modulate its metalloproteolytic activity.

  15. Active site determinants of substrate recognition by the metalloproteinases TACE and ADAM10

    PubMed Central

    Caescu, Cristina I.; Jeschke, Grace R.; Turk, Benjamin E.

    2009-01-01

    The metalloproteinases TACE (ADAM17) and ADAM10 are the primary enzymes responsible for catalyzing release of membrane anchored proteins from the cell surface in metazoan organisms. While the repertoire of protein substrates for these two proteases is partially overlapping, each one appears to target a subset of unique proteins in vivo. The mechanisms by which the two proteases achieve specificity for particular substrates are not completely understood. We have used peptide libraries to define the cleavage site selectivity of TACE and ADAM10. The two proteases have distinct primary sequence requirements at multiple positions surrounding the cleavage site in their substrates, which allowed us to generate peptide substrates that are highly specific for each of these proteases. The major difference between the two protease specificities maps to the P1′ position (immediately downstream of the cleavage site) of the substrate. At this position, TACE is selective for smaller aliphatic residues, while ADAM10 can accommodate aromatic amino acids. Using mutagenesis we identify three residues in the S1′ pockets of these enzymes that dramatically influence specificity for both peptide and protein substrates. Our results suggest that substrate selectivity of TACE and ADAM10 can be at least partly rationalized by specific features of their active sites. PMID:19715556

  16. ADAM17 is regulated by a rapid and reversible mechanism that controls access to its catalytic site

    PubMed Central

    Le Gall, Sylvain M.; Maretzky, Thorsten; Issuree, Priya D. A.; Niu, Xiao-Da; Reiss, Karina; Saftig, Paul; Khokha, Rama; Lundell, Daniel; Blobel, Carl P.

    2010-01-01

    Protein ectodomain shedding is crucial for cell–cell interactions because it controls the bioavailability of soluble tumor necrosis factor-α (TNFα) and ligands of the epidermal growth factor (EGF) receptor, and the release of many other membrane proteins. Various stimuli can rapidly trigger ectodomain shedding, yet much remains to be learned about the identity of the enzymes that respond to these stimuli and the mechanisms underlying their activation. Here, we demonstrate that the membrane-anchored metalloproteinase ADAM17, but not ADAM10, is the sheddase that rapidly responds to the physiological signaling pathways stimulated by thrombin, EGF, lysophosphatidic acid and TNFα. Stimulation of ADAM17 is swift and quickly reversible, and does not depend on removal of its inhibitory pro-domain by pro-protein convertases, or on dissociation of an endogenous inhibitor, TIMP3. Moreover, activation of ADAM17 by physiological stimuli requires its transmembrane domain, but not its cytoplasmic domain, arguing against inside–out signaling via cytoplasmic phosphorylation as the underlying mechanism. Finally, experiments with the tight binding hydroxamate inhibitor DPC333, used here to probe the accessibility of the active site of ADAM17, demonstrate that this inhibitor can quickly bind to ADAM17 in stimulated, but not quiescent cells. These findings support the concept that activation of ADAM17 involves a rapid and reversible exposure of its catalytic site. PMID:20980382

  17. Cleavage Site Localization Differentially Controls Interleukin-6 Receptor Proteolysis by ADAM10 and ADAM17

    PubMed Central

    Riethmueller, Steffen; Ehlers, Johanna C.; Lokau, Juliane; Düsterhöft, Stefan; Knittler, Katharina; Dombrowsky, Gregor; Grötzinger, Joachim; Rabe, Björn; Rose-John, Stefan; Garbers, Christoph

    2016-01-01

    Limited proteolysis of the Interleukin-6 Receptor (IL-6R) leads to the release of the IL-6R ectodomain. Binding of the cytokine IL-6 to the soluble IL-6R (sIL-6R) results in an agonistic IL-6/sIL-6R complex, which activates cells via gp130 irrespective of whether the cells express the IL-6R itself. This signaling pathway has been termed trans-signaling and is thought to mainly account for the pro-inflammatory properties of IL-6. A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 are the major proteases that cleave the IL-6R. We have previously shown that deletion of a ten amino acid long stretch within the stalk region including the cleavage site prevents ADAM17-mediated cleavage, whereas the receptor retained its full biological activity. In the present study, we show that deletion of a triple serine (3S) motif (Ser-359 to Ser-361) adjacent to the cleavage site is sufficient to prevent IL-6R cleavage by ADAM17, but not ADAM10. We find that the impaired shedding is caused by the reduced distance between the cleavage site and the plasma membrane. Positioning of the cleavage site in greater distance towards the plasma membrane abrogates ADAM17-mediated shedding and reveals a novel cleavage site of ADAM10. Our findings underline functional differences in IL-6R proteolysis by ADAM10 and ADAM17. PMID:27151651

  18. John Quincy Adams's rhetorical crusade for astronomy.

    PubMed

    Portolano, M

    2000-09-01

    Astronomy thrived in Europe during the early nineteenth century, but in the United States a utilitarian mind-set opposed it. John Quincy Adams's oratory in support of American astronomical discovery reached its peak during congressional debate over the Smithsonian Institution (1838-1846). During this debate Adams countered proposals to found a university with plans for an observatory. His addresses to congressional and public audiences about observatories and astronomy were intended to foster interest in the science and encourage the growing astronomical community in America. Although the U.S. Naval Observatory in Washington, D.C., was established before the Smithsonian debate ended, many considered Adams its political father. Adams composed his speeches on astronomy in a systematic manner, following neoclassical principles of rhetoric that he had taught at Harvard University. His speeches both in and outside of Congress show evidence of the rhetorical principles he conscientiously used in the service of astronomy.

  19. Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing▿

    PubMed Central

    Kwun, Hyun Jin; da Silva, Suzane Ramos; Shah, Ishita M.; Blake, Neil; Moore, Patrick S.; Chang, Yuan

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses. PMID:17522213

  20. ADAMs family and relatives in cardiovascular physiology and pathology.

    PubMed

    Zhang, Pu; Shen, Mengcheng; Fernandez-Patron, Carlos; Kassiri, Zamaneh

    2016-04-01

    A disintegrin and metalloproteinases (ADAMs) are a family of membrane-bound proteases. ADAM-TSs (ADAMs with thrombospondin domains) are a close relative of ADAMs that are present in soluble form in the extracellular space. Dysregulated production or function of these enzymes has been associated with pathologies such as cancer, asthma, Alzheimer's and cardiovascular diseases. ADAMs contribute to angiogenesis, hypertrophy and apoptosis in a stimulus- and cell type-dependent manner. Among the ADAMs identified so far (34 in mouse, 21 in human), ADAMs 8, 9, 10, 12, 17 and 19 have been shown to be involved in cardiovascular development or cardiomyopathies; and among the 19 ADAM-TSs, ADAM-TS1, 5, 7 and 9 are important in development of the cardiovascular system, while ADAM-TS13 can contribute to vascular disorders. Meanwhile, there remain a number of ADAMs and ADAM-TSs whose function in the cardiovascular system has not been yet explored. The current knowledge about the role of ADAMs and ADAM-TSs in the cardiovascular pathologies is still quite limited. The most detailed studies have been performed in other cell types (e.g. cancer cells) and organs (nervous system) which can provide valuable insight into the potential functions of ADAMs and ADAM-TSs, their mechanism of action and therapeutic potentials in cardiomyopathies. Here, we review what is currently known about the structure and function of ADAMs and ADAM-TSs, and their roles in development, physiology and pathology of the cardiovascular system. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Epilepsy-related ligand/receptor complex LGI1 and ADAM22 regulate synaptic transmission.

    PubMed

    Fukata, Yuko; Adesnik, Hillel; Iwanaga, Tsuyoshi; Bredt, David S; Nicoll, Roger A; Fukata, Masaki

    2006-09-22

    Abnormally synchronized synaptic transmission in the brain causes epilepsy. Most inherited forms of epilepsy result from mutations in ion channels. However, one form of epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF), is characterized by mutations in a secreted neuronal protein, LGI1. We show that ADAM22, a transmembrane protein that when mutated itself causes seizure, serves as a receptor for LGI1. LGI1 enhances AMPA receptor-mediated synaptic transmission in hippocampal slices. The mutated form of LGI1 fails to bind to ADAM22. ADAM22 is anchored to the postsynaptic density by cytoskeletal scaffolds containing stargazin. These studies in rat brain indicate possible avenues for understanding human epilepsy.

  2. A Conversation with Adam Heller.

    PubMed

    Heller, Adam; Cairns, Elton J

    2015-01-01

    Adam Heller, Ernest Cockrell Sr. Chair in Engineering Emeritus of the John J. McKetta Department of Chemical Engineering at The University of Texas at Austin, recalls his childhood in the Holocaust and his contributions to science and technology that earned him the US National Medal of Technology and Innovation in a conversation with Elton J. Cairns, Professor of Chemical and Biomolecular Engineering at the University of California, Berkeley. Dr. Heller, born in 1933, describes the enslavement of his father by Hungarians in 1942; the confiscation of his family's home, business, and all its belongings in 1944; and his incarceration in a brick factory with 18,000 Jews who were shipped by the Hungarians to be gassed by Germans in Auschwitz. Dr. Heller and his immediate family survived the Holocaust and arrived in Israel in 1945. He studied under Ernst David Bergmann at the Hebrew University, and then worked at Bell Laboratories and GTE Laboratories, where he headed Bell Lab's Electronic Materials Research Department. At GTE Laboratories, he built in 1966 the first neodymium liquid lasers and in 1973 with Jim Auborn conceived and engineered the lithium thionyl chloride battery, one of the first to be manufactured lithium batteries, which is still in use. After joining the faculty of engineering of The University of Texas at Austin, he cofounded with his son Ephraim Heller TheraSense, now a major part of Abbott Diabetes Care, which produced a microcoulometer that made the monitoring of glucose painless by accurately measuring the blood glucose concentration in 300 nL of blood. He also describes the electrical wiring of enzymes, the basis for Abbott's state-of-the-art continuous glucose monitoring system. He discusses his perspective of reducing the risk of catastrophic global warming in a wealth-accumulating, more-energy-consuming world and provides advice for students entering careers in science or engineering.

  3. ADAM17 limits the expression of CSF1R on murine hematopoietic progenitors

    PubMed Central

    Becker, Amy M.; Walcheck, Bruce; Bhattacharya, Deepta

    2014-01-01

    All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through post-transcriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of Csf1r transcripts than their upstream precursors, yet show limited cell surface protein expression of CSF1R. ALPs and other hematopoietic progenitors deficient in ADAM17, a metalloprotease that can cleave CSF1R, display elevated cell surface CSF1R expression. Adam17−/− ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, Adam17−/− ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of M-CSF. Mice with hematopoietic-specific deletion of Adam17 have grossly normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential. PMID:25308957

  4. ADAM17 limits the expression of CSF1R on murine hematopoietic progenitors.

    PubMed

    Becker, Amy M; Walcheck, Bruce; Bhattacharya, Deepta

    2015-01-01

    All-lymphoid progenitors (ALPs) yield few myeloid cells in vivo, but readily generate such cells in vitro. The basis for this difference remains unknown. We hypothesized that ALPs limit responsiveness to in vivo concentrations of myeloid-promoting cytokines by reducing expression of the corresponding receptors, potentially through posttranscriptional mechanisms. Consistent with such a mechanism, ALPs express higher levels of CSF1R transcripts than their upstream precursors, yet show limited cell-surface protein expression of colony-stimulating factor 1 receptor (CSF1R). All-lymphoid progenitors and other hematopoietic progenitors deficient in A disintegrin and metalloproteinase domain 17 (ADAM17), display elevated cell surface CSF1R expression. ADAM17(-/-) ALPs, however, fail to yield myeloid cells upon transplantation into irradiated recipients. Moreover, ADAM17(-/-) ALPs yield fewer macrophages in vitro than control ALPs at high concentrations of macrophage colony stimulating factor. Mice with hematopoietic-specific deletion of ADAM17 have normal numbers of myeloid and lymphoid progenitors and mature cells in vivo. These data demonstrate that ADAM17 limits CSF1R protein expression on hematopoietic progenitors, but that compensatory mechanisms prevent elevated CSF1R levels from altering lymphoid progenitor potential. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  5. ADAM9 promotes lung cancer metastases to brain by a plasminogen activator-based pathway.

    PubMed

    Lin, Chen-Yuan; Chen, Hung-Jen; Huang, Cheng-Chung; Lai, Liang-Chuan; Lu, Tzu-Pin; Tseng, Guan-Chin; Kuo, Ting-Ting; Kuok, Qian-Yu; Hsu, Jennifer L; Sung, Shian-Ying; Hung, Mien-Chie; Sher, Yuh-Pyng

    2014-09-15

    The transmembrane cell adhesion protein ADAM9 has been implicated in cancer cell migration and lung cancer metastasis to the brain, but the underpinning mechanisms are unclear and clinical support has been lacking. Here, we demonstrate that ADAM9 enhances the ability of tissue plasminogen activator (tPA) to cleave and stimulate the function of the promigratory protein CDCP1 to promote lung metastasis. Blocking this mechanism of cancer cell migration prolonged survival in tumor-bearing mice and cooperated with dexamethasone and dasatinib (a dual Src/Abl kinase inhibitor) treatment to enhance cytotoxic treatment. In clinical specimens, high levels of ADAM9 and CDCP1 correlated with poor prognosis and high risk of mortality in patients with lung cancer. Moreover, ADAM9 levels in brain metastases derived from lung tumors were relatively higher than the levels observed in primary lung tumors. Our results show how ADAM9 regulates lung cancer metastasis to the brain by facilitating the tPA-mediated cleavage of CDCP1, with potential implications to target this network as a strategy to prevent or treat brain metastatic disease. ©2014 American Association for Cancer Research.

  6. Chromosomal mapping, sequence and transcription analysis of the porcine fertilin beta gene (ADAM2).

    PubMed

    Day, A E; Quilter, C R; Sargent, C A; Mileham, A J

    2003-10-01

    Fertilin beta (ADAM2) forms a part of the heterodimeric surface protein fertilin, found on the plasma membrane of mammalian sperm, and has been implicated in the process of sperm-egg fusion. Analysis of cDNA products obtained from adult porcine testis mRNA has presented a sequence corresponding to 2620 bp of the ADAM2 gene. This sequence contained an open reading frame encoding a 735-amino acid protein and homologous to ADAM2 genes known in other mammalian species. Polymerase chain reaction (PCR) analysis of genomic DNA showed that the 2620 bp of cDNA sequence comprises at least 21 exons and spans approximately 76 kb of genomic DNA, with its size and structure being relatively conserved between mouse, human and pig. Fluorescence in situ hybridization was used to map ADAM2 to chromosome 15 of the pig, using a bacterial artificial chromosome clone from the PigE BAC library. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Analysis of nine mRNA samples, by reverse transcriptase-PCR, from different porcine tissues has also suggested that expression of ADAM2 is limited to the testis, a finding that is consistent with other mammalian species.

  7. Functional recognition of the modified human tRNALys3(UUU) anticodon domain by HIV's nucleocapsid protein and a peptide mimic.

    PubMed

    Graham, William D; Barley-Maloney, Lise; Stark, Caren J; Kaur, Amarpreet; Stolarchuk, Christina; Stolyarchuk, Khrystyna; Sproat, Brian; Leszczynska, Grazyna; Malkiewicz, Andrzej; Safwat, Nedal; Mucha, Piotr; Guenther, Richard; Agris, Paul F

    2011-07-22

    The HIV-1 nucleocapsid protein, NCp7, facilitates the use of human tRNA(Lys3)(UUU) as the primer for reverse transcription. NCp7 also remodels the htRNA's amino acid accepting stem and anticodon domains in preparation for their being annealed to the viral genome. To understand the possible influence of the htRNA's unique composition of post-transcriptional modifications on NCp7 recognition of htRNA(Lys3)(UUU), the protein's binding and functional remodeling of the human anticodon stem and loop domain (hASL(Lys3)) were studied. NCp7 bound the hASL(Lys3)(UUU) modified with 5-methoxycarbonylmethyl-2-thiouridine at position-34 (mcm(5)s(2)U(34)) and 2-methylthio-N(6)-threonylcarbamoyladenosine at position-37 (ms(2)t(6)A(37)) with a considerably higher affinity than the unmodified hASL(Lys3)(UUU) (K(d)=0.28±0.03 and 2.30±0.62 μM, respectively). NCp7 denatured the structure of the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) more effectively than that of the unmodified hASL(Lys3)(UUU). Two 15 amino acid peptides selected from phage display libraries demonstrated a high affinity (average K(d)=0.55±0.10 μM) and specificity for the ASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37) comparable to that of NCp7. The peptides recognized a t(6)A(37)-modified ASL with an affinity (K(d)=0.60±0.09 μM) comparable to that for hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37), indicating a preference for the t(6)A(37) modification. Significantly, one of the peptides was capable of relaxing the hASL(Lys3)(UUU)-mcm(5)s(2)U(34);ms(2)t(6)A(37);Ψ(39) structure in a manner similar to that of NCp7, and therefore could be used to further study protein recognition of RNA modifications. The post-transcriptional modifications of htRNA(Lys3)(UUU) have been found to be important determinants of NCp7's recognition prior to the tRNA(Lys3)(UUU) being annealed to the viral genome as the primer of reverse transcription. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

    PubMed

    Cura, Vincent; Troffer-Charlier, Nathalie; Wurtz, Jean Marie; Bonnefond, Luc; Cavarelli, Jean

    2014-09-01

    Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

  9. The disintegrin domain of ADAM9: a ligand for multiple β1 renal integrins

    PubMed Central

    2004-01-01

    Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the β1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595–603]. Discrete segments of the nephron express several defined β1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell–matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell–matrix binding. To define the specific renal tubular β1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to α1, α3, α6, αv and β1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the α3, α6, αv and β1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the β1 class. PMID:15361064

  10. The disintegrin domain of ADAM9: a ligand for multiple beta1 renal integrins.

    PubMed

    Mahimkar, Rajeev M; Visaya, Orvin; Pollock, Allan S; Lovett, David H

    2005-01-15

    Renal tubular epithelial cells in all nephron segments express a distinct member of the metalloprotease-disintegrin family, ADAM9 (a disintegrin and metalloprotease 9), in a punctate basolateral distribution co-localized to the beta1 integrin chain [Mahimkar, Baricos, Visaya, Pollock and Lovett (2000) J. Am. Soc. Nephrol. 11, 595-603]. Discrete segments of the nephron express several defined beta1 integrins, suggesting that ADAM9 interacts with multiple renal integrins and thereby regulates epithelial cell-matrix interactions. Intact ADAM9 and a series of deletion constructs sequentially lacking the metalloprotease domain and the disintegrin domain were assembled as chimaeras with a C-terminal GFP (green fluorescent protein) tag. Stable expression of the ADAM9/GFP protein on the surface of HEK-293 cells (human embryonic kidney 293 cells) significantly decreased adhesion to types I and IV collagen, vitronectin and laminin, but had little effect on adhesion to fibronectin. Expression of the disintegrin/cysteine-rich/GFP construct yielded a similar, but more marked pattern of decreased adhesion. Expression of the cysteine-rich/GFP construct had no effect on adhesion, indicating that the disintegrin domain was responsible for the competitive inhibition of cell-matrix binding. To define the specific renal tubular beta1 integrins interacting with the ADAM9 disintegrin domain, a recombinant GST (glutathione S-transferase)-disintegrin protein was used as a substrate in adhesion assays in the presence or absence of specific integrin-blocking antibodies. Inclusion of antibodies to alpha1, alpha3, alpha6, alphav and beta1 blocked adhesion of HEK-293 cells to GST-disintegrin protein. Immobilized GST-disintegrin domain perfused with renal cortical lysates specifically recovered the alpha3, alpha6, alphav and beta1 integrin chains by Western analysis. It is concluded that ADAM9 is a polyvalent ligand, through its disintegrin domain, for multiple renal integrins of the beta1

  11. Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum†

    PubMed Central

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-01-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes. PMID:16227584

  12. A Staphylococcus aureus Pore-Forming Toxin Subverts the Activity of ADAM10 to Cause Lethal Infection

    PubMed Central

    Inoshima, Ichiro; Inoshima, Naoko; Wilke, Georgia; Powers, Michael; Frank, Karen; Wang, Yang; Wardenburg, Juliane Bubeck

    2011-01-01

    Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone 1,2. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia 3–5. α-hemolysin injures epithelial cells by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 6. We show that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage; similarly, E-cadherin proteolysis and barrier disruption is attenuated in ADAM10 knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that Hla can usurp the metalloprotease activity of its receptor reveals a novel mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury, and defines ADAM10 inhibition as a strategy for disease modification. PMID:21926978

  13. An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth

    PubMed Central

    Saha, Nayanendu; Eissman, Moritz F.; Xu, Kai; Llerena, Carmen; Kusebauch, Ulrike; Ding, Bi-Sen; Cao, Zhongwei; Rafii, Shahin; Ernst, Matthias; Scott, Andrew M.; Nikolov, Dimitar B.; Lackmann, Martin

    2016-01-01

    The transmembrane metalloprotease ADAM10 sheds a range of cell surface proteins, including ligands and receptors of the Notch, Eph, and erbB families, thereby activating signaling pathways critical for tumor initiation and maintenance. ADAM10 is thus a promising therapeutic target. Although widely expressed, its activity is normally tightly regulated. We now report prevalence of an active form of ADAM10 in tumors compared with normal tissues, in mouse models and humans, identified by our conformation-specific antibody mAb 8C7. Structure/function experiments indicate mAb 8C7 binds an active conformation dependent on disulfide isomerization and oxidative conditions, common in tumors. Moreover, this active ADAM10 form marks cancer stem-like cells with active Notch signaling, known to mediate chemoresistance. Importantly, specific targeting of active ADAM10 with 8C7 inhibits Notch activity and tumor growth in mouse models, particularly regrowth after chemotherapy. Our results indicate targeted inhibition of active ADAM10 as a potential therapy for ADAM10-dependent tumor development and drug resistance. PMID:27503072

  14. Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-11-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

  15. Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients

    PubMed Central

    Álvarez-Fernández, Sheila María; Barbariga, Marco; Cannizzaro, Luca; Cannistraci, Carlo Vittorio; Hurley, Laura; Zanardi, Alan; Conti, Antonio; Sanvito, Francesca; Innocenzi, Anna; Pecorelli, Nicolò; Braga, Marco; Alessio, Massimo

    2016-01-01

    A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease. PMID:27517630

  16. ADAM: automated data management for research datasets

    PubMed Central

    Woodbridge, Mark; Tomlinson, Christopher D.; Butcher, Sarah A.

    2013-01-01

    Existing repositories for experimental datasets typically capture snapshots of data acquired using a single experimental technique and often require manual population and continual curation. We present a storage system for heterogeneous research data that performs dynamic automated indexing to provide powerful search, discovery and collaboration features without the restrictions of a structured repository. ADAM is able to index many commonly used file formats generated by laboratory assays and therefore offers specific advantages to the experimental biology community. However, it is not domain specific and can promote sharing and re-use of working data across scientific disciplines. Availability and implementation: ADAM is implemented using Java and supported on Linux. It is open source under the GNU General Public License v3.0. Installation instructions, binary code, a demo system and virtual machine image and are available at http://www.imperial.ac.uk/bioinfsupport/resources/software/adam. Contact: m.woodbridge@imperial.ac.uk PMID:23109181

  17. Proceedings of the 1989 ADAM Workshop

    NASA Astrophysics Data System (ADS)

    Chipperfield, Alan

    ADAM is now a major software project; it provides a fully integrated environment for both data reduction and data acquisition. It is being used in Hawaii, Australia and the Canary Islands, as well as the UK, and has been adopted by Starlink as the environment in which Starlink data reduction software should run. One of the most remarkable things about ADAM is that it has been developed as a co-operative effort between groups that are spread across the world. Although the initial system came out of RGO, and ROE provided by far the major effort in designing and implementing the VAX version, various parts of what is now regarded as 'ADAM' have also come from other establishments. Co-ordinating a project being developed in this way is not an easy job, but the somewhat varied parentage of ADAM - although sometimes an administrative nightmare - is also one of its strengths; it is not a system developed in one place to serve the specific needs of that one place. One way in which this development is co-ordinated is by a series of workshops. These have taken place at about 18 month intervals since the first one in late 1985. The workshops are attended by people actively developing and/or making extensive use of ADAM, and provide a forum for detailed discussion of the problems in the current system and plans for its extension. The 1989 ADAM Workshop was held at Cosener's House, Abingdon from 3rd to 7th July 1989. An 'Open Meeting' was held on Friday 30th June at RAL to enable members of the Starlink community to provide input to the Workshop discussions. Before the previous workshop, in Hawaii, a trend had started to emerge for different establishments to plug the gaps in ADAM (which at the time was missing a number of important facilities) with local solutions. The Hawaii Workshop consolidated these local extensions, adopting some and rejecting others. As a result, ADAM, as reviewed by this third workshop, was a much more complete and uniform system, and it was possible to

  18. The ADAM workshops and meeting summary

    NASA Astrophysics Data System (ADS)

    Chipperfield, Alan J.

    1990-01-01

    ADAM is now a major software project; it provides a fully integrated environment for both data reduction and data acquisition. It is being used in Hawaii, Australia and the Canary Islands, as well as the UK, and has been adopted by Starlink as the environment in which Starlink data reduction software should run. One of the most remarkable things about ADAM is that it has been developed as a co-operative effort between groups that are spread across the world. Although the initial system came out of RGO, and ROE provided by far the major effort in designing and implementing the VAX version, various parts of what is now regarded as 'ADAM' have also come from other establishments. Co-ordinating a project being developed in this way is not an easy job, but the somewhat varied parentage of ADAM - although sometimes an administrative nightmare - is also one of its strengths; it is not a system developed in one place to serve the specific needs of that one place. One way in which this development is co-ordinated is by a series of workshops. These have taken place at about 18 month intervals since the first one in late 1985. The workshops are attended by people actively developing and/or making extensive use of ADAM, and provide a forum for detailed discussion of the problems in the current system and plans for its extension. The 1989 ADAM Workshop was held at Cosener's House, Abingdon from 3rd to 7th July 1989. An 'Open Meeting' was held on Friday 30th June at RAL to enable members of the Starlink community to provide input to the Workshop discussions. Before the previous workshop, in Hawaii, a trend had started to emerge for different establishments to plug the gaps in ADAM (which at the time was missing a number of important facilities) with local solutions. The Hawaii Workshop consolidated these local extensions, adopting some and rejecting others. As a result, ADAM, as reviewed by this third workshop, was a much more complete and uniform system, and it was possible to

  19. Glioma-Derived ADAM10 Induces Regulatory B Cells to Suppress CD8+ T Cells

    PubMed Central

    Li, Wen-sheng; Luo, Lun; Huang, Zhen-chao; Guo, Ying

    2014-01-01

    CD8+ T cells play an important role in the anti-tumor activities of the body. The dysfunction of CD8+ T cells in glioma is unclear. This study aims to elucidate the glioma cell-derived ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in the suppression of CD8+ effector T cells by the induction of regulatory B cells. In this study, glioma cells were isolated from surgically removed glioma tissue and stimulated by Phorbol myristate acetage (PMA) in the culture. The levels of ADAM10 in the culture were determined by enzyme-linked immunosorbent assay. Immune cells were assessed by flow cytometry. The results showed that the isolated glioma cells express ADAM10, which was markedly up regulated after stimulated with PMA. The glioma-derived ADAM10 induced activated B cells to differentiate into regulatory B cells, the later suppressed CD8+ T cell proliferation as well as the induced regulatory T cells, which also showed the immune suppressor effect on CD8+ effector T cell proliferation. In conclusion, glioma cells produce ADAM10 to induce Bregs; the latter suppresses CD8+ T cells and induces Tregs. PMID:25127032

  20. Positive selection at reproductive ADAM genes with potential intercellular binding activity.

    PubMed

    Glassey, Barb; Civetta, Alberto

    2004-05-01

    Many genes with a role in reproduction, including those implicated in fertilization and spermatogenesis, have been shown to evolve at a faster rate relative to genes associated with other functions and tissues. These survey studies usually group a wide variety of genes with different characteristics and evolutionary histories as reproductive genes based on their site of expression or function. We have examined the molecular evolution of the ADAM (a disintegrin and metalloprotease) gene family, a structurally and functionally diverse group of genes expressed in reproductive and somatic tissue to test whether a variety of protein characteristics such as phylogenetic clusters, tissue of expression, and proteolytic and adhesive function can group fast evolving ADAM genes. We found that all genes were evolving under purifying selection (d(N)/d(S) < 1), although reproductive ADAMs, including those implicated in fertilization and spermatogenesis, evolved at the fastest rate. Genes with a role in binding to cell receptors in endogenous tissue appear to be evolving under purifying selection, regardless of the tissue of expression. In contrast, positive selection of codon sites in the disintegrin/cysteine-rich adhesion domains was detected exclusively in ADAMs 2 and 32, two genes expressed in the testis with a potential role in sperm-egg adhesion. Positive selection was detected in the transmembrane/cytosolic tail region of ADAM genes expressed in a variety of tissues.

  1. Do benzodiazepines mimic reverse-turn structures?

    NASA Astrophysics Data System (ADS)

    Hata, Masayuki; Marshall, Garland R.

    2006-05-01

    The role of benzodiazepine derivatives (BZD) as a privileged scaffold that mimics β-turn structures (Ripka et al. (1993) Tetrahedron 49:3593-3608) in peptide/protein recognition was reexamined in detail. Stable BZD ring conformers were determined with MM3, and experimental reverse-turn structures were extracted from the basis set of protein crystal structures previously defined by Ripka et al. Ideal β-turns were also modeled and similarly compared with BZD conformers. Huge numbers of conformers were generated by systematically scanning the torsional degrees of freedom for BZDs, as well as those of ideal β-turns for comparison. Using these structures, conformers of BZDs were fit to experimental structures as suggested by Ripka et al., or modeled classical β-turn conformers, and the root-mean-square deviation (RMSD) values were calculated for each pairwise comparison. Pairs of conformers with the smallest RMSD values for overlap of the four α-β side-chain orientations were selected. All overlaps of BZD conformers with experimental β-turns yielded one or more comparisons where the least RMSD was significantly small, 0.48-0.86 Å, as previously suggested. Utilizing a different methodology, the overall conclusion that benzodiazepines could serve as reverse-turn mimetics of Ripka et al. is justified. The least RMSD values for the overlap of BZDs and modeled classical β-turns were also less than 1 Å. When comparing BZDs with experimental or classical β-turns, the set of experimental β-turns selected by Ripka et al. fit the BZD scaffolds better than modeled classical β-turns; however, all the experimental β-turns did not fit a particular BZD scaffold better. A single BZD ring conformation, and/or chiral orientation, can mimic some, but not all, of the experimental β-turn structures. BZD has two central ring conformations and one chiral center that explains why the four variations of the BZD scaffold can mimic all types of β-turn structure examined. It was

  2. Role of ADAM17 in the non-cell autonomous effects of oncogene-induced senescence.

    PubMed

    Morancho, Beatriz; Martínez-Barriocanal, Águeda; Villanueva, Josep; Arribas, Joaquín

    2015-08-12

    Cellular senescence is a terminal cell proliferation arrest that can be triggered by oncogenes. One of the traits of oncogene-induced senescence (OIS) is the so-called senescence-associated secretory phenotype or senescence secretome. Depending on the context, the non-cell autonomous effects of OIS may vary from tumor suppression to promotion of metastasis. Despite being such a physiological and pathologically relevant effector, the mechanisms of generation of the senescence secretome are largely unknown. We analyzed by label-free proteomics the secretome of p95HER2-induced senescent cells and compared the levels of the membrane-anchored proteins with their transcript levels. Then, protein and RNA levels of ADAM17 were evaluated by using Western blot and reverse transcription-polymerase chain reaction, its localization by using biotin labeling and immunofluorescence, and its activity by using alkaline phosphatase-tagged substrates. The p95HER2-expressing cell lines, senescent MCF7 and proliferating MCF10A, were analyzed to study ADAM17 regulation. Finally, we knocked down ADAM17 to determine its contribution to the senescence-associated secretome. The effect of this secretome was evaluated in migration assays in vitro and in nude mice by assessing the metastatic ability of orthotopically co-injected non-senescent cells. Using breast cancer cells expressing p95HER2, a constitutively active fragment of the proto-oncogene HER2 that induces OIS, we show that the extracellular domains of a variety of membrane-bound proteins form part of the senescence secretome. We determine that these proteins are regulated transcriptionally and, in addition, that their shedding is limited by the protease ADAM17. The activity of the sheddase is constrained, at least in part, by the accumulation of cellular cholesterol. The blockade of ADAM17 abrogates several prometastatic effects of the p95HER2-induced senescence secretome, both in vitro and in vivo. Considering these findings, we

  3. Women in History--Abigail Adams: Life, Accomplishments, and Ideas

    ERIC Educational Resources Information Center

    Kenan, Sharon K.

    2008-01-01

    This article profiles the life, accomplishments, and ideas of Abigail Adams. Born in 1944, Adams lacked a formal education, but she more than made up for that shortcoming with her love of reading, especially literature, and her interests in politics and events surrounding the young colonies. Adams was supportive of the advancement of women. She…

  4. Women in History--Abigail Adams: Life, Accomplishments, and Ideas

    ERIC Educational Resources Information Center

    Kenan, Sharon K.

    2008-01-01

    This article profiles the life, accomplishments, and ideas of Abigail Adams. Born in 1944, Adams lacked a formal education, but she more than made up for that shortcoming with her love of reading, especially literature, and her interests in politics and events surrounding the young colonies. Adams was supportive of the advancement of women. She…

  5. A Disintegrin and Metalloprotease (ADAM): Historical Overview of Their Functions

    PubMed Central

    Giebeler, Nives; Zigrino, Paola

    2016-01-01

    Since the discovery of the first disintegrin protein from snake venom and the following identification of a mammalian membrane-anchored metalloprotease-disintegrin implicated in fertilization, almost three decades of studies have identified additional members of these families and several biochemical mechanisms regulating their expression and activity in the cell. Most importantly, new in vivo functions have been recognized for these proteins including cell partitioning during development, modulation of inflammatory reactions, and development of cancers. In this review, we will overview the a disintegrin and metalloprotease (ADAM) family of proteases highlighting some of the major research achievements in the analysis of ADAMs’ function that have underscored the importance of these proteins in physiological and pathological processes over the years. PMID:27120619

  6. Paraprofessional of the Year 2009: Tina Adams

    ERIC Educational Resources Information Center

    Berry, John N., III

    2009-01-01

    There is no doubt among the staff and managers at North Carolina State University (NCSU) Libraries, Raleigh, that advanced library technician Tina Adams deserves to be the winner of the "Library Journal's "Paraprofessional of the Year Award for 2009." "Certainly this library has never seen anyone like her before, not in my nine…

  7. Adam Smith, Religion, and Tuition Tax Credits.

    ERIC Educational Resources Information Center

    Alexander, Kern

    1983-01-01

    Examines tuition tax credit programs in framework of Adam Smith's ideas on the economic impact of established churches. Finds that tuition tax credits would amount to state expenditures to relieve the financial burden of parochial school parents and would allow churches to invest commercially to maintain their charitable functions. (JW)

  8. Adam Smith and the Rhetoric of Style.

    ERIC Educational Resources Information Center

    Moran, Michael G.

    Historians of rhetoric have generally accepted the view that Adam Smith rejected the principles of classical rhetoric. However, while there can be no doubt that Smith greatly truncated the five classical arts of rhetoric (invention, arrangement, style, memory, and delivery) by reducing his concerns largely to style and arrangement, he did not…

  9. Propagandist of the Revolution: Samuel Adams.

    ERIC Educational Resources Information Center

    Scanlon, Thomas M.

    This paper explores Samuel Adam's role as perhaps the most important propagandist of the American Revolution and his efforts to exploit Great Britain's mistakes and to engender in the American colonists a love of liberty and a fear that Great Britain, if not resisted, would replace that liberty with tyranny. Suggesting that the Revolutionary War…

  10. Propagandist of the Revolution: Samuel Adams.

    ERIC Educational Resources Information Center

    Scanlon, Thomas M.

    This paper explores Samuel Adam's role as perhaps the most important propagandist of the American Revolution and his efforts to exploit Great Britain's mistakes and to engender in the American colonists a love of liberty and a fear that Great Britain, if not resisted, would replace that liberty with tyranny. Suggesting that the Revolutionary War…

  11. ADAM: another database of abbreviations in MEDLINE.

    PubMed

    Zhou, Wei; Torvik, Vetle I; Smalheiser, Neil R

    2006-11-15

    Abbreviations are an important type of terminology in the biomedical domain. Although several groups have already created databases of biomedical abbreviations, these are either not public, or are not comprehensive, or focus exclusively on acronym-type abbreviations. We have created another abbreviation database, ADAM, which covers commonly used abbreviations and their definitions (or long-forms) within MEDLINE titles and abstracts, including both acronym and non-acronym abbreviations. A model of recognizing abbreviations and their long-forms from titles and abstracts of MEDLINE (2006 baseline) was employed. After grouping morphological variants, 59 405 abbreviation/long-form pairs were identified. ADAM shows high precision (97.4%) and includes most of the frequently used abbreviations contained in the Unified Medical Language System (UMLS) Lexicon and the Stanford Abbreviation Database. Conversely, one-third of abbreviations in ADAM are novel insofar as they are not included in either database. About 19% of the novel abbreviations are non-acronym-type and these cover at least seven different types of short-form/long-form pairs. A free, public query interface to ADAM is available at http://arrowsmith.psych.uic.edu, and the entire database can be downloaded as a text file.

  12. Adam Smith, Religion, and Tuition Tax Credits.

    ERIC Educational Resources Information Center

    Alexander, Kern

    1983-01-01

    Examines tuition tax credit programs in framework of Adam Smith's ideas on the economic impact of established churches. Finds that tuition tax credits would amount to state expenditures to relieve the financial burden of parochial school parents and would allow churches to invest commercially to maintain their charitable functions. (JW)

  13. Possible Binary Lightcurve for 3145 Walter Adams

    NASA Astrophysics Data System (ADS)

    Owings, Larry E.; Warner, Brian D.; Pravec, Petr; Kusnirak, Peter

    2011-01-01

    Lightcurve observations have yielded period determinations for asteroid 3145 Walter Adams. A primary period of 2.7113 ± 0.0001 hr, amplitude of 0.10 ± 0.05, and a possible secondary period of about 17.4 hr. The estimated diameter ratio is D2/D1 = 0.22 ± 0.02.

  14. ADAM10 mediates N-cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina.

    PubMed

    Paudel, Sharada; Kim, Yeoun-Hee; Huh, Man-Il; Kim, Song-Ja; Chang, Yongmin; Park, Young Jeung; Lee, Kyoo Won; Jung, Jae-Chang

    2013-04-01

    Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro-form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10-specific siRNA, the level of mature ADAM10 decreased. Phase-contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X-treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X-treated cultures. N-cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N-cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N-cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation.

  15. Illnesses of the brain in John Quincy Adams.

    PubMed

    Paulson, George

    2004-12-01

    John Quincy Adams, the sixth and perhaps most scholarly American president, served courageously despite familial essential tremor, depression, and cerebrovascular disease. His cousin Samuel Adams and his father John Adams also had essential tremor, which the later called "quiveration". Alcoholism and depression affected several members of J.Q. Adams's family. Following his own time as president, J.Q. Adams returned to duty as the congressman who most assiduously fought slavery, a fight he continued even after he had suffered a major left hemispheric stroke. His fatal collapse in Congress, protesting the Mexican War, is legendary among the final illnesses of American statesmen.

  16. Heparan sulfate regulates ADAM12 through a molecular switch mechanism.

    PubMed

    Sørensen, Hans Peter; Vivès, Romain R; Manetopoulos, Christina; Albrechtsen, Reidar; Lydolph, Magnus C; Jacobsen, Jonas; Couchman, John R; Wewer, Ulla M

    2008-11-14

    The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

  17. ADAM-9 is a novel mediator of tenascin-C-stimulated invasiveness of brain tumor–initiating cells

    PubMed Central

    Sarkar, Susobhan; Zemp, Franz J.; Senger, Donna; Robbins, Stephen M.; Yong, V. Wee

    2015-01-01

    Background Tenascin-C (TNC), an extracellular matrix protein overexpressed in malignant gliomas, stimulates invasion of conventional glioma cell lines (U251, U87). However, there is a dearth of such information on glioma stemlike cells. Here, we have addressed whether and how TNC may regulate the invasiveness of brain tumor–initiating cells (BTICs) that give rise to glioma progenies. Methods Transwell inserts coated with extracellular matrix proteins were used to determine the role of TNC in BTIC invasion. Microarray analysis, lentiviral constructs, RNA interference-mediated knockdown, and activity assay ascertained the role of proteases in TNC-stimulated BTIC invasion in culture. Involvement of proteases was validated using orthotopic brain xenografts in mice. Results TNC stimulated BTIC invasiveness in a metalloproteinase-dependent manner. A global gene expression screen identified the metalloproteinase ADAM-9 as a potential regulator of TNC-stimulated BTIC invasiveness, and this was corroborated by an increase of ADAM-9 protein in 4 glioma patient–derived BTIC lines. Notably, RNA interference to ADAM-9, as well as inhibition of mitogen-activated protein kinase 8 (c-Jun NH2-terminal kinase), attenuated TNC-stimulated ADAM-9 expression, proteolytic activity, and BTIC invasiveness. The relevance of ADAM-9 to tumor invasiveness was validated using resected human glioblastoma specimens and orthotopic xenografts where elevation of ADAM-9 and TNC expression was prominent at the invasive front of the tumor. Conclusions This study has identified TNC as a promoter of the invasiveness of BTICs through a mechanism involving ADAM-9 proteolysis via the c-Jun NH2-terminal kinase pathway. PMID:25646025

  18. ADAM10 Cell Surface Expression but Not Activity Is Critical for Staphylococcus aureus α-Hemolysin-Mediated Activation of the NLRP3 Inflammasome in Human Monocytes.

    PubMed

    Ezekwe, Ejiofor A D; Weng, Chengyu; Duncan, Joseph A

    2016-03-30

    The Staphylococcus aureus toxin, α-hemolysin, is an important and well-studied virulence factor in staphylococcal infection. It is a soluble monomeric protein that, once secreted by the bacterium, forms a heptameric pore in the membrane of a broad range of host cell types. Hemolysin was recently discovered to bind and activate a disintegrin and metalloprotease 10 (ADAM10). In epithelial and endothelial cells, ADAM10 activation is required for the toxin's activity against these cells. In host monocytic cells, α-hemolysin activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome leading to production of pro-inflammatory cytokines and cell death. We now show that ADAM10 is critical for α-hemolysin-mediated activation of the NLRP3 inflammasome in human monocytes as siRNA knockdown or chemical blockade of ADAM10-α-hemolysin interaction leads to diminished inflammasome activation and cell death by reducing the available ADAM10 on the cell surface. Unlike epithelial cell and endothelial cell damage, which requires α-hemolysin induced ADAM10 activation, ADAM10 protease activity was not required for NLRP3 inflammasome activation. This work confirms the importance of ADAM10 in immune activation by α-hemolysin, but indicates that host cell signal induction by the toxin is different between host cell types.

  19. A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

    PubMed Central

    Prakasam, H. Sandeep; Gallo, Luciana I.; Li, Hui; Ruiz, Wily G.; Hallows, Kenneth R.; Apodaca, Gerard

    2014-01-01

    Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A1 adenosine receptors (A1ARs) triggers a Gi-Gβγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A1AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17S811A mutant or expression of a tail-minus construct inhibits A1AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17S811A in bladder tissues impairs A1AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain. PMID:25232008

  20. ADAM10 Cell Surface Expression but Not Activity Is Critical for Staphylococcus aureus α-Hemolysin-Mediated Activation of the NLRP3 Inflammasome in Human Monocytes

    PubMed Central

    Ezekwe, Ejiofor A.D.; Weng, Chengyu; Duncan, Joseph A.

    2016-01-01

    The Staphylococcus aureus toxin, α-hemolysin, is an important and well-studied virulence factor in staphylococcal infection. It is a soluble monomeric protein that, once secreted by the bacterium, forms a heptameric pore in the membrane of a broad range of host cell types. Hemolysin was recently discovered to bind and activate a disintegrin and metalloprotease 10 (ADAM10). In epithelial and endothelial cells, ADAM10 activation is required for the toxin’s activity against these cells. In host monocytic cells, α-hemolysin activates the nucleotide-binding domain and leucine-rich repeat containing gene family, pyrin domain containing 3 (NLRP3) inflammasome leading to production of pro-inflammatory cytokines and cell death. We now show that ADAM10 is critical for α-hemolysin-mediated activation of the NLRP3 inflammasome in human monocytes as siRNA knockdown or chemical blockade of ADAM10-α-hemolysin interaction leads to diminished inflammasome activation and cell death by reducing the available ADAM10 on the cell surface. Unlike epithelial cell and endothelial cell damage, which requires α-hemolysin induced ADAM10 activation, ADAM10 protease activity was not required for NLRP3 inflammasome activation. This work confirms the importance of ADAM10 in immune activation by α-hemolysin, but indicates that host cell signal induction by the toxin is different between host cell types. PMID:27043625

  1. Cloning and expression of ADAM-related metalloproteases in equine laminitis.

    PubMed

    Coyne, Michael J; Cousin, Hélène; Loftus, John P; Johnson, Philip J; Belknap, James K; Gradil, Carlos M; Black, Samuel J; Alfandari, Dominique

    2009-06-15

    Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.

  2. Engineering hydrogels as extracellular matrix mimics

    PubMed Central

    Geckil, Hikmet; Xu, Feng; Zhang, Xiaohui; Moon, SangJun

    2010-01-01

    Extracellular matrix (ECM) is a complex cellular environment consisting of proteins, proteoglycans, and other soluble molecules. ECM provides structural support to mammalian cells and a regulatory milieu with a variety of important cell functions, including assembling cells into various tissues and organs, regulating growth and cell–cell communication. Developing a tailored in vitro cell culture environment that mimics the intricate and organized nanoscale meshwork of native ECM is desirable. Recent studies have shown the potential of hydrogels to mimic native ECM. Such an engineered native-like ECM is more likely to provide cells with rational cues for diagnostic and therapeutic studies. The research for novel biomaterials has led to an extension of the scope and techniques used to fabricate biomimetic hydrogel scaffolds for tissue engineering and regenerative medicine applications. In this article, we detail the progress of the current state-of-the-art engineering methods to create cell-encapsulating hydrogel tissue constructs as well as their applications in in vitro models in biomedicine. PMID:20394538

  3. Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

    PubMed Central

    Martin, Ana Carolina Baptista Moreno; Cardoso, Ana Carolina Ferreira; Selistre-de-Araujo, Heloisa Sobreiro; Cominetti, Márcia Regina

    2015-01-01

    One of the most important features of malignant cells is their capacity to invade adjacent tissues and metastasize to distant organs. This process involves the creation, by tumor and stroma cells, of a specific microenvironment, suitable for proliferation, migration and invasion of tumor cells. The ADAM family of proteins has been involved in these processes. This work aimed to investigate the role of the recombinant disintegrin domain of the human ADAM9 (rADAM9D) on the adhesive and mobility properties of DU145 prostate tumor cells. rADAM9D was able to support DU145 cell adhesion, inhibit the migration of DU145 cells, as well as the invasion of this cell line through matrigel in vitro. Overall this work demonstrates that rADAM9D induces specific cellular migratory properties when compared with different constructs having additional domains, specially those of metalloproteinase and cysteine-rich domains. Furthermore, we showed that rADAM9D was able to inhibit cell adhesion, migration and invasion mainly through interacting with α6β1 in DU145 tumor cell line. These results may contribute to the development of new therapeutic strategies for prostate cancer. PMID:26211476

  4. Molecular Signals Elicited by GPCR Agonists in Hypertension, Cardiovascular Remodeling: Are MMPs and ADAMs Elusive Therapeutic Targets?

    PubMed Central

    Wang, Xiang; Bosonea, Ana-Maria; Odenbach, Jeffrey; Fernandez-Patron, Carlos

    2014-01-01

    Hypertension, the condition characterized by sustained elevated blood pressure, affects over 25% of adults in developed countries and is accompanied by pathological cardiac remodeling (i.e., hypertrophy and fibrosis), thus being a major risk factor for cardiac failure. Life style, the environment, genetic factors, diabetes or obesity can all promote development and progression of hypertension associated cardiovascular disease in part because these conditions induce an excess production of pro-hypertensive, pro-hypertrophic and pro-fibrotic agonists. Here we review signaling pathways shared by major agonists including angiotensin II, catecholamines and endothelins. At the cellular level, these agonists initiate disease signaling by activating cognate G protein-coupled receptors (GPCRs). Early events in agonist-signaling include Ca2+ release from intracellular stores, Ca2+ uptake from extracellular millieu into cells and reactive oxygen species (ROS) generation by NADPH oxidase. ROS production in turn contributes to activation of matrix metalloproteinases (MMPs) and ‘a disintegrin and metalloproteinases’ (ADAMs). Activated MMPs and ADAMs cleave growth factors, cytokines as well as cell surface receptors, including GPCRs. Excessive activation of MMPs and ADAMs links agonist receptors with transcription and translation of disease-associated genes, including those of MMPs and ADAMs. Recent research indicates a complex and dynamic regulation of MMPs and ADAMs activity and expression by agonists, which poses a significant challenge to strategies aiming at targeting specific MMPs or ADAMs in cardiovascular disease. PMID:24976815

  5. IL-1β and ADAM17 are central regulators of β-defensin expression in Candida esophagitis.

    PubMed

    Pahl, Rene; Brunke, Gabriele; Steubesand, Nadine; Schubert, Sabine; Böttner, Martina; Wedel, Thilo; Jürgensen, Christian; Hampe, Jochen; Schäfer, Heiner; Zeissig, Sebastian; Schreiber, Stefan; Rosenstiel, Philip; Reiss, Karina; Arlt, Alexander

    2011-04-01

    Candida albicans resides on epithelial surfaces as part of the physiological microflora. However, under certain conditions, it may cause life-threatening infections, including Candida sepsis. We have recently shown that human β-defensins (hBDs) hBD-2 and hBD-3 are upregulated in Candida esophagitis and that this antifungal host response is distinctly regulated by NF-κB and MAPK/activator protein-1 (AP-1) pathways. Here, we show that C. albicans induces hBD-2 through an autocrine IL-1β loop and that activation of the epidermal growth factor receptor (EGFR) by endogenous transforming growth factor-α (TGF-α) is a crucial event in the induction of hBD-3. To further dissect upstream signaling events, we investigated expression of the central sheddases for EGFR ligands ADAM10 and ADAM17 in the healthy and infected esophagus. Next, we used pharmaceutical inhibitors and small-interfering RNA-mediated knock down of ADAM10 and ADAM17 to reveal that ADAM17-induced shedding of TGF-α is a crucial step in the induction of hBD-3 expression in response to Candida infection. In conclusion, we describe for the first time an autocrine IL-1β loop responsible for the induction of hBD-2 expression and an ADAM17-TGF-α-EGFR-MAPK/AP-1 pathway leading to hBD-3 upregulation in the course of a Candida infection of the esophagus.

  6. Disintegrin Metalloprotease (ADAM) 10 Regulates Endothelial Permeability and T Cell Transmigration by Proteolysis of Vascular Endothelial Cadherin

    PubMed Central

    Schulz, Beate; Pruessmeyer, Jessica; Maretzky, Thorsten; Ludwig, Andreas; Blobel, Carl P.; Saftig, Paul; Reiss, Karina

    2009-01-01

    Vascular endothelial (VE)-cadherin is the major adhesion molecule of endothelial adherens junctions. It plays an essential role in controlling endothelial permeability, vascular integrity, leukocyte transmigration, and angiogenesis. Elevated levels of soluble VE-cadherin are associated with diseases like coronary atherosclerosis. Previous data showed that the extracellular domain of VE-cadherin is released by an unknown metalloprotease activity during apoptosis. In this study, we used gain of function analyses, inhibitor studies and RNA interference experiments to analyze the proteolytic release of VE-cadherin in human umbilical vein endothelial cells (HUVECs). We found that VE-cadherin is specifically cleaved by the disintegrin and metalloprotease ADAM10 in its ectodomain releasing a soluble fragment and generating a carboxyterminal membrane bound stub, which is a substrate for a subsequent γ-secretase cleavage. This ADAM10-mediated proteolysis could be induced by Ca2+-influx and staurosporine treatment, indicating that ADAM10-mediated VE-cadherin cleavage contributes to the dissolution of adherens junctions during endothelial cell activation and apoptosis, respectively. In contrast, protein kinase C activation or inhibition did not modulate VE-cadherin processing. Increased ADAM10 expression was functionally associated with an increase in endothelial permeability. Remarkably, our data indicate that ADAM10 activity also contributes to the thrombin-induced decrease of endothelial cell-cell adhesion. Moreover, knockdown of ADAM10 in HUVECs as well as in T cells by small interfering RNA impaired T cell transmigration. Taken together our data identify ADAM10 as a novel regulator of vascular permeability and demonstrate a hitherto unknown function of ADAM10 in the regulation of VE-cadherin-dependent endothelial cell functions and leukocyte transendothelial migration. PMID:18420943

  7. Lysophosphatidic acid-induced ADAM12 expression mediates human adipose tissue-derived mesenchymal stem cell-stimulated tumor growth.

    PubMed

    Do, Eun Kyoung; Kim, Young Mi; Heo, Soon Chul; Kwon, Yang Woo; Shin, Sang Hun; Suh, Dong-Soo; Kim, Ki-Hyung; Yoon, Man-Soo; Kim, Jae Ho

    2012-11-01

    Lysophosphatidic acid (LPA) is involved in mesenchymal stem cell-stimulated tumor growth in vivo. However, the molecular mechanism by which mesenchymal stem cells promote tumorigenesis remains elusive. In the present study, we demonstrate that conditioned medium from A549 human lung adenocarcinoma cells (A549 CM) induced the expression of ADAM12, a disintegrin and metalloproteases family member, in human adipose tissue-derived mesenchymal stem cells (hASCs). A549 CM-stimulated ADAM12 expression was abrogated by pretreatment of hASCs with the LPA receptor 1 inhibitor Ki16425 or by small interfering RNA-mediated silencing of LPA receptor 1, suggesting a key role for the LPA-LPA receptor 1 signaling axis in A549 CM-stimulated ADAM12 expression. Silencing of ADAM12 expression using small interfering RNA or short hairpin RNA abrogated LPA-induced expression of both α-smooth muscle actin, a marker of carcinoma-associated fibroblasts, and ADAM12 in hASCs. Using a xenograft transplantation model of A549 cells, we demonstrated that silencing of ADAM12 inhibited the hASC-stimulated in vivo growth of A549 xenograft tumors and the differentiation of transplanted hASCs to α-smooth muscle actin-positive carcinoma-associated fibroblasts. LPA-conditioned medium from hASCs induced the adhesion of A549 cells and silencing of ADAM12 inhibited LPA-induced expression of extracellular matrix proteins, periostin and βig-h3, in hASCs and LPA-conditioned medium-stimulated adhesion of A549 cells. These results suggest a pivotal role for LPA-stimulated ADAM12 expression in tumor growth and the differentiation of hASCs to carcinoma-associated fibroblasts expressing α-smooth muscle actin, periostin, and βig-h3.

  8. The emerging role of matrix metalloproteases of the ADAM family in male germ cell apoptosis

    PubMed Central

    Urriola-Muñoz, Paulina; Lagos-Cabré, Raúl

    2011-01-01

    Constitutive germ cell apoptosis during mammalian spermatogenesis is a key process for controlling sperm output and to eliminate damaged or unwanted cells. An increase or decrease in the apoptosis rate has deleterious consequences and leads to low sperm production. Apoptosis in spermatogenesis has been widely studied, but the mechanism by which it is induced under physiological or pathological conditions has not been clarified. We have recently identified the metalloprotease ADAM17 (TACE) as a putative physiological inducer of germ cell apoptosis. The mechanisms involved in regulating the shedding of the ADAM17 extracellular domain are still far from being understood, although they are important in order to understand cell-cell communications. Here, we review the available data regarding apoptosis during mammalian spermatogenesis and the localization of ADAM proteins in the male reproductive tract. We propose an integrative working model where ADAM17, p38 MAPK, protein kinase C (PKC) and the tyrosine kinase c-Abl participate in the physiological signalling cascade inducing apoptosis in germ cells. In our model, we also propose a role for the Sertoli cell in regulating the Fas/FasL system in order to induce the extrinsic pathway of apoptosis in germ cells. This working model could be applied to further understand constitutive apoptosis in spermatogenesis and in pathological conditions (e.g., varicocele) or following environmental toxicants exposure (e.g., genotoxicity or xenoestrogens). PMID:22319668

  9. Pore-forming bacterial toxins and antimicrobial peptides as modulators of ADAM function.

    PubMed

    Reiss, Karina; Bhakdi, Sucharit

    2012-11-01

    Membrane-perturbating proteins and peptides are widespread agents in biology. Pore-forming bacterial toxins represent major virulence factors of pathogenic microorganisms. Membrane-damaging peptides constitute important antimicrobial effectors of innate immunity. Membrane perturbation can incur multiple responses in mammalian cells. The present discussion will focus on the interplay between membrane-damaging agents and the function of cell-bound metalloproteinases of the ADAM family. These transmembrane enzymes have emerged as the major proteinase family that mediate the proteolytic release of membrane-associated proteins, a process designated as "shedding". They liberate a large spectrum of functionally active molecules including inflammatory cytokines, growth factor receptors and cell adhesion molecules, thereby regulating such vital cellular functions as cell-cell adhesion, cell proliferation and cell migration. ADAM activation may constitute part of the cellular recovery machinery on the one hand, but likely also promotes inflammatory processes on the other. The mechanisms underlying ADAM activation and the functional consequences thereof are currently the subject of intensive research. Attention here is drawn to the possible involvement of purinergic receptors and ceramide generation in the context of ADAM activation following membrane perturbation by membrane-active agents.

  10. α-Lipoic acid reduces neurogenic hypertension by blunting oxidative stress-mediated increase in ADAM17.

    PubMed

    de Queiroz, Thyago M; Xia, Huijing; Filipeanu, Catalin M; Braga, Valdir A; Lazartigues, Eric

    2015-09-01

    We previously reported that type 2 angiotensin-converting enzyme (ACE2) compensatory activity is impaired by the disintegrin and metalloprotease 17 (ADAM17), and lack of ACE2 is associated with oxidative stress in neurogenic hypertension. To investigate the relationship between ADAM17 and oxidative stress, Neuro2A cells were treated with ANG II (100 nM) 24 h after vehicle or α-lipoic acid (LA, 500 μM). ADAM17 expression was increased by ANG II (120.5 ± 9.1 vs. 100.2 ± 0.8%, P < 0.05) and decreased after LA (69.0 ± 0.3 vs. 120.5 ± 9.1%, P < 0.05). In another set of experiments, LA reduced ADAM17 (92.9 ± 5.3 vs. 100.0 ± 11.2%, P < 0.05) following its overexpression. Moreover, ADAM17 activity was reduced by LA in ADAM17-overexpressing cells [109.5 ± 19.8 vs. 158.0 ± 20.0 fluorescence units (FU)·min(-1)·μg protein(-1), P < 0.05], in which ADAM17 overexpression increased oxidative stress (114.1 ± 2.5 vs. 101.0 ± 1.0%, P < 0.05). Conversely, LA-treated cells attenuated ADAM17 overexpression-induced oxidative stress (76.0 ± 9.1 vs. 114.1 ± 2.5%, P < 0.05). In deoxycorticosterone acetate (DOCA)-salt hypertensive mice, a model in which ADAM17 expression and activity are increased, hypertension was blunted by pretreatment with LA (119.0 ± 2.4 vs. 131.4 ± 2.2 mmHg, P < 0.05). In addition, LA improved dysautonomia and baroreflex sensitivity. Furthermore, LA blunted the increase in NADPH oxidase subunit expression, as well as the increase in ADAM17 and decrease in ACE2 activity in the hypothalamus of DOCA-salt hypertensive mice. Taken together, these data suggest that LA might preserve ACE2 compensatory activity by breaking the feedforward cycle between ADAM17 and oxidative stress, resulting in a reduction of neurogenic hypertension. Copyright © 2015 the American Physiological Society.

  11. Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain.

    PubMed

    Miller, Miles A; Moss, Marcia L; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S; Griffith, Linda G; Lauffenburger, Douglas A

    2015-10-19

    Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, "decoy" antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

  12. Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

    NASA Astrophysics Data System (ADS)

    Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

    2015-10-01

    Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

  13. Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

    PubMed Central

    Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

    2015-01-01

    Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling. PMID:26477568

  14. Catalytic properties of ADAM12 and its domain deletion mutants.

    PubMed

    Jacobsen, Jonas; Visse, Robert; Sørensen, Hans Peter; Enghild, Jan J; Brew, Keith; Wewer, Ulla M; Nagase, Hideaki

    2008-01-15

    Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.

  15. A facile method to synthesize histones with posttranslational modification mimics.

    PubMed

    Wang, Zhiyong U; Wang, Yane-Shih; Pai, Pei-Jing; Russell, William K; Russell, David H; Liu, Wenshe R

    2012-07-03

    Using an evolved pyrrolysyl-tRNA synthetase-tRNA(Pyl) pair, a Se-alkylselenocysteine was genetically incorporated into histone H3 with a high protein expression yield. Quantitative oxidative elimination of Se-alkylselenocysteine followed by Michael addition reactions with various thiol nucleophiles generated biologically active mimics of H3 with posttranslational modifications including lysine methylation, lysine acetylation, and serine phosphorylation.

  16. Iterative Mechanism Solutions with Scenario and ADAMS

    NASA Technical Reports Server (NTRS)

    Rhoades, Daren

    2006-01-01

    This slide presentation reviews the use of iterative solutions using Scenario for Motion (UG NX 2 Motion) to assist in designing the Mars Science Laboratory (MSL). The MSL will have very unique design requirements, and in order to meet these requirements the system must have the ability to design for static stability, simulate mechanism kinematics, simulate dynamic behaviour and be capable of reconfiguration, and iterations as designed. The legacy process used on the Mars Exploration rovers worked, but it was cumbersome using multiple tools, limited configuration control, with manual process and communication, and multiple steps. The aim is to develop a mechanism that would reduce turn around time, and make more reiterations possible, to improve the quality and quantity of data, and to enhance configuration control. Currently for NX Scenario for Motion uses are in the articulation studies, the simulations of traverse motions,and subsystem simulations. The design of the Rover landing model requires accurate results, flexible elements, such as beams, and the use of the full ADAMS solver has been used. In order to achieve this, when required, there has been a direct translation from Scenario to ADAMS, with additional data in ascii format. The process that has been designed to move from Scenario to ADAMS is reviewed.

  17. Iterative Mechanism Solutions with Scenario and ADAMS

    NASA Technical Reports Server (NTRS)

    Rhoades, Daren

    2006-01-01

    This slide presentation reviews the use of iterative solutions using Scenario for Motion (UG NX 2 Motion) to assist in designing the Mars Science Laboratory (MSL). The MSL will have very unique design requirements, and in order to meet these requirements the system must have the ability to design for static stability, simulate mechanism kinematics, simulate dynamic behaviour and be capable of reconfiguration, and iterations as designed. The legacy process used on the Mars Exploration rovers worked, but it was cumbersome using multiple tools, limited configuration control, with manual process and communication, and multiple steps. The aim is to develop a mechanism that would reduce turn around time, and make more reiterations possible, to improve the quality and quantity of data, and to enhance configuration control. Currently for NX Scenario for Motion uses are in the articulation studies, the simulations of traverse motions,and subsystem simulations. The design of the Rover landing model requires accurate results, flexible elements, such as beams, and the use of the full ADAMS solver has been used. In order to achieve this, when required, there has been a direct translation from Scenario to ADAMS, with additional data in ascii format. The process that has been designed to move from Scenario to ADAMS is reviewed.

  18. Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha).

    PubMed

    Aydin, Cumhur; Aytan, Nurgul; Mahon, Mathew J; Tawfeek, Hesham A W; Kowall, Neil W; Dedeoglu, Alpaslan; Bastepe, Murat

    2009-08-01

    GNAS gives rise to multiple imprinted gene products, including the alpha-subunit of the stimulatory G protein (Gs(alpha)) and its variant XL(alpha)s. Based on genomic sequence, the translation of XL(alpha)s begins from the middle of a long open reading frame, suggesting the existence of an N-terminally extended variant termed extralarge XLalphas (XXL(alpha)s). Although XXL(alpha), like Gs(alpha) and XL(alpha)s, would be affected by most disease-causing GNAS mutations, its authenticity and biological significance remained unknown. Here we identified a mouse cDNA clone that comprises the entire open reading frame encoding XXL(alpha)s. Whereas XXL(alpha)s mRNA was readily detected in mouse heart by RT-PCR, it appeared virtually absent in insulinoma-derived INS-1 cells. By Northern blots and RT-PCR, XXL(alpha)s mRNA was detected primarily in the mouse brain, cerebellum, and spleen. Immunohistochemistry using a specific anti-XXL(alpha)s antibody demonstrated XXL(alpha)s protein in multiple brain areas, including dorsal hippocampus and cortex. In transfected cells, full-length human XXL(alpha)s was localized to the plasma membrane and mediated isoproterenol- and cholera toxin-stimulated cAMP accumulation. XXL(alpha)s-R844H, which bears a mutation analogous to that in the constitutively active Gs(alpha) mutant Gs(alpha)-R201H (gsp oncogene), displayed elevated basal signaling. However, unlike Gs(alpha)-R201H, which mostly remains in the cytoplasm, both XXL(alpha)s-R844H and a constitutively active XL(alpha)s mutant localized to the plasma membrane. Hence, XXL(alpha)s is a distinct GNAS product and can mimic Gs(alpha), but the constitutively active XXL(alpha)s and Gs(alpha) mutants differ from each other regarding subcellular targeting. Our findings suggest that XXL(alpha)s deficiency or hyperactivity may contribute to the pathogenesis of diseases caused by GNAS mutations.

  19. ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

    PubMed

    Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

    2016-05-01

    ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells.

  20. Inhibition of ADAM10 promotes the clearance of Aβ across the BBB by reducing LRP1 ectodomain shedding.

    PubMed

    Shackleton, B; Crawford, F; Bachmeier, C

    2016-08-08

    Transport across the blood-brain barrier (BBB) is an important mediator of beta-amyloid (Aβ) accumulation in the brain and a contributing factor in the pathogenesis of Alzheimer's disease (AD). One of the receptors responsible for the transport of Aβ in the BBB is the low density lipoprotein receptor-related protein 1 (LRP1). LRP1 is susceptible to proteolytic shedding at the cell surface, which prevents endocytic transport of ligands. Previously, we reported a strong inverse correlation between LRP1 shedding in the brain and Aβ transit across the BBB. Several proteases contribute to the ectodomain shedding of LRP1 including the α-secretase, a desintegrin and metalloproteinase domain containing protein 10 (ADAM10). The role of ADAM10 in the shedding of LRP1 and Aβ BBB clearance was assessed through pharmacological inhibition of ADAM10 in an in vitro model of the BBB and through the use of ADAM10 endothelial specific knock-out mice. In addition, an acute treatment paradigm with an ADAM10 inhibitor was also tested in an AD mouse model to assess the effect of ADAM10 inhibition on LRP1 shedding and Aβbrain accumulation. In the current studies, inhibition of ADAM10 reduced LRP1 shedding in brain endothelial cultures and increased Aβ42 transit across an in vitro model of the BBB. Similarly, transgenic ADAM10 endothelial knockout mice displayed lower LRP1 shedding in the brain and significantly enhanced Aβ clearance across the BBB compared to wild-type animals. Acute treatment with the ADAM10-selective inhibitor GI254023X in an AD mouse model substantially reduced brain LRP1 shedding and increased Aβ40 levels in the plasma, indicating enhanced Aβ transit from the brain to the periphery. Furthermore, both soluble and insoluble Aβ40 and Aβ42 brain levels were decreased following GI254023X treatment, but these effects lacked statistical significance. These studies demonstrate a role for ADAM10 in the ectodomain shedding of LRP1 in the brain and the clearance of

  1. Internal architecture of the proximal femur--Adam's or Adams' arch? Historical mystery.

    PubMed

    Bartonícek, J

    2002-12-01

    The designation 'Adam Bogen' describing the thick medial cortex of the femoral neck is an incorrect term. This arch was described by Robert Adams (1795-1871), an outstanding Irish anatomist and surgeon. He was famous mainly for his book on gout and the description of disorders of cardiac rhythm, the so-called Adams-Stokes syndrome. He published his original description in the today unfortunately almost forgotten Cyclopaedia of Anatomy and Physiology, Vol. II (London, Longman, 1836-1839). The main editor of this monumental six-volume work was the famous anatomist and surgeon R.B.Todd. This book represents a significant source of information on diseases and injuries of the great joints (shoulder, elbow, wrist, knee, ankle).

  2. Shedding of Collagen XVII/BP180 in Skin Depends on Both ADAM10 and ADAM9*

    PubMed Central

    Franzke, Claus-Werner; Bruckner-Tuderman, Leena; Blobel, Carl P.

    2009-01-01

    Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9−/− keratinocytes, and (d) H2O2 enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin. PMID:19574220

  3. Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.

    PubMed

    Franzke, Claus-Werner; Bruckner-Tuderman, Leena; Blobel, Carl P

    2009-08-28

    Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10(-/-) cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9(-/-) keratinocytes, and (d) H(2)O(2) enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9(-/-) mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin.

  4. Bone tumor mimics: avoiding misdiagnosis.

    PubMed

    Gould, C Frank; Ly, Justin Q; Lattin, Grant E; Beall, Douglas P; Sutcliffe, Joseph B

    2007-01-01

    Whether discovered incidentally or as part of a focused diagnostic evaluation, the finding of a benign osseous lesion that has radiologic features resembling a bone tumor is not uncommon. Some of the more common benign and nonneoplastic entities that can sometimes be confused with tumors are the following: cortical desmoid, Brodie abscess, synovial herniation pit, pseudocyst, enostosis, intraosseous ganglion cyst, fibrous dysplasia, stress fracture, avulsion fracture (healing stage), bone infarct, myositis ossificans, brown tumor, and subchondral cyst. Accurate diagnosis and management of these lesions require a basic understanding of their epidemiology, clinical presentations, anatomic distributions, imaging features, differential considerations, and therapeutic options. This in-depth review of 13 potential bone tumor mimics will assist the radiologist in correctly identifying these benign lesions and in avoiding misdiagnosis and related morbidity. This review will also aid the radiologist in making appropriate recommendations to the referring physician for management or further imaging.

  5. Adam12 plays a role during uterine decidualization in mice.

    PubMed

    Zhang, Li; Guo, Weixiang; Chen, Qi; Fan, Xiujun; Zhang, Ying; Duan, Enkui

    2009-12-01

    In mouse, decidualization is characterized by the proliferation of stromal cells and their differentiation into specialized type of cells (decidual cells) with polyploidy, surrounding the implanting blastocyst. However, the mechanisms involved in these processes remain poorly understood. Using multiple approaches, we have examined the role of Adam12 in decidualization during early pregnancy in mice. Adam12 is spatiotemporally expressed in decidualizing stromal cells in intact pregnant females and in pseudopregnant mice undergoing artificially induced decidualization. In the ovariectomized mouse uterus, the expression of Adam12 is upregulated after progesterone treatment, which is primarily mediated by nuclear progesterone receptor. In a stromal cell culture model, the expression of Adam12 gradually rises with the progression of stromal decidualization, whereas the attenuated expression of Adam12 after siRNA knockdown significantly blocks the progression of decidualization. Our study suggests that Adam12 is involved in promoting uterine decidualization during pregnancy.

  6. Novel Gbeta Mimic Kelch Proteins Gpb1 and Gpb2 Connect G-Protein Signaling to Ras Via Yeast Neurofibromin Homologs Ira 1 and Ira 2: A Model for Human NF1

    DTIC Science & Technology

    2005-03-01

    in a decrease of the RasGAP Iral/2 proteins and consequently to an increase in the GTP bound form of Ras, which is the active form of Ras and...essential for membrane localization and function fusion protein exhibited a decreased level of myristoylation and required for palmitoylation, and...or when Gpbl /2 were overexpressed. As a!., 2000). Ethanol and glycerol are structurally unrelated F3 shown in Figure 3, A and B, Gpa2 membrane

  7. ADAM10 inhibition of human CD30 shedding increases specificity of targeted immunotherapy in vitro.

    PubMed

    Eichenauer, Dennis A; Simhadri, Vijaya Lakshmi; von Strandmann, Elke Pogge; Ludwig, Andreas; Matthews, Vance; Reiners, Katrin S; von Tresckow, Bastian; Saftig, Paul; Rose-John, Stefan; Engert, Andreas; Hansen, Hinrich P

    2007-01-01

    CD30 is a transmembrane protein selectively overexpressed on many human lymphoma cells and therefore an interesting target for antibody-based immunotherapy. However, binding of therapeutic antibodies stimulates a juxtamembrane cleavage of CD30 leading to a loss of target antigen and an enhanced release of the soluble ectodomain of CD30 (sCD30). Here, we show that sCD30 binds to CD30 ligand (CD153)-expressing non-target cells. Because antibodies bind to sCD30, this results in unwanted antibody binding to these cells via sCD30 bridging. To overcome shedding-dependent damage of normal cells in CD30-specific immunotherapy, we analyzed the mechanism involved in the release. Shedding of CD30 can be enhanced by protein kinase C (PKC) activation, implicating the disintegrin metalloproteinase ADAM17 but not free cytoplasmic calcium. However, antibody-induced CD30 shedding is calcium dependent and PKC independent. This shedding involved the related metalloproteinase ADAM10 as shown by the use of the preferential ADAM10 inhibitor GI254023X and by an ADAM10-deficient cell line generated from embryonically lethal ADAM10(-/-) mouse. In coculture experiments, the antibody-induced transfer of sCD30 from the human Hodgkin's lymphoma cell line L540 to the CD30-negative but CD153-expressing human mast cell line HMC-1 was inhibited by GI254023X. These findings suggest that selective metalloproteinase inhibitors blocking antibody-induced shedding of target antigens could be of therapeutic value to increase the specificity and reduce side effects of immunotherapy with monoclonal antibodies.

  8. Up-regulation of the alpha-secretase ADAM10 by retinoic acid receptors and acitretin.

    PubMed

    Tippmann, Frank; Hundt, Jana; Schneider, Anja; Endres, Kristina; Fahrenholz, Falk

    2009-06-01

    Late-onset Alzheimer's disease is often connected with nutritional misbalance, such as enhanced cholesterol intake, deficiency in polyunsaturated fatty acids, or hypovitaminosis. The alpha-secretase ADAM10 has been found to be regulated by retinoic acid, the bioreactive metabolite of vitamin A. Here we show that retinoids induce gene expression of ADAM10 and alpha-secretase activity by nonpermissive retinoid acid receptor/retinoid X receptor (RAR/RXR) heterodimers, whereby alpha- and beta-isotypes of RAR play a major role. However, ligands of other RXR binding partners, such as the vitamin D receptor, do not stimulate alpha-secretase activity. On the basis of these findings, we examined the effect of synthetic retinoids and found a strong enhancement of nonamyloidogenic processing of the amyloid precursor protein by the vitamin A analog acitretin: it stimulated ADAM10 promoter activity with an EC(50) of 1.5 microM and led to an increase of mature ADAM10 protein that resulted in a two- to three-fold increase of the ratio between alpha- and beta-secretase activity in neuroblastoma cells. The alpha-secretase stimulation by acitretin was completely inhibited by the ADAM10-specific inhibitor GI254023X. Intracerebral injection of acitretin in APP/PS1-21 transgenic mice led to a reduction of Abeta(40) and Abeta(42). The results of this study may have clinical relevance because acitretin has been approved for the treatment of psoriasis since 1997 and found generally safe for long-term use in humans.

  9. Dysfunctional ADAM22 implicated in progressive encephalopathy with cortical atrophy and epilepsy

    PubMed Central

    Muona, Mikko; Fukata, Yuko; Anttonen, Anna-Kaisa; Laari, Anni; Palotie, Aarno; Pihko, Helena; Lönnqvist, Tuula; Valanne, Leena; Somer, Mirja; Fukata, Masaki

    2016-01-01

    Objective: To identify the molecular genetic basis of a syndrome characterized by rapidly progressing cerebral atrophy, intractable seizures, and intellectual disability. Methods: We performed exome sequencing in the proband and whole-genome single nucleotide polymorphism genotyping (copy number variant analysis) in the proband-parent trio. We used heterologous expression systems to study the functional consequences of identified mutations. Results: The search for potentially deleterious recessive or de novo variants yielded compound heterozygous missense (c.1202G>A, p.Cys401Tyr) and frameshift deletion (c.2396delG, p.Ser799IlefsTer96) mutations in ADAM22, which encodes a postsynaptic receptor for LGI1. The deleterious effect of the mutations was observed in cell surface binding and immunoprecipitation assays, which revealed that both mutant proteins failed to bind to LGI1. Furthermore, immunoprecipitation assays showed that the frameshift mutant ADAM22 also did not bind to the postsynaptic scaffolding protein PSD-95. Conclusions: The mutations identified abolish the LGI1-ADAM22 ligand-receptor complex and are thus a likely primary cause of the proband's epilepsy syndrome, which is characterized by unusually rapidly progressing cortical atrophy starting at 3–4 months of age. These findings are in line with the implicated role of the LGI1-ADAM22 complex as a key player in nervous system development, specifically in functional maturation of postnatal synapses. Because the frameshift mutation affects an alternatively spliced exon with highest expression in postnatal brain, the combined effect of the mutations is likely to be hypomorphic rather than complete loss of function. This is compatible with the longer survival of the patient compared to Lgi1−/− and Adam22−/− mice, which develop lethal seizures during the first postnatal weeks. PMID:27066583

  10. Molecular Mimics of the Tumour Antigen MUC1

    PubMed Central

    James, Tharappel C.; Bond, Ursula

    2012-01-01

    A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as “self”, and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as ‘proof of principle’ we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1) from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides. PMID:23166757

  11. Cytoskeletal confinement of CX3CL1 limits its susceptibility to proteolytic cleavage by ADAM10

    PubMed Central

    Wong, Harikesh S.; Jaumouillé, Valentin; Heit, Bryan; Doodnauth, Sasha A.; Patel, Sajedabanu; Huang, Yi-Wei; Grinstein, Sergio; Robinson, Lisa A.

    2014-01-01

    CX3CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX3CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX3CL1. We observed that the majority of cell surface CX3CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX3CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX3CL1 confinement and increased CX3CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand. PMID:25253723

  12. ADAM12 as a marker of trisomy 18 in the first and second trimester of pregnancy.

    PubMed

    Spencer, Kevin; Cowans, Nicholas J

    2007-09-01

    ADAM12 (a disintegrin and metalloprotease 12) is a placentally derived glycoprotein that appears to be involved in growth and differentiation. The maternal serum concentration of ADAM12 appears to be a very good marker of trisomy 21 in the early first trimester when levels are reduced, and in the second trimester around 16-18 weeks levels are elevated. One small preliminary study of first trimester pregnancies with trisomy 18 found reduced levels in the maternal serum, and we examine herein the potential of ADAM12 as a marker of trisomy 18 in both the first and second trimester of pregnancy. The concentration of ADAM12 was determined by a time-resolved immunofluorometric assay in 132 first and 12 second trimester cases of trisomy 18, and 389 first and 341 second trimester gestational age-matched control pregnancies. Medians of normal pregnancies were established by polynomial regression and used to determine the population distribution parameters for the trisomy 18 and control groups. Correlation with previously established pregnancy-associated plasma protein-A (PAPP-A) and free beta-human chorionic gonadotropin (beta-hCG) multiples of the median (MoMs) and nuchal translucency thickness (NT) MoM were determined and used to model the performance of first trimester screening with ADAM12 in combination with other first trimester markers. The maternal serum concentration of ADAM12 in the first trimester was significantly reduced with a median MoM of 0.829 (p < 0.001) and a mean log10 MoM SD of 0.2663 compared to 0.3353 in the controls. In the second trimester small series ADAM12 was significantly increased with a median MoM of 2.09 (p = 0.001) and a mean log10 MoM SD of 0.2607 compared to 0.4318 in controls. There was a significant correlation of ADAM12 MoM with gestational age (r = 0.510) in trisomy 18 cases, and the median MoM increased from 0.51 at 10 weeks to 1.28 at 13 weeks and 2.09 across the 14-18 week window. ADAM12 was correlated with PAPP-A (r = 0.1918) in

  13. ADAM Proteases: Ligand Processing and Modulation of the Notch Pathway

    PubMed Central

    Zolkiewska, Anna

    2009-01-01

    ADAM metalloproteases play important roles in development and disease. One of the key functions of ADAMs is the proteolytic processing of Notch receptors and their ligands. ADAM-mediated cleavage of Notch represents the first step of the regulated intramembrane proteolysis of the receptor, leading to activation of the Notch pathway. Recent reports indicate that the transmembrane Notch ligands also undergo ADAM-mediated processing in cultured cells and in vivo. The proteolytic processing of Notch ligands modulates the strength and duration of Notch signals, leads to generation of soluble intracellular domains of the ligands, and may support a bi-directional signaling between cells. PMID:18344021

  14. ADAM12 and PAPP-A: Candidate regulators of trophoblast invasion and first trimester markers of healthy trophoblasts.

    PubMed

    Christians, Julian K; Beristain, Alexander G

    2016-03-03

    Proper placental development and function is crucial for a healthy pregnancy, and there has been substantial research to identify markers of placental dysfunction for the early detection of pregnancy complications. Low first-trimester levels of a disintegrin and metalloproteinase 12 (ADAM12) and pregnancy-associated plasma protein-A (PAPP-A) have been consistently associated with the subsequent development of preeclampsia and fetal growth restriction. These molecules are both metalloproteinases secreted by the placenta that cleave insulin-like growth factor binding proteins (IGFBPs), although ADAM12 also has numerous other substrates. Recent work has identified ADAM12, and particularly its shorter variant, ADAM12S, as a regulator of the migration and invasion of trophoblasts into the lining of the uterus, a critical step in normal placental development. While the mechanisms underlying this regulation are not yet clear, they may involve the liberation of heparin-binding EGF-like growth factor (HB-EGF) and/or IGFs from IGFBPs. In contrast, there has been relatively little functional work examining PAPP-A or the IGFBP substrates of ADAM12 and PAPP-A. Understanding the functions of these markers and the mechanisms underlying their association with disease could improve screening strategies and enable the development of new therapeutic interventions.

  15. Novel Gbeta Mimic Kelch Proteins (Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira1 and Ira2. A Model for Human NF1

    DTIC Science & Technology

    2007-03-01

    half of Kel1 (Kelch-repeat protein 1) is homologous to S. pombe or C . neoformans Ral2. S. cerevisiae Kel1 is involved in cell morphology and mating...Based on BLAST searches, C . neoformans has genes encoding hypothetical proteins homologous to the kelch-repeat containing amino terminus of...RAL2), KEM2(KEL1), KEL2) in the C . neoformans H99 strain background and found that Kem1 (Ral2) and Kem2 (Kel1), but not Kel2, are in part involved in

  16. An RF Performance Sensitivity and Process Yield Model for MIMIC CAD applications. MIMIC Program. Phase 3

    DTIC Science & Technology

    1991-09-16

    AD-A242 266 --III ! I I I 1111 i~tll An RF Performance Sensitivity and Process Yield Model for MIMIC CAD Applications MIMIC Program, Phase 3 Final...DAAL01-89-K-0906-4 September 16, 1991 An RF Performance Sensitivity and Process Yield Model for MIMIC CAD Applications MIMIC Program, Phase 3 Final...Yield Model MIMIC Phase 3 for MIMIC CAD Applications Contract No. DAALO1-89-K-0906-4 6. AUTHOR(S) R.J. Trew, D.E. Stoneking, P.Gilmore, C.T. Kelley

  17. Batesian mimics influence mimicry ring evolution.

    PubMed Central

    Franks, Daniel W.; Noble, Jason

    2004-01-01

    Mathematical models of mimicry typically involve artificial prey species with fixed colorations or appearances; this enables a comparison of predation rates to demonstrate the level of protection a mimic might be afforded. Fruitful theoretical results have been produced using this method, but it is also useful to examine the possible evolutionary consequences of mimicry. To that end, we present individual-based evolutionary simulation models where prey colorations are free to evolve. We use the models to examine the effect of Batesian mimics on Müllerian mimics and mimicry rings. Results show that Batesian mimics can potentially incite Müllerian mimicry relationships and encourage mimicry ring convergence. PMID:15058397

  18. Batesian mimics influence mimicry ring evolution.

    PubMed

    Franks, Daniel W; Noble, Jason

    2004-01-22

    Mathematical models of mimicry typically involve artificial prey species with fixed colorations or appearances; this enables a comparison of predation rates to demonstrate the level of protection a mimic might be afforded. Fruitful theoretical results have been produced using this method, but it is also useful to examine the possible evolutionary consequences of mimicry. To that end, we present individual-based evolutionary simulation models where prey colorations are free to evolve. We use the models to examine the effect of Batesian mimics on Müllerian mimics and mimicry rings. Results show that Batesian mimics can potentially incite Müllerian mimicry relationships and encourage mimicry ring convergence.

  19. From Adam Swift to Adam Smith: How the "Invisible Hand" Overcomes Middle Class Hypocrisy

    ERIC Educational Resources Information Center

    Tooley, James

    2007-01-01

    This paper challenges Richard Pring's suggestion that parents using private education may be undermining the desire for social justice and equality, using recent arguments of Adam Swift as a springboard. Swift's position on the banning of private schools, which uses a Rawlsian "veil of ignorance" argument, is explored, and it is suggested that, if…

  20. From Adam Swift to Adam Smith: How the "Invisible Hand" Overcomes Middle Class Hypocrisy

    ERIC Educational Resources Information Center

    Tooley, James

    2007-01-01

    This paper challenges Richard Pring's suggestion that parents using private education may be undermining the desire for social justice and equality, using recent arguments of Adam Swift as a springboard. Swift's position on the banning of private schools, which uses a Rawlsian "veil of ignorance" argument, is explored, and it is suggested that, if…

  1. Which polyesters can mimic polyethylene?

    PubMed

    Stempfle, Florian; Ortmann, Patrick; Mecking, Stefan

    2013-01-11

    Self-metathesis of erucic acid by [(PCy(3))(η-C-C(3)H(4)N(2)Mes(2))Cl(2)Ru = CHPh] (Grubbs second- generation catalyst) followed by catalytic hydrogenation and purification via the ester yields 1,26-hexacosanedioate (>99% purity). Polyesterification with 1,26-hexacosanediol, generated from the diester, affords polyester-26,26, which features a T(m) of 114 °C (T(c) = 92 °C, ΔH(m) = 160 J g(-1)). Ultralong-chain model polyesters-38,23 (T(m) = 109 °C) and -44,23 (T(m) = 111 °C), generated via multistep procedures including acyclic diene metathesis polymerization, underline that melting points of such aliphatic polyesters do not gradually increase with methylene sequence chain length. Available data suggest that to mimic linear polyethylenes thermal properties, even longer sequences, amounting to at least four times a fatty acid chain, fully incorporated in a linear fashion are required.

  2. Neuron-Derived ADAM10 Production Stimulates Peripheral Nerve Injury-Induced Neuropathic Pain by Cleavage of E-Cadherin in Satellite Glial Cells.

    PubMed

    Li, Jian; Ouyang, Qing; Chen, Cheng-Wen; Chen, Qian-Bo; Li, Xiang-Nan; Xiang, Zheng-Hua; Yuan, Hong-Bin

    2017-09-01

    Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. β-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of β-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells.

  3. Novel Gbeta Mimic Kelch Proteins (Gpb1 and Gpb2 Connect G-Protein Signaling to Ras via Yeast Neurofibromin Homologs Ira1 and Ira2: A Model for Human NF1

    DTIC Science & Technology

    2008-03-01

    the level of pigmentation produced following 2 or 4 days incubation compared to the isogenic wild type strains and mutants with defects in the enzyme...coupling, Gpb1/2 serve as potent molecular brakes to constrain signaling via the PKA signaling pathway during both vegetative growth and dimorphic...In ad- dition to regulating Ras activity, neurofibromin also gov- erns G protein-mediated adenylyl cyclase activity in the fruit fly Drosophila

  4. Once a Batesian mimic, not always a Batesian mimic: mimic reverts back to ancestral phenotype when the model is absent.

    PubMed

    Prudic, Kathleen L; Oliver, Jeffrey C

    2008-05-22

    Batesian mimics gain protection from predation through the evolution of physical similarities to a model species that possesses anti-predator defences. This protection should not be effective in the absence of the model since the predator does not identify the mimic as potentially dangerous and both the model and the mimic are highly conspicuous. Thus, Batesian mimics should probably encounter strong predation pressure outside the geographical range of the model species. There are several documented examples of Batesian mimics occurring in locations without their models, but the evolutionary responses remain largely unidentified. A mimetic species has four alternative evolutionary responses to the loss of model presence. If predation is weak, it could maintain its mimetic signal. If predation is intense, it is widely presumed the mimic will go extinct. However, the mimic could also evolve a new colour pattern to mimic another model species or it could revert back to its ancestral, less conspicuous phenotype. We used molecular phylogenetic approaches to reconstruct and test the evolution of mimicry in the North American admiral butterflies (Limenitis: Nymphalidae). We confirmed that the more cryptic white-banded form is the ancestral phenotype of North American admiral butterflies. However, one species, Limenitis arthemis, evolved the black pipevine swallowtail mimetic form but later reverted to the white-banded more cryptic ancestral form. This character reversion is strongly correlated with the geographical absence of the model species and its host plant, but not the host plant distribution of L. arthemis. Our results support the prediction that a Batesian mimic does not persist in locations without its model, but it does not go extinct either. The mimic can revert back to its ancestral, less conspicuous form and persist.

  5. ADAM 23/MDC3, a Human Disintegrin That Promotes Cell Adhesion via Interaction with the αvβ3 Integrin through an RGD-independent Mechanism

    PubMed Central

    Cal, Santiago; Freije, José M.P.; López, José M.; Takada, Yoshikazu; López-Otín, Carlos

    2000-01-01

    ADAM 23 (a disintegrin and metalloproteinase domain)/MDC3 (metalloprotease, disintegrin, and cysteine-rich domain) is a member of the disintegrin family of proteins expressed in fetal and adult brain. In this work we show that the disintegrin-like domain of ADAM 23 produced in Escherichia coli and immobilized on culture dishes promotes attachment of different human cells of neural origin, such as neuroblastoma cells (NB100 and SH-Sy5y) or astrocytoma cells (U373 and U87 MG). Analysis of ADAM 23 binding to integrins revealed a specific interaction with αvβ3, mediated by a short amino acid sequence present in its putative disintegrin loop. This sequence lacks any RGD motif, which is a common structural determinant supporting αvβ3-mediated interactions of diverse proteins, including other disintegrins. αvβ3 also supported adhesion of HeLa cells transfected with a full-length cDNA for ADAM 23, extending the results obtained with the recombinant protein containing the disintegrin domain of ADAM 23. On the basis of these results, we propose that ADAM 23, through its disintegrin-like domain, may function as an adhesion molecule involved in αvβ3-mediated cell interactions occurring in normal and pathological processes, including progression of malignant tumors from neural origin. PMID:10749942

  6. ADAM8 as a drug target in Pancreatic Cancer

    PubMed Central

    Schlomann, Uwe; Koller, Garrit; Conrad, Catharina; Ferdous, Taheera; Golfi, Panagiota; Garcia, Adolfo Molejon; Höfling, Sabrina; Parsons, Maddy; Costa, Patricia; Soper, Robin; Bossard, Maud; Hagemann, Thorsten; Roshani, Rozita; Sewald, Norbert; Ketchem, Randal R.; Moss, Marcia L.; Rasmussen, Fred H.; Miller, Miles A.; Lauffenburger, Douglas A.; Tuveson, David A.; Nimsky, Christopher; Bartsch, Jörg W.

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with less than 5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease-disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK 1/2 and higher MMP activities. For biological function, ADAM8 requires multimerisation and associates with β1-integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerisation. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK 1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a KrasG12D-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy. PMID:25629724

  7. The Failed Educations of John Stuart Mill and Henry Adams.

    ERIC Educational Resources Information Center

    Crossley, Robert

    1979-01-01

    Analyzes and contrasts Mill's "Autobiography" and Adams'"The Education of Henry Adams" in order to present two approaches to the nature of education and of failure. Maintains that their perspectives may serve as catalysts and cautions for contemporary theories of education and its utility and relevance. (CAM)

  8. The Failed Educations of John Stuart Mill and Henry Adams.

    ERIC Educational Resources Information Center

    Crossley, Robert

    1979-01-01

    Analyzes and contrasts Mill's "Autobiography" and Adams'"The Education of Henry Adams" in order to present two approaches to the nature of education and of failure. Maintains that their perspectives may serve as catalysts and cautions for contemporary theories of education and its utility and relevance. (CAM)

  9. BAFF- and TACI-Dependent Processing of BAFFR by ADAM Proteases Regulates the Survival of B Cells.

    PubMed

    Smulski, Cristian R; Kury, Patrick; Seidel, Lea M; Staiger, Hannah S; Edinger, Anna K; Willen, Laure; Seidl, Maximilan; Hess, Henry; Salzer, Ulrich; Rolink, Antonius G; Rizzi, Marta; Schneider, Pascal; Eibel, Hermann

    2017-02-28

    B cell activating factor (BAFF) provides B cells with essential survival signals. It binds to three receptors: BAFFR, TACI, and BCMA that are differentially expressed by B cell subsets. BAFFR is early expressed in circulating B cells and provides key signals for further maturation. Here, we report that highly regulated BAFFR processing events modulate BAFF responses. BAFFR processing is triggered by BAFF binding in B cells co-expressing TACI and it is executed by the metalloproteases ADAM10 and ADAM17. The degree of BAFF oligomerization, the expression of ADAM proteins in different B cell subsets, and the activation status of the cell determine the proteases involved in BAFFR processing. Inhibition of ADAM10 augments BAFF-dependent survival of primary human B cells, whereas inhibition of ADAM17 increases BAFFR expression levels on germinal center B cells. Therefore, BAFF-induced processing of BAFFR regulates BAFF-mediated B cell responses in a TACI-dependent manner. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Postnatal disruption of the disintegrin/metalloproteinase ADAM10 in brain causes epileptic seizures, learning deficits, altered spine morphology, and defective synaptic functions.

    PubMed

    Prox, Johannes; Bernreuther, Christian; Altmeppen, Hermann; Grendel, Jasper; Glatzel, Markus; D'Hooge, Rudi; Stroobants, Stijn; Ahmed, Tariq; Balschun, Detlef; Willem, Michael; Lammich, Sven; Isbrandt, Dirk; Schweizer, Michaela; Horré, Katrien; De Strooper, Bart; Saftig, Paul

    2013-08-07

    The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid β (Aβ) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPβ and endogenous Aβ peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.

  11. ADAM17 Transactivates EGFR Signaling during Embryonic Eyelid Closure

    PubMed Central

    Hassemer, Eryn L.; Endres, Bradley; Toonen, Joseph A.; Ronchetti, Adam; Dubielzig, Richard; Sidjanin, Duska J.

    2013-01-01

    Purpose. During mammalian embryonic eyelid closure ADAM17 has been proposed to play a role as a transactivator of epidermal growth factor receptor (EGFR) signaling by shedding membrane bound EGFR ligands. However, ADAM17 also sheds numerous other ligands, thus implicating ADAM17 in additional molecular pathways. The goal of this study was to experimentally establish the role of ADAM17 and determine ADAM17-mediated pathways essential for the embryonic eyelid closure. Methods. Wild-type (WT) and woe mice, carrying a hypomorphic mutation in Adam17, were evaluated using H&E and scanning electron microscopy. Expressions of ADAM17, EGFR, and the phosphorylated form EGFR-P were evaluated using immunohistochemistry. BrdU and TUNEL assays were used to evaluate cell proliferation and apoptosis, respectively. In vitro scratch assays of primary cultures were used to evaluate cell migration. Clinical and histologic analyses established if the hypermorphic EgfrDsk5 allele can rescue the woe embryonic eyelid closure. Results. woe mice exhibited a failure to develop the leading edge of the eyelid and consequently failure of the embryonic eyelid closure. Expression of ADAM17 was identified in the eyelid epithelium in the cells of the leading edge. ADAM17 is essential for epithelial cell migration, but does not play a role in proliferation and apoptosis. EGFR was expressed in both WT and woe eyelid epithelium, but the phosphorylated EGFR-P form was detected only in WT. The EgfrDsk5 allele rescued woe eyelid closure defects, but also rescued woe anterior segment defects and the absence of meibomian glands. Conclusions. We provide in vivo genetic evidence that the role of ADAM17 during embryonic eyelid closure is to transactivate EGFR signaling. PMID:23211830

  12. Helping Eve overcome ADAM: G-quadruplexes in the ADAM-15 promoter as new molecular targets for breast cancer therapeutics.

    PubMed

    Brown, Robert V; Gaerig, Vanessa C; Simmons, Taesha; Brooks, Tracy A

    2013-12-05

    ADAM-15, with known zymogen, secretase, and disintegrin activities, is a catalytically active member of the ADAM family normally expressed in early embryonic development and aberrantly expressed in various cancers, including breast, prostate and lung. ADAM-15 promotes extracellular shedding of E-cadherin, a soluble ligand for the HER2/neu receptor, leading to activation, increased motility, and proliferation. Targeted downregulation of both ADAM-15 and HER2/neu function synergistically kills breast cancer cells, but to date there are no therapeutic options for decreasing ADAM-15 function or expression. In this vein, we have examined a unique string of guanine-rich DNA within the critical core promoter of ADAM-15. This region of DNA consists of seven contiguous runs of three or more consecutive guanines, which, under superhelical stress, can relax from duplex DNA to form an intrastrand secondary G-quadruplex (G4) structure. Using biophysical and biological techniques, we have examined the G4 formation within the entire and various truncated regions of the ADAM-15 promoter, and demonstrate strong intrastrand G4 formation serving to function as a biological silencer element. Characterization of the predominant G4 species formed within the ADAM-15 promoter will allow for specific drug targeting and stabilization, and the further development of novel, targeted therapeutics.

  13. IL-13-induced proliferation of airway epithelial cells: mediation by intracellular growth factor mobilization and ADAM17

    PubMed Central

    Booth, Brian W; Sandifer, Tracy; Martin, Erika L; Martin, Linda D

    2007-01-01

    Background The pleiotrophic cytokine interleukin (IL)-13 features prominently in allergic and inflammatory diseases. In allergic asthma, IL-13 is well established as an inducer of airway inflammation and tissue remodeling. We demonstrated previously that IL-13 induces release of transforming growth factor-α (TGFα) from human bronchial epithelial cells, with proliferation of these cells mediated by the autocrine/paracrine action of this growth factor. TGFα exists as an integral membrane protein and requires proteolytic processing to its mature form, with a disintegrin and metalloproteinase (ADAM)17 responsible for this processing in a variety of tissues. Methods In this study, normal human bronchial epithelial (NHBE) cells grown in air/liquid interface (ALI) culture were used to examine the mechanisms whereby IL-13 induces release of TGFα and cellular proliferation. Inhibitors and antisense RNA were used to examine the role of ADAM17 in these processes, while IL-13-induced changes in the intracellular expression of TGFα and ADAM17 were visualized by confocal microscopy. Results IL-13 was found to induce proliferation of NHBE cells, and release of TGFα, in an ADAM17-dependent manner; however, this IL-13-induced proliferation did not appear to result solely from ADAM17 activation. Rather, IL-13 induced a change in the location of TGFα expression from intracellular to apical regions of the NHBE cells. The apical region was also found to be a site of significant ADAM17 expression, even prior to IL-13 stimulation. Conclusion Results from this study indicate that ADAM17 mediates IL-13-induced proliferation and TGFα shedding in NHBE cells. Furthermore, they provide the first example wherein a cytokine (IL-13) induces a change in the intracellular expression pattern of a growth factor, apparently inducing redistribution of intracellular stores of TGFα to the apical region of NHBE cells where expression of ADAM17 is prominent. Thus, IL-13-induced, ADAM17-mediated

  14. Gene-specific cell labeling using MiMIC transposons

    PubMed Central

    Gnerer, Joshua P.; Venken, Koen J. T.; Dierick, Herman A.

    2015-01-01

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family. PMID:25712101

  15. Gene-specific cell labeling using MiMIC transposons.

    PubMed

    Gnerer, Joshua P; Venken, Koen J T; Dierick, Herman A

    2015-04-30

    Binary expression systems such as GAL4/UAS, LexA/LexAop and QF/QUAS have greatly enhanced the power of Drosophila as a model organism by allowing spatio-temporal manipulation of gene function as well as cell and neural circuit function. Tissue-specific expression of these heterologous transcription factors relies on random transposon integration near enhancers or promoters that drive the binary transcription factor embedded in the transposon. Alternatively, gene-specific promoter elements are directly fused to the binary factor within the transposon followed by random or site-specific integration. However, such insertions do not consistently recapitulate endogenous expression. We used Minos-Mediated Integration Cassette (MiMIC) transposons to convert host loci into reliable gene-specific binary effectors. MiMIC transposons allow recombinase-mediated cassette exchange to modify the transposon content. We developed novel exchange cassettes to convert coding intronic MiMIC insertions into gene-specific binary factor protein-traps. In addition, we expanded the set of binary factor exchange cassettes available for non-coding intronic MiMIC insertions. We show that binary factor conversions of different insertions in the same locus have indistinguishable expression patterns, suggesting that they reliably reflect endogenous gene expression. We show the efficacy and broad applicability of these new tools by dissecting the cellular expression patterns of the Drosophila serotonin receptor gene family.

  16. Global characterization of the SRC-1 transcriptome identifies ADAM22 as an ER-independent mediator of endocrine resistant breast cancer

    PubMed Central

    McCartan, Damian; Bolger, Jarlath; Fagan, Aílis; Byrne, Christopher; Hao, Yuan; Qin, Li; McIlroy, Marie; Xu, Jianming; Hill, Arnold; Gaora, Peadar Ó; Young, Leonie

    2013-01-01

    The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other non-steroidal transcription factors, drives this tumor adaptability. In this discovery study we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct ER-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular and in vivo studies. ADAM22 functioned in cellular migration and differentiation and its levels were increased endocrine resistant tumors compared to endocrine sensitive tumors in a mouse xenograft models of human breast cancer. Clinically ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer. PMID:22072566

  17. Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand–dependent terminal keratinocyte differentiation

    PubMed Central

    Cobzaru, Cristina; Triantafyllopoulou, Antigoni; Löffek, Stefanie; Horiuchi, Keisuke; Threadgill, David W.; Kurz, Thomas; van Rooijen, Nico; Bruckner-Tuderman, Leena

    2012-01-01

    ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal growth factor receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in keratinocytes (A17ΔKC) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident 2 d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17ΔKC skin, and topical treatment of A17ΔKC mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in keratinocytes (EgfrΔKC) closely resembled A17ΔKC mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17–EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects. PMID:22565824

  18. John Adams and CERN: Personal Recollections

    NASA Astrophysics Data System (ADS)

    Brianti, G.; Plane, D. E.

    2014-02-01

    By any standards, John Adams had a most remarkable career. He was involved in three important, emerging technologies, radar, particle accelerators and controlled fusion, and had an outstanding impact on the last two. Without a university education, he attained hierarchical positions of the highest level in prestigious national and international organizations. This article covers the CERN part of his career, by offering some personal insights into the different facets of his contributions to major accelerator projects, from the first strong-focusing synchrotron, the PS, to the SPS and its conversion to a proton--antiproton collider. In particular, it outlines his abilities as a leader of an international collaboration, which has served as an example for international initiatives in other disciplines.

  19. Adam Paulsen, a Pioneer in Auroral Research

    NASA Astrophysics Data System (ADS)

    Jørgensen, Torben S.; Rasmussen, Ole

    2006-02-01

    The 20 to 30 years following the first International Polar Year in 1882-1883 was a period of quickly advancing knowledge and understanding of auroral phenomena. This was the time when hypotheses of aurora being due to, for example, reflections of fires from the interior of the Earth or sunlight from ice particles were abandoned and replaced by the mechanism of precipitating electrons. One of the auroral researchers at that time was the Dane Adam Frederik Wivet Paulsen (1833-1907). However, when reading literature about auroral history, his ideas and work do not seem to have attracted much interest outside his own and neighboring countries. For example, in his sweeping historical account Majestic Lights: The Aurora in Science, History, and the Arts [1980], author Robert Eather only referred to Paulsen in a couple of lines.

  20. ADAM-17: The Enzyme That Does It All

    PubMed Central

    Gooz, Monika

    2010-01-01

    This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme or TACE, ADAM-17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion molecules are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer’s disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiology of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme. PMID:20184396

  1. EphrinB2 Affects Apical Constriction in Xenopus Embryos and is Regulated by ADAM10 and Flotillin-1

    PubMed Central

    Ji, Yon Ju; Hwang, Yoo-Seok; Mood, Kathleen; Cho, Hee-Jun; Lee, Hyun-Shik; Winterbottom, Emily; Cousin, Hèléne; Daar, Ira O.

    2014-01-01

    The Eph/ephrin signaling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed upon the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels upon the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo. PMID:24662724

  2. EphrinB2 affects apical constriction in Xenopus embryos and is regulated by ADAM10 and flotillin-1

    NASA Astrophysics Data System (ADS)

    Ji, Yon Ju; Hwang, Yoo-Seok; Mood, Kathleen; Cho, Hee-Jun; Lee, Hyun-Shik; Winterbottom, Emily; Cousin, Hélène; Daar, Ira O.

    2014-03-01

    The Eph/ephrin signalling pathways have a critical function in cell adhesion and repulsion, and thus play key roles in various morphogenetic events during development. Here we show that a decrease in ephrinB2 protein causes neural tube closure defects during Xenopus laevis embryogenesis. Such a decrease in ephrinB2 protein levels is observed on the loss of flotillin-1 scaffold protein, a newly identified ephrinB2-binding partner. This dramatic decline in ephrinB2 protein levels on the absence of flotillin-1 expression is specific, and is partly the result of an increased susceptibility to cleavage by the metalloprotease ADAM10. These findings indicate that flotillin-1 regulates ephrinB2 protein levels through ADAM10, and is required for appropriate neural tube morphogenesis in the Xenopus embryo.

  3. Control design and simulation of systems modeled using ADAMS

    NASA Technical Reports Server (NTRS)

    Sohoni, Vikram N.

    1989-01-01

    A technique for control design and simulation using the ADAMS software and a control design software package is presented. For design of control systems ADAMS generates a minimum realization linear time invariant (LTI), state space representation of multi-body models. This LTI representation can be produced in formats for input to several commercial control design packages. The user can exercise various design strategies in the control design software to arrive at a suitable compensator. The resulting closed loop model can then be simulated using ADAMS. This procedure is illustrated with two examples.

  4. Maternal serum ADAM12s as a potential marker of trisomy 21 prior to 10 weeks of gestation.

    PubMed

    Spencer, Kevin; Vereecken, Annie; Cowans, Nicholas J

    2008-03-01

    ADAM12s (a disintegrin and metalloprotease) is a placenta-derived glycoprotein that is involved in growth and differentiation and has been shown to be a potential first-trimester and second-trimester marker of trisomy 21 and other aneuploides. Maternal ADAM12s concentrations show a considerable temporal variation with gestational age and in the initial study levels were found to be significantly reduced in the early first trimester. Here we study the levels prior to 10 weeks of gestation to establish further the effectiveness or otherwise of ADAM12s as an early screening marker. Samples collected as part of routine first-trimester screening were retrieved from storage. In total, ten samples from singleton pregnancies with trisomy 21 were identified and were collected between the 8th and 9th weeks of gestation-of these 80% had been identified by combined first-trimester screening. A series of 62 gestational age-matched samples from singleton pregnancies collected during the same period formed the control group. ADAM12s was measured by a new DELFIA assay incorporating two monoclonals (6E6 and 8F8). Results were expressed as multiples of the median (MoM). The median MoM ADAM12s at a median gestation of 9.3 weeks was 0.61 which was significantly lower than in the controls (p = 0.011) when compared by the Mann-Whitney test. The corresponding median pregnancy associated plasma protein (PAPP-A) was 0.30 and free beta-human chorionic gonadotropin (beta-hCG) 2.02. Combining the data from this study and from the only other published study with data prior to 10 weeks suggests that ADAM12s may have the potential as an early screening marker for trisomy 21, but may not be as reduced as first thought. Copyright (c) 2008 John Wiley & Sons, Ltd.

  5. microRNA-590 suppresses the tumorigenesis and invasiveness of non-small cell lung cancer cells by targeting ADAM9.

    PubMed

    Wang, Fei-Fei; Wang, Song; Xue, Wen-Hua; Cheng, Jing-Liang

    2016-12-01

    microRNAs (miRNAs), a family of small non-coding RNA molecules, are implicated in cancer growth and progression. In the present study, we examined the expression and biological roles of miR-590 in non-small cell lung cancer (NSCLC). Compared to normal lung tissues, miR-590 expression was downregulated in primary NSCLCs and, to a greater extent, in corresponding brain metastases. NSCLC cell lines with high metastatic potential had significantly (P < 0.05) lower levels of miR-590 than those with low metastatic potential. Re-expression of miR-590 suppressed NSCLC cell proliferation, colony formation, migration, and invasion in vitro and tumorigenesis in vivo. In contrast, inhibition of miR-590 enhanced the migration and invasion of NSCLC cells. Mechanistic studies revealed that a disintegrin and metalloproteinase 9 (ADAM9) was a direct target of miR-590. Delivery of miR-590 mimic was found to decrease endogenous ADAM9 expression in NSCLC cells. Enforced expression of a miRNA-resistant form of ADAM9 significantly restored the aggressive behaviors in miR-590-overexpressing NSCLC cells. Taken together, our data reveal miR-590 as a tumor suppressor in NSCLC, which is at least partially mediated through targeting of ADAM9. Restoration of miR-590 may provide a promising therapeutic strategy for NSCLC.

  6. Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1

    PubMed Central

    Petersen, Jens E. V.; Mkumbaye, Sixbert I.; Vaaben, Anna V.; Manjurano, Alphaxard; Lyimo, Eric; Kavishe, Reginald A.; Mwakalinga, Steven B.; Mosha, Jacklin; Minja, Daniel T. R.; Lusingu, John P. A.; Theander, Thor G.; Lavstsen, Thomas; Wang, Christian W.

    2016-01-01

    The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloproteinase 17 (ADAM17) is a protein responsible for releasing cytokines, including Tumor Necrosis Factor α (TNFα), and shedding of adhesion proteins. In this study, we show that plasma levels of ADAM17 are increased in Tanzanian children hospitalized with a malaria infection compared with asymptomatic children but similar to children hospitalized with other infectious diseases. The plasma levels of ADAM17 decreased during recovery after an acute malaria episode. Plasma levels of Ang2 were associated with markers of malaria severity and levels of var transcripts encoding P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) containing Cysteine Rich Inter Domain Region α1 (CIDRα1) domains predicted to bind Endothelial Protein C receptor (EPCR). ADAM17 levels were not associated with expression of var genes encoding different PfEMP1 types when controlling for age. These data are the first to report ADAM17 plasma levels in malaria-exposed individuals, and support the notion that parasite sequestration mediated by EPCR-binding PfEMP1 is associated with endothelial activation and pathology in severe paediatric malaria. PMID:27784899

  7. Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1.

    PubMed

    Petersen, Jens E V; Mkumbaye, Sixbert I; Vaaben, Anna V; Manjurano, Alphaxard; Lyimo, Eric; Kavishe, Reginald A; Mwakalinga, Steven B; Mosha, Jacklin; Minja, Daniel T R; Lusingu, John P A; Theander, Thor G; Lavstsen, Thomas; Wang, Christian W

    2016-10-27

    The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloproteinase 17 (ADAM17) is a protein responsible for releasing cytokines, including Tumor Necrosis Factor α (TNFα), and shedding of adhesion proteins. In this study, we show that plasma levels of ADAM17 are increased in Tanzanian children hospitalized with a malaria infection compared with asymptomatic children but similar to children hospitalized with other infectious diseases. The plasma levels of ADAM17 decreased during recovery after an acute malaria episode. Plasma levels of Ang2 were associated with markers of malaria severity and levels of var transcripts encoding P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) containing Cysteine Rich Inter Domain Region α1 (CIDRα1) domains predicted to bind Endothelial Protein C receptor (EPCR). ADAM17 levels were not associated with expression of var genes encoding different PfEMP1 types when controlling for age. These data are the first to report ADAM17 plasma levels in malaria-exposed individuals, and support the notion that parasite sequestration mediated by EPCR-binding PfEMP1 is associated with endothelial activation and pathology in severe paediatric malaria.

  8. Whole-exome sequencing identifies ADAM10 mutations as a cause of reticulate acropigmentation of Kitamura, a clinical entity distinct from Dowling-Degos disease.

    PubMed

    Kono, Michihiro; Sugiura, Kazumitsu; Suganuma, Mutsumi; Hayashi, Masahiro; Takama, Hiromichi; Suzuki, Tamio; Matsunaga, Kayoko; Tomita, Yasushi; Akiyama, Masashi

    2013-09-01

    Reticulate acropigmentation of Kitamura (RAK) is a rare genetic disorder of cutaneous pigmentation with an autosomal dominant pattern of inheritance and a high penetration rate. The characteristic skin lesions are reticulate, slightly depressed pigmented macules mainly affecting the dorsa of the hands and feet, which first appear before puberty and subsequently expand to the proximal limb and the trunk. To identify mutations that cause RAK, we performed exome sequencing of four family members in a pedigree with RAK. Fifty-three SNV/Indels were considered as candidate mutations after some condition narrowing. We confirmed the mutation status in each candidate gene of four other members in the same pedigree to find the gene that matched the mutation status and phenotype of each member. A mutation in ADAM10 encoding a zinc metalloprotease, a disintegrin and metalloprotease domain-containing protein 10 (ADAM10), was identified in the RAK family. ADAM10 is known to be involved in the ectodomain shedding of various substrates in the skin. Sanger sequencing of four additional unrelated RAK patients revealed four additional ADAM10 mutations. We identified a total of three truncating mutations, a splice site mutation and a missense mutation in ADAM10. We searched for mutations in the KRT5 gene, a causative gene for the similar pigmentation disorder Dowling-Degos disease (DDD), in all the patients and found no KRT5 mutation. These results reveal that mutations in ADAM10 are a cause of RAK and that RAK is an independent clinical entity distinct from DDD.

  9. ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas.

    PubMed

    Fan, Xing; Wang, Yongheng; Zhang, Chuanbao; Liu, Li; Yang, Sen; Wang, Yinyan; Liu, Xing; Qian, Zenghui; Fang, Shengyu; Qiao, Hui; Jiang, Tao

    2016-08-26

    The A disintegrin and metalloproteinase 9 (ADAM9) protein has been suggested to promote carcinoma invasion and appears to be overexpressed in various human cancers. However, its role has rarely been investigated in gliomas and, thus, in the current study we have evaluated ADAM9 expression in gliomas and examined the relevance of its expression in the prognosis of glioma patients. Clinical characteristics, RNA sequence data, and the case follow-ups were reviewed for 303 patients who had histological, confirmed gliomas. The ADAM9 expression between lower-grade glioma (LGG) and glioblastoma (GBM) patients was compared and its association with progression-free survival (PFS) and overall survival (OS) was assessed to evaluate its prognostic value. Our data suggested that GBM patients had significantly higher expression of ADAM9 in comparison to LGG patients (p < 0.001, t-test). In addition, among the LGG patients, aggressive astrocytic tumors displayed significantly higher ADAM9 expression than oligodendroglial tumors (p < 0.001, t-test). Moreover, high ADAM9 expression also correlated with poor clinical outcome (p < 0.001 and p < 0.001, log-rank test, for PFS and OS, respectively) in LGG patients. Further, multivariate analysis suggested ADAM9 expression to be an independent marker of poor survival (p = 0.002 and p = 0.003, for PFS and OS, respectively). These results suggest that ADAM9 mRNA expression is associated with tumor grade and histological type in gliomas and can serve as an independent prognostic factor, specifically in LGG patients.

  10. ADAM9 Expression Is Associate with Glioma Tumor Grade and Histological Type, and Acts as a Prognostic Factor in Lower-Grade Gliomas

    PubMed Central

    Fan, Xing; Wang, Yongheng; Zhang, Chuanbao; Liu, Li; Yang, Sen; Wang, Yinyan; Liu, Xing; Qian, Zenghui; Fang, Shengyu; Qiao, Hui; Jiang, Tao

    2016-01-01

    The A disintegrin and metalloproteinase 9 (ADAM9) protein has been suggested to promote carcinoma invasion and appears to be overexpressed in various human cancers. However, its role has rarely been investigated in gliomas and, thus, in the current study we have evaluated ADAM9 expression in gliomas and examined the relevance of its expression in the prognosis of glioma patients. Clinical characteristics, RNA sequence data, and the case follow-ups were reviewed for 303 patients who had histological, confirmed gliomas. The ADAM9 expression between lower-grade glioma (LGG) and glioblastoma (GBM) patients was compared and its association with progression-free survival (PFS) and overall survival (OS) was assessed to evaluate its prognostic value. Our data suggested that GBM patients had significantly higher expression of ADAM9 in comparison to LGG patients (p < 0.001, t-test). In addition, among the LGG patients, aggressive astrocytic tumors displayed significantly higher ADAM9 expression than oligodendroglial tumors (p < 0.001, t-test). Moreover, high ADAM9 expression also correlated with poor clinical outcome (p < 0.001 and p < 0.001, log-rank test, for PFS and OS, respectively) in LGG patients. Further, multivariate analysis suggested ADAM9 expression to be an independent marker of poor survival (p = 0.002 and p = 0.003, for PFS and OS, respectively). These results suggest that ADAM9 mRNA expression is associated with tumor grade and histological type in gliomas and can serve as an independent prognostic factor, specifically in LGG patients. PMID:27571068

  11. 12. Looking northwest along Adams Dam Road toward State Routes ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. Looking northwest along Adams Dam Road toward State Routes 92 and 100 intersection, stone wall on right. - Winterthur Farms, Intersection State Routes 92 & 100, Intersection State Routes 92 & 100, Winterthur, New Castle County, DE

  12. Role of ADAM and ADAMTS metalloproteinases in airway diseases

    PubMed Central

    2009-01-01

    Lungs are exposed to the outside environment and therefore to toxic and infectious agents or allergens. This may lead to permanent activation of innate immune response elements. A Disintegrin And Metalloproteinases (ADAMs) and ADAMs with Thrombospondin motifs (ADAMTS) are proteinases closely related to Matrix Metalloproteinases (MMPs). These multifaceted molecules bear metalloproteinase and disintegrin domains endowing them with features of both proteinases and adhesion molecules. Proteinases of the ADAM family are associated to various physiological and pathological processes and display a wide spectrum of biological effects encompassing cell fusion, cell adhesion, "shedding process", cleavage of various substrates from the extracellular matrix, growth factors or cytokines... This review will focus on the putative roles of ADAM/ADAMTS proteinases in airway diseases such as asthma and COPD. PMID:20034386

  13. 7. Historic American Buildings Survey, C. C. Adams, Photographer August ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. Historic American Buildings Survey, C. C. Adams, Photographer August 1931, SEED PACKING ROOM, Gift of New York State Department of Education. - Shaker North Family Washhouse (first), Shaker Road, New Lebanon, Columbia County, NY

  14. iRHOM2-dependent regulation of ADAM17 in cutaneous disease and epidermal barrier function.

    PubMed

    Brooke, Matthew A; Etheridge, Sarah L; Kaplan, Nihal; Simpson, Charlotte; O'Toole, Edel A; Ishida-Yamamoto, Akemi; Marches, Olivier; Getsios, Spiro; Kelsell, David P

    2014-08-01

    iRHOM2 is a highly conserved, catalytically inactive member of the Rhomboid family, which has recently been shown to regulate the maturation of the multi-substrate ectodomain sheddase enzyme ADAM17 (TACE) in macrophages. Dominant iRHOM2 mutations are the cause of the inherited cutaneous and oesophageal cancer-susceptibility syndrome tylosis with oesophageal cancer (TOC), suggesting a role for this protein in epithelial cells. Here, using tissues derived from TOC patients, we demonstrate that TOC-associated mutations in iRHOM2 cause an increase in the maturation and activity of ADAM17 in epidermal keratinocytes, resulting in significantly upregulated shedding of ADAM17 substrates, including EGF-family growth factors and pro-inflammatory cytokines. This activity is accompanied by increased EGFR activity, increased desmosome processing and the presence of immature epidermal desmosomes, upregulated epidermal transglutaminase activity and heightened resistance to Staphylococcal infection in TOC keratinocytes. Many of these features are consistent with the presence of a constitutive wound-healing-like phenotype in TOC epidermis, which may shed light on a novel pathway in skin repair, regeneration and inflammation.

  15. An evolutionary recent neuroepithelial cell adhesion function of huntingtin implicates ADAM10-Ncadherin.

    PubMed

    Lo Sardo, Valentina; Zuccato, Chiara; Gaudenzi, Germano; Vitali, Barbara; Ramos, Catarina; Tartari, Marzia; Myre, Michael A; Walker, James A; Pistocchi, Anna; Conti, Luciano; Valenza, Marta; Drung, Binia; Schmidt, Boris; Gusella, James; Zeitlin, Scott; Cotelli, Franco; Cattaneo, Elena

    2012-05-01

    The Huntington's disease gene product, huntingtin, is indispensable for neural tube formation, but its role is obscure. We studied neurulation in htt-null embryonic stem cells and htt-morpholino zebrafish embryos and found a previously unknown, evolutionarily recent function for this ancient protein. We found that htt was essential for homotypic interactions between neuroepithelial cells; it permitted neurulation and rosette formation by regulating metalloprotease ADAM10 activity and Ncadherin cleavage. This function was embedded in the N terminus of htt and was phenocopied by treatment of htt knockdown zebrafish with an ADAM10 inhibitor. Notably, in htt-null cells, reversion of the rosetteless phenotype occurred only with expression of evolutionarily recent htt heterologues from deuterostome organisms. Conversely, all of the heterologues that we tested, including htt from Drosophila melanogaster and Dictyostelium discoideum, exhibited anti-apoptotic activity. Thus, anti-apoptosis may have been one of htt’s ancestral function(s), but, in deuterostomes, htt evolved to acquire a unique regulatory activity for controlling neural adhesion via ADAM10-Ncadherin, with implications for brain evolution and development.

  16. ADAM33 gene polymorphisms are associated with the risk of idiopathic pulmonary fibrosis.

    PubMed

    Uh, Soo-Taek; Jang, An-Soo; Park, Sung-Woo; Park, Jong-Sook; Min, Chang-Gi; Kim, Yong Hoon; Park, Byung-Lae; Shin, Hyoung Doo; Kim, Dong Soon; Park, Choon-Sik

    2014-08-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive dyspnea and worsening lung function due to remodeling of the lung, including epithelial mesenchymal transition. ADAM33 is a disintegrin and metalloprotease domain-containing protein, which may be related to lung fibrosis by exerting angiogenesis and remodeling of the lung. Thus, we evaluated the association of single-nucleotide polymorphisms (SNPs) of ADAM33 with the risk of IPF. A total of 237 patients with IPF and 183 healthy subjects participated in the present study. Nine polymorphisms were selected. Genotyping was performed by single-base extension. Polymorphisms and haplotypes were analyzed for associations with the risk of IPF using multiple logistic regression models controlling for age, gender, and smoking status as covariates. All SNPs were in Hardy-Weinberg equilibrium. The minor allele frequency (MAF) of rs628977G>A in intron 21 was significantly lower in subjects with surgical IPF than in normal controls in the recessive model [33.2 vs. 38.0 %, p = 0.02, OR = 0.40 (0.19-0.84)]. When the subjects with clinical IPF were included, the difference in MAF persisted with a p value of 0.03 [OR = 0.50 (0.27-0.94)]. ADAM33 rs628977G>A was marginally associated with a decreased risk of IPF in a recessive model.

  17. iRHOM2-dependent regulation of ADAM17 in cutaneous disease and epidermal barrier function

    PubMed Central

    Brooke, Matthew A.; Etheridge, Sarah L.; Kaplan, Nihal; Simpson, Charlotte; O'Toole, Edel A.; Ishida-Yamamoto, Akemi; Marches, Olivier; Getsios, Spiro; Kelsell, David P.

    2014-01-01

    iRHOM2 is a highly conserved, catalytically inactive member of the Rhomboid family, which has recently been shown to regulate the maturation of the multi-substrate ectodomain sheddase enzyme ADAM17 (TACE) in macrophages. Dominant iRHOM2 mutations are the cause of the inherited cutaneous and oesophageal cancer-susceptibility syndrome tylosis with oesophageal cancer (TOC), suggesting a role for this protein in epithelial cells. Here, using tissues derived from TOC patients, we demonstrate that TOC-associated mutations in iRHOM2 cause an increase in the maturation and activity of ADAM17 in epidermal keratinocytes, resulting in significantly upregulated shedding of ADAM17 substrates, including EGF-family growth factors and pro-inflammatory cytokines. This activity is accompanied by increased EGFR activity, increased desmosome processing and the presence of immature epidermal desmosomes, upregulated epidermal transglutaminase activity and heightened resistance to Staphylococcal infection in TOC keratinocytes. Many of these features are consistent with the presence of a constitutive wound-healing-like phenotype in TOC epidermis, which may shed light on a novel pathway in skin repair, regeneration and inflammation. PMID:24643277

  18. Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs. Role of the alpha(6) integrin subunit.

    PubMed

    Bigler, D; Takahashi, Y; Chen, M S; Almeida, E A; Osbourne, L; White, J M

    2000-04-21

    Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin beta) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D+C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D+C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 microM, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (approximately 75 versus approximately 55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfunction-blocking anti-alpha(6) monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2.

  19. ADAM17 substrate release in proximal tubule drives kidney fibrosis

    PubMed Central

    Kefaloyianni, Eirini; Muthu, Muthu Lakshmi; Kaeppler, Jakob; Sun, Xiaoming; Sabbisetti, Venkata; Chalaris, Athena; Rose-John, Stefan; Wong, Eitan; Sagi, Irit; Waikar, Sushrut S.; Rennke, Helmut; Bonventre, Joseph V.

    2016-01-01

    Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates’ receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor–treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis. PMID:27642633

  20. The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

    PubMed Central

    Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

    2015-01-01

    ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

  1. The extracellular metalloprotease AdamTS-A anchors neural lineages in place within and preserves the architecture of the central nervous system.

    PubMed

    Skeath, James B; Wilson, Beth A; Romero, Selena E; Snee, Mark J; Zhu, Yi; Lacin, Haluk

    2017-09-01

    The extracellular matrix (ECM) regulates cell migration and sculpts organ shape. AdamTS proteins are extracellular metalloproteases known to modify ECM proteins and promote cell migration, but demonstrated roles for AdamTS proteins in regulating CNS structure and ensuring cell lineages remain fixed in place have not been uncovered. Using forward genetic approaches in Drosophila, we find that reduction of AdamTS-A function induces both the mass exodus of neural lineages out of the CNS and drastic perturbations to CNS structure. Expressed and active in surface glia, AdamTS-A acts in parallel to perlecan and in opposition to viking/collagen IV and βPS-integrin to keep CNS lineages rooted in place and to preserve the structural integrity of the CNS. viking/collagen IV and βPS-integrin are known to promote tissue stiffness and oppose the function of perlecan, which reduces tissue stiffness. Our work supports a model in which AdamTS-A anchors cells in place and preserves CNS architecture by reducing tissue stiffness. © 2017. Published by The Company of Biologists Ltd.

  2. TACE (ADAM17) inhibits Schwann cell myelination.

    PubMed

    La Marca, Rosa; Cerri, Federica; Horiuchi, Keisuke; Bachi, Angela; Feltri, M Laura; Wrabetz, Lawrence; Blobel, Carl P; Quattrini, Angelo; Salzer, James L; Taveggia, Carla

    2011-06-12

    Tumor necrosis factor-α-converting enzyme (TACE; also known as ADAM17) is a proteolytic sheddase that is responsible for the cleavage of several membrane-bound molecules. We report that TACE cleaves neuregulin-1 (NRG1) type III in the epidermal growth factor domain, probably inactivating it (as assessed by deficient activation of the phosphatidylinositol-3-OH kinase pathway), and thereby negatively regulating peripheral nervous system (PNS) myelination. Lentivirus-mediated knockdown of TACE in vitro in dorsal root ganglia neurons accelerates the onset of myelination and results in hypermyelination. In agreement, motor neurons of conditional knockout mice lacking TACE specifically in these cells are significantly hypermyelinated, and small-caliber fibers are aberrantly myelinated. Further, reduced TACE activity rescues hypomyelination in NRG1 type III haploinsufficient mice in vivo. We also show that the inhibitory effect of TACE is neuron-autonomous, as Schwann cells lacking TACE elaborate myelin of normal thickness. Thus, TACE is a modulator of NRG1 type III activity and is a negative regulator of myelination in the PNS.

  3. "ADAM' or "EVE'?--a toxicological conundrum.

    PubMed

    Cox, D E; Williams, K R

    1996-01-12

    The 3,4-methylenedioxy ring-substituted amphetamines, including "ADAM' and "EVE', are currently popular drugs of abuse. Adverse reactions are reported in the clinical literature but few fatal cases are documented and little toxicological data is available to guide those determining the cause or manner of death in such cases. We report two deaths presenting in a similar manner and with similar clinical features. Various body fluid samples were analysed for amphetamines by gas chromatography/mass spectrometry. In one case, amphetamine alone was detected at levels of 1.54 mg/l and 1.47 mg/l in postmortem blood and admission serum, respectively. The other involved several 3,4-methylenedioxy ring-substituted amphetamines, namely MDA, MDMA and MDEA, at levels of 0.25 mg/l, 0.43 mg/l and 0.3 mg/l, respectively in postmortem femoral blood and 0.24 mg/l, 0.55 mg/l and 0.49 mg/l in admission blood. The interpretation of these toxicological results and some novel legal issues are discussed.

  4. Adam Politzer-Father of Modern Otology.

    PubMed

    Dhungat, J V Pai; Gore, Geeta

    2015-09-01

    Adam Politzer (1835-1920) was born in Alberti near the city of Budapest in Hungary. He studied medicine at the University of Vienna and obtained his Doctorate degree in 1859. Some of his teachers belonged to the famous second "Vienna School" such as Joseph Skoda, Karl Rokitansky, Von Hebra, Josef Hyrtil, Johann Von Oppolzer and famous physiologist Carl Ludwig -who took special interest in him and was influential in his subsequent career. Politzer showed unusual interest in diseases of the ear and started to work in Carl Ludwig's laboratory. His interest at that time was mainly the physics of the auditory system. He studied the innervations of the intrinsic muscles of the ear There he was the first to demonstrate that the innervations of the tensor tympani muscle was by trigeminal nerve and that of the stapedial muscle was by facial nerve. He studied the air movement in the Eustachian tube and variation of air pressure in the tympanic cavity by connecting two manometers- one placed in the external auditory canal meatus, and another in the pharynx. He showed valve near the opening into the middle ear which controls the process. It is usually closed to keep the bacteria and other things away from the mouth and nose.

  5. CRADA Final Report: Mucin Mimic and Glycopeptide Synthesis

    SciTech Connect

    Bertozzi, Carolyn R.

    2002-10-22

    Mucus has several constituents but the most important are the mucins, heavily O-glycosylated proteins characterized by long stretches of tandem repeat sequences rich in glycosylated serine and threonine residues, with N- and C-terminal domains that have determined to a large extent by the viscous and viscoelastic properties of mucin glycoproteins. Indeed, these properties are evident in reconstituted purified mucin glycoproteins. Oligomeric mucin can be deconstructed into its monomeric components and then further into the domains that comprise each mucin molecule. There are two major domain types. "Glycodomains" are defined by stretches of the tandemly repeated Thr/Ser-rich segments that bear the characteristic O-linked glycans of the mucin molecule. The goal of this project is to synthesize polymeric materials that mimic mucin glycodomains. In order to mimic the central features of mucin, these materials should have dense clusters of glycans that bear a similar structure to those found in native mucins, and a fairly rigid polymer backbone. Four different polymers bearing ketone groups for the attachment of sugars were synthesized. GalNAc{alpha}-ONH{sub 2} and Sia{alpha}2,6GaINAc{alpha}·ONH{sub 2} both of which could be ligated to the polymer scaffolds were synthesized. Mucin glycodomain mimics were successfully synthesized by ligation of glycans to polymers.

  6. Benign breast lesions that mimic malignancy.

    PubMed

    Torous, Vanda F; Schnitt, Stuart J; Collins, Laura C

    2017-02-01

    Many benign and reactive lesions of the breast show morphological overlap with malignant lesions. These benign mimics of malignancy often present diagnostic challenges to even the most experienced pathologists. This review focuses on several benign lesions of the breast that mimic malignant entities. For each of these lesions, we describe the key morphological and immunohistochemical features, potential diagnostic pitfalls, and our approach to arriving at the correct diagnosis. Copyright © 2016 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

  7. A-Disintegrin and Metalloprotease (ADAM) 10 and 17 promote self-renewal of brain tumor sphere forming cells.

    PubMed

    Bulstrode, Harry; Jones, Louise M; Siney, Elodie J; Sampson, Jessica M; Ludwig, Andreas; Gray, William P; Willaime-Morawek, Sandrine

    2012-12-29

    It has been proposed that gliomas contain a subpopulation of 'Brain Tumor Stem Cells' (BTSCs), which demonstrate resistance to conventional therapies. A potential component of the environment governing the behavior of these BTSCs is a class of transmembrane proteins with structural and signaling functions, the A-Disintegrin And Metalloproteases (ADAMs). In this study we confirm overexpression of ADAM10 and 17 in human glioma tissue compared to human controls, and especially in tumor sphere cultures thought to enrich for BTSCs. Inhibition of ADAM10/17 function impairs the growth of tumor spheres with evidence of depletion of the sphere forming cell population. This results from a combination of reduced proliferation, cell death and a switch of sphere-forming cells away from symmetric self-renewal division towards neuronal differentiation. A developing appreciation of the role of ADAMs in BTSC promises insights into pathophysiology and potential therapeutic avenues in this intractable group of tumors. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. A bivariate genome-wide association study identifies ADAM12 as a novel susceptibility gene for Kashin-Beck disease

    PubMed Central

    Hao, Jingcan; Wang, Wenyu; Wen, Yan; Xiao, Xiao; He, Awen; Guo, Xiong; Yang, Tielin; Liu, Xiaogang; Shen, Hui; Chen, Xiangding; Tian, Qing; Deng, Hong-Wen; Zhang, Feng

    2016-01-01

    Kashin-Beck disease (KBD) is a chronic osteoarthropathy, which manifests as joint deformities and growth retardation. Only a few genetic studies of growth retardation associated with the KBD have been carried out by now. In this study, we conducted a two-stage bivariate genome-wide association study (BGWAS) of the KBD using joint deformities and body height as study phenotypes, totally involving 2,417 study subjects. Articular cartilage specimens from 8 subjects were collected for immunohistochemistry. In the BGWAS, ADAM12 gene achieved the most significant association (rs1278300 p-value = 9.25 × 10−9) with the KBD. Replication study observed significant association signal at rs1278300 (p-value = 0.007) and rs1710287 (p-value = 0.002) of ADAM12 after Bonferroni correction. Immunohistochemistry revealed significantly decreased expression level of ADAM12 protein in the KBD articular cartilage (average positive chondrocyte rate = 47.59 ± 7.79%) compared to healthy articular cartilage (average positive chondrocyte rate = 64.73 ± 5.05%). Our results suggest that ADAM12 gene is a novel susceptibility gene underlying both joint destruction and growth retardation of the KBD. PMID:27545300

  9. A bivariate genome-wide association study identifies ADAM12 as a novel susceptibility gene for Kashin-Beck disease.

    PubMed

    Hao, Jingcan; Wang, Wenyu; Wen, Yan; Xiao, Xiao; He, Awen; Guo, Xiong; Yang, Tielin; Liu, Xiaogang; Shen, Hui; Chen, Xiangding; Tian, Qing; Deng, Hong-Wen; Zhang, Feng

    2016-08-22

    Kashin-Beck disease (KBD) is a chronic osteoarthropathy, which manifests as joint deformities and growth retardation. Only a few genetic studies of growth retardation associated with the KBD have been carried out by now. In this study, we conducted a two-stage bivariate genome-wide association study (BGWAS) of the KBD using joint deformities and body height as study phenotypes, totally involving 2,417 study subjects. Articular cartilage specimens from 8 subjects were collected for immunohistochemistry. In the BGWAS, ADAM12 gene achieved the most significant association (rs1278300 p-value = 9.25 × 10(-9)) with the KBD. Replication study observed significant association signal at rs1278300 (p-value = 0.007) and rs1710287 (p-value = 0.002) of ADAM12 after Bonferroni correction. Immunohistochemistry revealed significantly decreased expression level of ADAM12 protein in the KBD articular cartilage (average positive chondrocyte rate = 47.59 ± 7.79%) compared to healthy articular cartilage (average positive chondrocyte rate = 64.73 ± 5.05%). Our results suggest that ADAM12 gene is a novel susceptibility gene underlying both joint destruction and growth retardation of the KBD.

  10. ADAM-10 over-expression increases cortical synaptogenesis.

    PubMed

    Bell, Karen F S; Zheng, Luyu; Fahrenholz, Falk; Cuello, A Claudio

    2008-04-01

    Cortical cholinergic, glutamatergic and GABAergic terminals become upregulated during early stages of the transgenic amyloid pathology. Abundant evidence suggests that sAPP alpha, the product of the non-amyloidogenic alpha-secretase pathway, is neurotrophic both in vitro and when exogenously applied in vivo. The disintegrin metalloprotease ADAM-10 has been shown to have alpha-secretase activity in vivo. To determine whether sAPP alpha has an endogenous biological influence on cortical presynaptic boutons in vivo, we quantified cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities in either ADAM-10 moderate expressing (ADAM-10 mo) transgenic mice, which moderately overexpress ADAM-10, or age-matched non-transgenic controls. Both early and late ontogenic time points were investigated. ADAM-10 mo transgenic mice display significantly elevated cortical cholinergic, glutamatergic and GABAergic presynaptic bouton densities at the early time point (8 months). Only the cholinergic presynaptic bouton density remains significantly elevated in late-staged ADAM-10 mo transgenic animals (18 months). To confirm that the observed elevations were due to increased levels of endogenous murine sAPP alpha, exogenous human sAPP alpha was infused into the cortex of non-transgenic control animals for 1 week. Exogenous infusion of sAPP alpha led to significant elevations in the cholinergic, glutamatergic and GABAergic cortical presynaptic bouton populations. These results are the first to demonstrate an in vivo influence of ADAM-10 on neurotransmitter-specific cortical synaptic plasticity and further confirm the neurotrophic influence of sAPP alpha on cortical synaptogenesis.

  11. Taking Charge: Walter Sydney Adams and the Mount Wilson Observatory

    NASA Astrophysics Data System (ADS)

    Brashear, R.

    2004-12-01

    The growing preeminence of American observational astronomy in the first half of the 20th century is a well-known story and much credit is given to George Ellery Hale and his skill as an observatory-building entrepreneur. But a key figure who has yet to be discussed in great detail is Walter Sydney Adams (1876-1956), Hale's Assistant Director at Mount Wilson Observatory. Due to Hale's illnesses, Adams was Acting Director for much of Hale's tenure, and he became the second Director of Mount Wilson from 1923 to 1946. Behind his New England reserve Adams was instrumental in the growth of Mount Wilson and thus American astronomy in general. Adams was hand-picked by Hale to take charge of stellar spectroscopy work at Yerkes and Mount Wilson and the younger astronomer showed tremendous loyalty to Hale and Hale's vision throughout his career. As Adams assumed the leadership role at Mount Wilson he concentrated on making the observatory a place where researchers worked with great freedom but maintain a high level of cooperation. This paper will concentrate on Adams's early years and look at his growing relationship with Hale and how he came to be the central figure in the early history of Mount Wilson as both a solar and stellar observatory. His education, his years at Dartmouth and Yerkes (including his unfortunate encounter with epsilon Leonis), and his formative years on Mount Wilson are all important in learning how he shaped the direction of Mount Wilson and the development of American astronomy in the first half of the 20th century. This latter history cannot be complete until we bring Adams into better focus.

  12. Rapamycin promotes β-amyloid production via ADAM-10 inhibition

    PubMed Central

    Zhang, Sheqing; Salemi, Jon; Hou, Huayan; Zhu, Yuyan; Mori, Takashi; Giunta, Brian; Obregon, Demian; Tan, Jun

    2010-01-01

    Rapamycin is a well known immunosuppressant drug for rejection prevention in organ transplantation. Numerous clinical trials using rapamycin analogs, involving both children and adults with various disorders are currently ongoing worldwide. Most recently, rapamycin gained much attention for what appears to be life-span extending properties when administered to mice. The risk for Alzheimer disease (AD) is strongly and positively correlated with advancing age and is characterized by deposition of β-amyloid peptides (Aβ) as senile plaques in the brain. We report that rapamycin (2.5 μM), significantly increases Aβ generation in murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP). In concert with these observations, we found rapamycin significantly decreases the neuroprotective amino-terminal APP (amyloid precursor protein) cleavage product, soluble APP-α (sAPP-α) while increasing production of the β-carboxyl-terminal fragment of APP (β-CTF). These cleavage events are associated with decreased activation of a disintegrin and metallopeptidase domain-10 (ADAM-10), an important candidate α-secretase which opposes Aβ generation. To validate these findings in vivo, we intraperitoneal (i.p.) injected Tg2576 Aβ-overproducing transgenic mice with rapamycin (3 mg/kg/day) for 2 weeks. We found increased Aβ levels associated with decreased sAPP-α at an average rapamycin plasma concentration of 169.7 ± 23.5 ng/mL by high performance liquid chromatography (HPLC). These data suggest that although rapamycin may increase the lifespan in some mouse models, it may not decrease the risk for age-associated neurodegenerative disorders such as AD. PMID:20542014

  13. MIMIC Methods for Assessing Differential Item Functioning in Polytomous Items

    ERIC Educational Resources Information Center

    Wang, Wen-Chung; Shih, Ching-Lin

    2010-01-01

    Three multiple indicators-multiple causes (MIMIC) methods, namely, the standard MIMIC method (M-ST), the MIMIC method with scale purification (M-SP), and the MIMIC method with a pure anchor (M-PA), were developed to assess differential item functioning (DIF) in polytomous items. In a series of simulations, it appeared that all three methods…

  14. Reliability evaluation of I-123 ADAM SPECT imaging using SPM software and AAL ROI methods

    NASA Astrophysics Data System (ADS)

    Yang, Bang-Hung; Tsai, Sung-Yi; Wang, Shyh-Jen; Su, Tung-Ping; Chou, Yuan-Hwa; Chen, Chia-Chieh; Chen, Jyh-Cheng

    2011-08-01

    The level of serotonin was regulated by serotonin transporter (SERT), which is a decisive protein in regulation of serotonin neurotransmission system. Many psychiatric disorders and therapies were also related to concentration of cerebral serotonin. I-123 ADAM was the novel radiopharmaceutical to image SERT in brain. The aim of this study was to measure reliability of SERT densities of healthy volunteers by automated anatomical labeling (AAL) method. Furthermore, we also used statistic parametric mapping (SPM) on a voxel by voxel analysis to find difference of cortex between test and retest of I-123 ADAM single photon emission computed tomography (SPECT) images.Twenty-one healthy volunteers were scanned twice with SPECT at 4 h after intravenous administration of 185 MBq of 123I-ADAM. The image matrix size was 128×128 and pixel size was 3.9 mm. All images were obtained through filtered back-projection (FBP) reconstruction algorithm. Region of interest (ROI) definition was performed based on the AAL brain template in PMOD version 2.95 software package. ROI demarcations were placed on midbrain, pons, striatum, and cerebellum. All images were spatially normalized to the SPECT MNI (Montreal Neurological Institute) templates supplied with SPM2. And each image was transformed into standard stereotactic space, which was matched to the Talairach and Tournoux atlas. Then differences across scans were statistically estimated on a voxel by voxel analysis using paired t-test (population main effect: 2 cond's, 1 scan/cond.), which was applied to compare concentration of SERT between the test and retest cerebral scans.The average of specific uptake ratio (SUR: target/cerebellum-1) of 123I-ADAM binding to SERT in midbrain was 1.78±0.27, pons was 1.21±0.53, and striatum was 0.79±0.13. The cronbach's α of intra-class correlation coefficient (ICC) was 0.92. Besides, there was also no significant statistical finding in cerebral area using SPM2 analysis. This finding might help us

  15. Tough Ceramic Mimics Mother of Pearl

    ScienceCinema

    Ritchie, Robert

    2016-07-12

    Berkeley Lab scientists have mimicked the structure of mother of pearl to create what may well be the toughest ceramic ever produced. http://newscenter.lbl.gov/press-releases/2008/12/05/scientists-create-tough-ceramic-that-mimics-mother-of-pearl/

  16. Speciation: frog mimics prefer their own.

    PubMed

    Mallet, James

    2014-11-17

    Ranitomeya poison frogs in the Peruvian Amazon are a rare example of Müllerian mimicry in vertebrates. These frogs also prefer to court same-coloured mimics. This suggests that divergence in mimicry plays a role in reproductive isolation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Tough Ceramic Mimics Mother of Pearl

    SciTech Connect

    Ritchie, Robert

    2009-01-01

    Berkeley Lab scientists have mimicked the structure of mother of pearl to create what may well be the toughest ceramic ever produced. http://newscenter.lbl.gov/press-releases/2008/12/05/scientists-create-tough-ceramic-that-mimics-mother-of-pearl/

  18. Essential Role for ADAM19 in Cardiovascular Morphogenesis

    PubMed Central

    Zhou, Hong-Ming; Weskamp, Gisela; Chesneau, Valérie; Sahin, Umut; Vortkamp, Andrea; Horiuchi, Keisuke; Chiusaroli, Riccardo; Hahn, Rebecca; Wilkes, David; Fisher, Peter; Baron, Roland; Manova, Katia; Basson, Craig T.; Hempstead, Barbara; Blobel, Carl P.

    2004-01-01

    Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19−/− animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects. PMID:14673146

  19. ADAM12: a genetic modifier of preclinical peripheral arterial disease

    PubMed Central

    Chen, Lingdan; Okutsu, Mitsuharu; Farber, Charles R.; Hazarika, Surovi; Jones, W. Schuyler; Craig, Damian; Marchuk, Douglas A.; Lye, R. John; Shah, Svati H.; Annex, Brian H.

    2015-01-01

    In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2. PMID:26163448

  20. Phosphatidylserine exposure is required for ADAM17 sheddase function

    PubMed Central

    Sommer, Anselm; Kordowski, Felix; Büch, Joscha; Maretzky, Thorsten; Evers, Astrid; Andrä, Jörg; Düsterhöft, Stefan; Michalek, Matthias; Lorenzen, Inken; Somasundaram, Prasath; Tholey, Andreas; Sönnichsen, Frank D.; Kunzelmann, Karl; Heinbockel, Lena; Nehls, Christian; Gutsmann, Thomas; Grötzinger, Joachim; Bhakdi, Sucharit; Reiss, Karina

    2016-01-01

    ADAM17, a prominent member of the ‘Disintegrin and Metalloproteinase' (ADAM) family, controls vital cellular functions through cleavage of transmembrane substrates. Here we present evidence that surface exposure of phosphatidylserine (PS) is pivotal for ADAM17 to exert sheddase activity. PS exposure is tightly coupled to substrate shedding provoked by diverse ADAM17 activators. PS dependency is demonstrated in the following: (a) in Raji cells undergoing apoptosis; (b) in mutant PSA-3 cells with manipulatable PS content; and (c) in Scott syndrome lymphocytes genetically defunct in their capacity to externalize PS in response to intracellular Ca2+ elevation. Soluble phosphorylserine but not phosphorylcholine inhibits substrate cleavage. The isolated membrane proximal domain (MPD) of ADAM17 binds to PS but not to phosphatidylcholine liposomes. A cationic PS-binding motif is identified in this domain, replacement of which abrogates liposome-binding and renders the protease incapable of cleaving its substrates in cells. We speculate that surface-exposed PS directs the protease to its targets where it then executes its shedding function. PMID:27161080

  1. ADAM12: a genetic modifier of preclinical peripheral arterial disease.

    PubMed

    Dokun, Ayotunde O; Chen, Lingdan; Okutsu, Mitsuharu; Farber, Charles R; Hazarika, Surovi; Jones, W Schuyler; Craig, Damian; Marchuk, Douglas A; Lye, R John; Shah, Svati H; Annex, Brian H

    2015-09-01

    In prior studies from multiple groups, outcomes following experimental peripheral arterial disease (PAD) differed considerably across inbred mouse strains. Similarly, in humans with PAD, disease outcomes differ, even when there are similarities in risk factors, disease anatomy, arteriosclerotic burden, and hemodynamic measures. Previously, we identified a locus on mouse chromosome 7, limb salvage-associated quantitative trait locus 1 (LSq-1), which was sufficient to modify outcomes following experimental PAD. We compared expression of genes within LSq-1 in Balb/c mice, which normally show poor outcomes following experimental PAD, with that in C57Bl/6 mice, which normally show favorable outcomes, and found that a disintegrin and metalloproteinase gene 12 (ADAM12) had the most differential expression. Augmentation of ADAM12 expression in vivo improved outcomes following experimental PAD in Balb/c mice, whereas knockdown of ADAM12 made outcomes worse in C57Bl/6 mice. In vitro, ADAM12 expression modulates endothelial cell proliferation, survival, and angiogenesis in ischemia, and this appeared to be dependent on tyrosine kinase with Ig-like and EGF-like domain 2 (Tie2) activation. ADAM12 is sufficient to modify PAD severity in mice, and this likely occurs through regulation of Tie2.

  2. Hybrid mimics and hybrid vigor in Arabidopsis

    PubMed Central

    Wang, Li; Greaves, Ian K.; Groszmann, Michael; Wu, Li Min; Dennis, Elizabeth S.; Peacock, W. James

    2015-01-01

    F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor that form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants that have a F1-like phenotype, we have by recurrent selection produced pure breeding F5/F6 lines, hybrid mimics, in which the characteristics of the F1 hybrid are stabilized. These hybrid mimic lines, like the F1 hybrid, have larger leaves than the parent plant, and the leaves have increased photosynthetic cell numbers, and in some lines, increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 hybrid with those of eight hybrid mimic lines identified metabolic pathways altered in both; these pathways include down-regulation of defense response pathways and altered abiotic response pathways. F6 hybrid mimic lines are mostly homozygous at each locus in the genome and yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of transregulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding hybrid mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture. PMID:26283378

  3. Hybrid mimics and hybrid vigor in Arabidopsis.

    PubMed

    Wang, Li; Greaves, Ian K; Groszmann, Michael; Wu, Li Min; Dennis, Elizabeth S; Peacock, W James

    2015-09-01

    F1 hybrids can outperform their parents in yield and vegetative biomass, features of hybrid vigor that form the basis of the hybrid seed industry. The yield advantage of the F1 is lost in the F2 and subsequent generations. In Arabidopsis, from F2 plants that have a F1-like phenotype, we have by recurrent selection produced pure breeding F5/F6 lines, hybrid mimics, in which the characteristics of the F1 hybrid are stabilized. These hybrid mimic lines, like the F1 hybrid, have larger leaves than the parent plant, and the leaves have increased photosynthetic cell numbers, and in some lines, increased size of cells, suggesting an increased supply of photosynthate. A comparison of the differentially expressed genes in the F1 hybrid with those of eight hybrid mimic lines identified metabolic pathways altered in both; these pathways include down-regulation of defense response pathways and altered abiotic response pathways. F6 hybrid mimic lines are mostly homozygous at each locus in the genome and yet retain the large F1-like phenotype. Many alleles in the F6 plants, when they are homozygous, have expression levels different to the level in the parent. We consider this altered expression to be a consequence of transregulation of genes from one parent by genes from the other parent. Transregulation could also arise from epigenetic modifications in the F1. The pure breeding hybrid mimics have been valuable in probing the mechanisms of hybrid vigor and may also prove to be useful hybrid vigor equivalents in agriculture.

  4. Identification of ADAM10 as a major source of HER2 ectodomain sheddase activity in HER2 overexpressing breast cancer cells.

    PubMed

    Liu, Phillip C C; Liu, Xiangdong; Li, Yanlong; Covington, Maryanne; Wynn, Richard; Huber, Reid; Hillman, Milton; Yang, Gengjie; Ellis, Dawn; Marando, Cindy; Katiyar, Kamna; Bradley, Jodi; Abremski, Kenneth; Stow, Mark; Rupar, Mark; Zhuo, Jincong; Li, Yun-Long; Lin, Qiyan; Burns, David; Xu, Meizhong; Zhang, Colin; Qian, Ding-Quan; He, Chunhong; Sharief, Vaqar; Weng, Lingkai; Agrios, Costas; Shi, Eric; Metcalf, Brian; Newton, Robert; Friedman, Steven; Yao, Wenqing; Scherle, Peggy; Hollis, Gregory; Burn, Timothy C

    2006-06-01

    Overexpression and activating mutations of ErbB family members have been implicated in the development and progression of a variety of tumor types. Cleavage of the HER2 receptor by an as yet unidentified ectodomain sheddase has been shown to liberate the HER2 extracellular domain (ECD) leaving a fragment with constitutive kinase activity that can provide ligand-independent growth and survival signals to the cell. This process is clinically relevant since HER2 ECD serum levels in metastatic breast cancer patients are associated with a poorer prognosis. Thus, inhibition of the HER2 sheddase may provide a novel therapeutic approach for breast cancer. We describe the use of transcriptional profiling, pharmacological and in vitro approaches to identify the major source of HER2 sheddase activity. Real-time PCR was used to identify those ADAM family members which were expressed in HER2 shedding cell lines. siRNAs that selectively inhibited ADAM10 expression reduced HER2 shedding. In addition, we profiled over 1000 small molecules for in vitro inhibition of a panel of ADAM and MMP proteins; a positive correlation was observed only between ADAM10 inhibition and reduction of HER2 ECD shedding in a cell based assay. Finally, in vitro studies demonstrate that in combination with low doses of Herceptin, selective ADAM10 inhibitors decrease proliferation in HER2 overexpressing cell lines while inhibitors, that do not inhibit ADAM10, have no impact. These results are consistent with ADAM10 being a major determinant of HER2 shedding, the inhibition of which, may provide a novel therapeutic approach for treating a variety of cancers with active HER2 signaling.

  5. ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function

    PubMed Central

    Cui, Dan; Arima, Mitsuru; Takubo, Keiyo; Kimura, Tokuhiro; Horiuchi, Keisuke; Minagawa, Takuya; Matsuda, Satoshi; Ikeda, Eiji

    2015-01-01

    Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases. PMID:26242473

  6. ADAM12 and ADAM17 are essential molecules for hypoxia-induced impairment of neural vascular barrier function.

    PubMed

    Cui, Dan; Arima, Mitsuru; Takubo, Keiyo; Kimura, Tokuhiro; Horiuchi, Keisuke; Minagawa, Takuya; Matsuda, Satoshi; Ikeda, Eiji

    2015-08-05

    Neural vascular barrier is essential for the life of multicellular organisms, and its impairment by tissue hypoxia is known to be a central of pathophysiology accelerating the progression of various intractable neural diseases. Therefore, the molecules involved in hypoxia-induced impairment of vascular barrier can be the targets to establish new therapies for intractable diseases. Here, we demonstrate that a disintegrin and metalloproteinases (ADAMs) 12 and 17 expressed in endothelial cells are the molecules responsible for the impairment of neural vascular barrier by hypoxia. Brain microvascular endothelial cells in vitro lost their barrier properties immediately after hypoxic stimulation through diminished localization of claudin-5, a tight junction molecule, on cell membranes. Hypoxic disappearance of claudin-5 from cell membranes and the consequent loss of barrier properties were completely suppressed by inhibition of the metalloproteinase activity which was found to be attributed to ADAM12 and ADAM17. Inhibition of either ADAM12 or ADAM17 was sufficient to rescue the in vivo neural vasculature under hypoxia from the loss of barrier function. This is the first report to specify the molecules which are responsible for hypoxia-induced impairment of neural vascular barrier and furthermore can be the targets of new therapeutic strategies for intractable neural diseases.

  7. The Adam language: Ada extended with support for multiway activities

    NASA Technical Reports Server (NTRS)

    Charlesworth, Arthur

    1993-01-01

    The Adam language is an extension of Ada that supports multiway activities, which are cooperative activities involving two or more processes. This support is provided by three new constructs: diva procedures, meet statements, and multiway accept statements. Diva procedures are recursive generic procedures having a particular restrictive syntax that facilitates translation for parallel computers. Meet statements and multiway accept statements provide two ways to express a multiway rendezvous, which is an n-way rendezvous generalizing Ada's 2-way rendezvous. While meet statements tend to have simpler rules than multiway accept statements, the latter approach is a more straightforward extension of Ada. The only nonnull statements permitted within meet statements and multiway accept statements are calls on instantiated diva procedures. A call on an instantiated diva procedure is also permitted outside a multiway rendezvous; thus sequential Adam programs using diva procedures can be written. Adam programs are translated into Ada programs appropriate for use on parallel computers.

  8. 75 FR 51519 - Regional Transportation District-Acquisition Exemption-Union Pacific Railroad Company in Adams...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-20

    ... Railroad Company in Adams, Denver, and Jefferson Counties, CO Regional Transportation District (RTD) \\1... Subdivision extending approximately 8.96 miles, from milepost 628.50, in Adams County, CO., to milepost 637.46...

  9. Jefferson and Adams on the mind-body problem.

    PubMed

    Robinson, Daniel N

    2003-08-01

    Amidst the voluminous correspondence between Thomas Jefferson and John Adams are several letters pertaining to the material basis of mental life. These reveal in a most suggestive way the substantial differences between them. Well informed on prevailing scientific and philosophical perspectives, Jefferson and Adams used the issue to express their positions on the nature and limits of knowledge, the relative authority of scientific methods and speculations, and the larger question of human perfectibility. At the same time, their exchanges illuminate the prevailing and divergent perspectives on human psychology adopted by major leaders of thought in the New World.

  10. The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

    PubMed

    Jefferson, Tamara; Auf dem Keller, Ulrich; Bellac, Caroline; Metz, Verena V; Broder, Claudia; Hedrich, Jana; Ohler, Anke; Maier, Wladislaw; Magdolen, Viktor; Sterchi, Erwin; Bond, Judith S; Jayakumar, Arumugam; Traupe, Heiko; Chalaris, Athena; Rose-John, Stefan; Pietrzik, Claus U; Postina, Rolf; Overall, Christopher M; Becker-Pauly, Christoph

    2013-01-01

    The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

  11. Single-Walled Carbon Nanotubes: Mimics of Biological Ion Channels.

    PubMed

    Amiri, Hasti; Shepard, Kenneth L; Nuckolls, Colin; Hernández Sánchez, Raúl

    2017-02-08

    Here we report on the ion conductance through individual, small diameter single-walled carbon nanotubes. We find that they are mimics of ion channels found in natural systems. We explore the factors governing the ion selectivity and permeation through single-walled carbon nanotubes by considering an electrostatic mechanism built around a simplified version of the Gouy-Chapman theory. We find that the single-walled carbon nanotubes preferentially transported cations and that the cation permeability is size-dependent. The ionic conductance increases as the absolute hydration enthalpy decreases for monovalent cations with similar solid-state radii, hydrated radii, and bulk mobility. Charge screening experiments using either the addition of cationic or anionic polymers, divalent metal cations, or changes in pH reveal the enormous impact of the negatively charged carboxylates at the entrance of the single-walled carbon nanotubes. These observations were modeled in the low-to-medium concentration range (0.1-2.0 M) by an electrostatic mechanism that mimics the behavior observed in many biological ion channel-forming proteins. Moreover, multi-ion conduction in the high concentration range (>2.0 M) further reinforces the similarity between single-walled carbon nanotubes and protein ion channels.

  12. Single-Walled Carbon Nanotubes: Mimics of Biological Ion Channels

    PubMed Central

    2017-01-01

    Here we report on the ion conductance through individual, small diameter single-walled carbon nanotubes. We find that they are mimics of ion channels found in natural systems. We explore the factors governing the ion selectivity and permeation through single-walled carbon nanotubes by considering an electrostatic mechanism built around a simplified version of the Gouy–Chapman theory. We find that the single-walled carbon nanotubes preferentially transported cations and that the cation permeability is size-dependent. The ionic conductance increases as the absolute hydration enthalpy decreases for monovalent cations with similar solid-state radii, hydrated radii, and bulk mobility. Charge screening experiments using either the addition of cationic or anionic polymers, divalent metal cations, or changes in pH reveal the enormous impact of the negatively charged carboxylates at the entrance of the single-walled carbon nanotubes. These observations were modeled in the low-to-medium concentration range (0.1–2.0 M) by an electrostatic mechanism that mimics the behavior observed in many biological ion channel-forming proteins. Moreover, multi-ion conduction in the high concentration range (>2.0 M) further reinforces the similarity between single-walled carbon nanotubes and protein ion channels. PMID:28103039

  13. Mimics and chameleons in motor neurone disease

    PubMed Central

    Turner, Martin R; Talbot, Kevin

    2013-01-01

    The progression of motor neurone disease (MND) is currently irreversible, and the grave implications of diagnosis naturally fuels concern among neurologists over missing a potential mimic disorder. There is no diagnostic test for MND but in reality there are few plausible mimics in routine clinical practice. In the presence of a progressive pure motor disorder, signs such as florid fasciculations, bilateral tongue wasting, the ‘split hand’, head drop, emotionality, and cognitive or behavioural impairment carry high positive predictive value. MND is clinically heterogeneous, however, with some important chameleon-like presentations and considerable variation in clinical course. Lack of confidence about the scope of such variation, or an approach to diagnosis emphasising investigations over clinical common sense, has the potential to exacerbate diagnostic delay in MND and impede timely planning of the care which is essential to maximising quality of life. PMID:23616620

  14. Mimics and chameleons in motor neurone disease.

    PubMed

    Turner, Martin R; Talbot, Kevin

    2013-06-01

    The progression of motor neurone disease (MND) is currently irreversible, and the grave implications of diagnosis naturally fuels concern among neurologists over missing a potential mimic disorder. There is no diagnostic test for MND but in reality there are few plausible mimics in routine clinical practice. In the presence of a progressive pure motor disorder, signs such as florid fasciculations, bilateral tongue wasting, the 'split hand', head drop, emotionality, and cognitive or behavioural impairment carry high positive predictive value. MND is clinically heterogeneous, however, with some important chameleon-like presentations and considerable variation in clinical course. Lack of confidence about the scope of such variation, or an approach to diagnosis emphasising investigations over clinical common sense, has the potential to exacerbate diagnostic delay in MND and impede timely planning of the care which is essential to maximising quality of life.

  15. Environmental mimics of chemical warfare agents.

    PubMed

    Claborn, David M

    2004-12-01

    There are several natural and artificial factors that mimic the effects of chemical warfare agents, thereby causing unwarranted alarm and confusion on the battlefield. Symptoms associated with chemical warfare include paralysis, muscle tremors, heavy salivation, severe burns, blistering, and corrosive skin injuries among others. Similar symptoms can be produced from a variety of environmental sources, artificial and natural. This article reviews several published and unpublished examples of environmental factors that produce syndromes similar to those caused by these agents. Examples of such mimics include pesticides, blistering exudates from insects and plants, various types of bites, and naturally occurring diseases. The potential for confusion caused by these factors is discussed and means of discriminating between warfare agents and naturally occurring events are identified. Recommendations for the use of this information and for needed research are also discussed.

  16. The Role of ADAM9 in Tumor-Stromal Interactions in Breast Cancer

    DTIC Science & Technology

    2009-04-01

    endogenously express ADAM9-L and –S (Fig 1 ). Year two research focused on using isoform specific antibodies for immunocytochemistry in preparation for...migration. As outlined in the task 2 update, we have shown that the different isoforms of ADAM9 have opposing effects on breast cancer cell migration...both isoforms of ADAM-9, and our future work in this area will further define the relevant signaling pathways which participate in ADAM9 mediated

  17. Malware Mimics for Network Security Assessment

    DTIC Science & Technology

    2011-03-01

    Department of Defense, the use of Red Teams results in the most effective, realistic, and comprehensive training for network administrators. Our thesis...complex malware mimic behaviors. 15. NUMBER OF PAGES 129 14. SUBJECT TERMS Malware, Red Team , Computer Network Defense Training, Network...within the Department of Defense, the use of Red Teams results in the most effective, realistic, and comprehensive training for network administrators

  18. Reproducing Natural Spider Silks’ Copolymer Behavior in Synthetic Silk Mimics

    PubMed Central

    An, Bo; Jenkins, Janelle E.; Sampath, Sujatha; Holland, Gregory P.; Hinman, Mike; Yarger, Jeffery L.; Lewis, Randolph

    2012-01-01

    Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia, indicates that MaSp1 proteins are more easily formed into β-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure. PMID:23110450

  19. Reproducing natural spider silks' copolymer behavior in synthetic silk mimics.

    PubMed

    An, Bo; Jenkins, Janelle E; Sampath, Sujatha; Holland, Gregory P; Hinman, Mike; Yarger, Jeffery L; Lewis, Randolph

    2012-12-10

    Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia , indicates that MaSp1 proteins are more easily formed into β-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure.

  20. Reproducing Natural Spider Silks' Copolymer Behavior in Synthetic Silk Mimics

    SciTech Connect

    An, Bo; Jenkins, Janelle E; Sampath, Sujatha; Holland, Gregory P; Hinman, Mike; Yarger, Jeffery L; Lewis, Randolph

    2012-10-30

    Dragline silk from orb-weaving spiders is a copolymer of two large proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2). The ratio of these proteins is known to have a large variation across different species of orb-weaving spiders. NMR results from gland material of two different species of spiders, N. clavipes and A. aurantia, indicates that MaSp1 proteins are more easily formed into β-sheet nanostructures, while MaSp2 proteins form random coil and helical structures. To test if this behavior of natural silk proteins could be reproduced by recombinantly produced spider silk mimic protein, recombinant MaSp1/MaSp2 mixed fibers as well as chimeric silk fibers from MaSp1 and MaSp2 sequences in a single protein were produced based on the variable ratio and conserved motifs of MaSp1 and MaSp2 in native silk fiber. Mechanical properties, solid-state NMR, and XRD results of tested synthetic fibers indicate the differing roles of MaSp1 and MaSp2 in the fiber and verify the importance of postspin stretching treatment in helping the fiber to form the proper spatial structure.

  1. EGCG functions through estrogen receptor-mediated activation of ADAM10 in the promotion of non-amyloidogenic processing of APP

    PubMed Central

    Fernandez, Jamie Winderbaum; Rezai-Zadeh, Kavon; Obregon, Demian; Tan, Jun

    2010-01-01

    Estrogen depletion following menopause has been correlated with an increased risk of developing Alzheimer’s disease (AD). We previously explored the beneficial effect of (-)-epigallocatechin-3-gallate (EGCG) on AD mice and found increased non-amyloidogenic processing of amyloid precursor protein (APP) through the α-secretase a-disintegrin-and-metallopeptidase-domain 10 (ADAM10). Our results in this study suggest that EGCG-mediated enhancement of non-amyloidogenic processing of APP is mediated by the maturation of ADAM10 via an estrogen receptor-α (ERα)/PI3K/Akt dependent mechanism, independent of furin-mediated ADAM10 activation. These data support prior assertions that central selective estrogen receptor modulation could be a therapeutic target for AD and support the use of EGCG as a well-tolerated alternative to estrogen therapy in the prophylaxis and treatment of this disease. PMID:20849853

  2. A study of the association between the ADAM12 and SH3PXD2A (SH3MD1) genes and Alzheimer's disease.

    PubMed

    Laumet, Geoffroy; Petitprez, Vincent; Sillaire, Adeline; Ayral, Anne-Marie; Hansmannel, Franck; Chapuis, Julien; Hannequin, Didier; Pasquier, Florence; Scarpini, Elio; Galimberti, Daniela; Lendon, Corinne; Campion, Dominique; Amouyel, Philippe; Lambert, Jean-Charles

    2010-01-01

    Several observations suggest that neurotoxicity in Alzheimer's disease (AD) can be partly attributed to beta-amyloid (Abeta) and senile plaques. Recent work has suggested that the FISH (five SH3 domains) adapter protein and ADAM12 (a disintegrin and metalloprotease) may mediate the neurotoxic effect of Abeta. Both genes are located on chromosome 10, within a region linked to AD (for SH3PXD2A) or nearby (for ADAM12). A recent study reported a statistically significant interaction between 2 variants of these genes (rs3740473 for SH3PXD2A and rs11244787 for ADAM12) with respect to the risk of developing AD. With a view to replicating this observation, we genotyped the two SNPs in four European case-control cohorts of Caucasian origin (1913 cases and 1468 controls) but were unable to confirm the initial results.

  3. Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

    PubMed Central

    Chalaris, Athena; Adam, Nina; Sina, Christian; Rosenstiel, Philip; Lehmann-Koch, Judith; Schirmacher, Peter; Hartmann, Dieter; Cichy, Joanna; Gavrilova, Olga; Schreiber, Stefan; Jostock, Thomas; Matthews, Vance; Häsler, Robert; Becker, Christoph; Neurath, Markus F.; Reiß, Karina; Saftig, Paul

    2010-01-01

    The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target. PMID:20603312

  4. Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.

    PubMed

    Chaturvedi, Kaveri S; Hung, Chia S; Giblin, Daryl E; Urushidani, Saki; Austin, Anthony M; Dinauer, Mary C; Henderson, Jeffrey P

    2014-02-21

    Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst.

  5. Cupric Yersiniabactin Is a Virulence-Associated Superoxide Dismutase Mimic

    PubMed Central

    2013-01-01

    Many Gram-negative bacteria interact with extracellular metal ions by expressing one or more siderophore types. Among these, the virulence-associated siderophore yersiniabactin (Ybt) is an avid copper chelator, forming stable cupric (Cu(II)-Ybt) complexes that are detectable in infected patients. Here we show that Ybt-expressing E. coli are protected from intracellular killing within copper-replete phagocytic cells. This survival advantage is highly dependent upon the phagocyte respiratory burst, during which superoxide is generated by the NADPH oxidase complex. Chemical fractionation links this phenotype to a previously unappreciated superoxide dismutase (SOD)-like activity of Cu(II)-Ybt. Unlike previously described synthetic copper-salicylate (Cu(II)-SA) SOD mimics, the salicylate-based natural product Cu(II)-Ybt retains catalytic activity at physiologically plausible protein concentrations. These results reveal a new virulence-associated adaptation based upon spontaneous assembly of a non-protein catalyst. PMID:24283977

  6. Selective Loss of Presynaptic Potassium Channel Clusters at the Cerebellar Basket Cell Terminal Pinceau in Adam11 Mutants Reveals Their Role in Ephaptic Control of Purkinje Cell Firing

    PubMed Central

    Kole, Matthew J.; Qian, Jing; Waase, Marc P.; Klassen, Tara L.; Chen, Tim T.; Augustine, George J.

    2015-01-01

    A specialized axonal ending, the basket cell “pinceau,” encapsulates the Purkinje cell axon initial segment (AIS), exerting final inhibitory control over the integrated outflow of the cerebellar cortex. This nonconventional axo-axonic contact extends beyond the perisomatic chemical GABAergic synaptic boutons to the distal AIS, lacks both sodium channels and local exocytotic machinery, and yet contains a dense cluster of voltage-gated potassium channels whose functional contribution is unknown. Here, we show that ADAM11, a transmembrane noncatalytic disintegrin, is the first reported Kv1-interacting protein essential for localizing Kv1.1 and Kv1.2 subunit complexes to the distal terminal. Selective absence of these channels at the pinceau due to mutation of ADAM11 spares spontaneous GABA release from basket cells at the perisomatic synapse yet eliminates ultrarapid ephaptic inhibitory synchronization of Purkinje cell firing. Our findings identify a critical role for presynaptic K+ channels at the pinceau in ephaptic control over the speed and stability of spike rate coding at the Purkinje cell AIS in mice. SIGNIFICANCE STATEMENT This study identifies ADAM11 as the first essential molecule for the proper localization of potassium ion channels at presynaptic nerve terminals, where they modulate excitability and the release of neural transmitters. Genetic truncation of the transmembrane disintegrin and metalloproteinase protein ADAM11 resulted in the absence of Kv1 channels that are normally densely clustered at the terminals of basket cell axons in the cerebellar cortex. These specialized terminals are responsible for the release of the neurotransmitter GABA onto Purkinje cells and also display electrical signaling. In the ADAM11 mutant, GABAergic release was not altered, but the ultrarapid electrical signal was absent, demonstrating that the dense presynaptic cluster of Kv1 ion channels at these terminals mediate electrical transmission. Therefore, ADAM11 plays a

  7. Adam Smith and the Moral Economy of the Classroom System.

    ERIC Educational Resources Information Center

    Hamilton, D.

    1980-01-01

    Traces the development of mass schooling to its origins in 19th-century Glasgow. Its importance as an intellectual and economic center enabled Glasgow to invent a solution to the problem of urban schooling, while the association of scholars like Adam Smith with Glasgow University made Scottish educational theories acceptable around the world. (DB)

  8. Adam Smith and the Teaching of English Literature.

    ERIC Educational Resources Information Center

    Court, Franklin E.

    1985-01-01

    Adam Smith used selections from English literature in his classroom during the eighteenth century because he believed that vernacular literature could provide a ready context for the teaching of ideological, social, and moral lessons. He believed that higher education should prepare students for the real business of the real world. (RM)

  9. Father Knows Best: Using Adam Smith to Teach Transactions Costs

    ERIC Educational Resources Information Center

    Dupont, Brandon

    2014-01-01

    Adam Smith's moral philosophy can be used to introduce economics students to the important idea of transactions costs. The author provides a brief background in this article to Smith's moral philosophy and connects it to the costs of transacting in a way that fits easily into the standard principles of microeconomics classroom. By doing…

  10. The ADaptation and Anticipation Model (ADAM) of sensorimotor synchronization.

    PubMed

    van der Steen, M C Marieke; Keller, Peter E

    2013-01-01

    A constantly changing environment requires precise yet flexible timing of movements. Sensorimotor synchronization (SMS)-the temporal coordination of an action with events in a predictable external rhythm-is a fundamental human skill that contributes to optimal sensory-motor control in daily life. A large body of research related to SMS has focused on adaptive error correction mechanisms that support the synchronization of periodic movements (e.g., finger taps) with events in regular pacing sequences. The results of recent studies additionally highlight the importance of anticipatory mechanisms that support temporal prediction in the context of SMS with sequences that contain tempo changes. To investigate the role of adaptation and anticipatory mechanisms in SMS we introduce ADAM: an ADaptation and Anticipation Model. ADAM combines reactive error correction processes (adaptation) with predictive temporal extrapolation processes (anticipation) inspired by the computational neuroscience concept of internal models. The combination of simulations and experimental manipulations based on ADAM creates a novel and promising approach for exploring adaptation and anticipation in SMS. The current paper describes the conceptual basis and architecture of ADAM.

  11. The ADaptation and Anticipation Model (ADAM) of sensorimotor synchronization

    PubMed Central

    van der Steen, M. C. (Marieke); Keller, Peter E.

    2013-01-01

    A constantly changing environment requires precise yet flexible timing of movements. Sensorimotor synchronization (SMS)—the temporal coordination of an action with events in a predictable external rhythm—is a fundamental human skill that contributes to optimal sensory-motor control in daily life. A large body of research related to SMS has focused on adaptive error correction mechanisms that support the synchronization of periodic movements (e.g., finger taps) with events in regular pacing sequences. The results of recent studies additionally highlight the importance of anticipatory mechanisms that support temporal prediction in the context of SMS with sequences that contain tempo changes. To investigate the role of adaptation and anticipatory mechanisms in SMS we introduce ADAM: an ADaptation and Anticipation Model. ADAM combines reactive error correction processes (adaptation) with predictive temporal extrapolation processes (anticipation) inspired by the computational neuroscience concept of internal models. The combination of simulations and experimental manipulations based on ADAM creates a novel and promising approach for exploring adaptation and anticipation in SMS. The current paper describes the conceptual basis and architecture of ADAM. PMID:23772211

  12. What Ever Happened to . . . John Adams High School?

    ERIC Educational Resources Information Center

    Doremus, Richard R.

    1981-01-01

    First in a series, this article describes the rise and fall of John Adams High School, an experimental school in Portland (Oregon) that was recently closed after 12 years of operation. The experiences of those who tried to make the experiment work may help others interested in educational innovation. (WD)

  13. "The Adams Chronicles" and the American History Survey

    ERIC Educational Resources Information Center

    Rollins, Richard M.

    1977-01-01

    Film documentaries can be a valuable addition to introductory American history courses. Using "The Adams Chronicles" as an example, the author identifies flaws in the film and then explains how careful planning and analysis of the video and printed programs enabled him to incorporate it effectively into a freshman-level survey course at Ohio State…

  14. 5. Aerial view west, Adams Dam Road bottom center, State ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    5. Aerial view west, Adams Dam Road bottom center, State Route 100 center, duck pond and reservoir center, State Route 100 center right, State Route 92 below center right, Brandywine Creek State Park center bottom. - Winterthur Farms, Intersection State Routes 92 & 100, Intersection State Routes 92 & 100, Winterthur, New Castle County, DE

  15. 4. Aerial view southwest, Adams Dam Road bottom left, State ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    4. Aerial view southwest, Adams Dam Road bottom left, State Route 100 center, back gates to Winterthur and Wilmington Country Club upper center, duck pond and reservoir bottom right and center, and State Route 92 center bottom. - Winterthur Farms, Intersection State Routes 92 & 100, Intersection State Routes 92 & 100, Winterthur, New Castle County, DE

  16. 3. Aerial view southeast, State Route 92 bottom left, Adams ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. Aerial view southeast, State Route 92 bottom left, Adams Dam Road center, Brandywine Creek State Park and J. Chandler Farm in center left, duck pond bottom right and reservoir bottom left. - Winterthur Farms, Intersection State Routes 92 & 100, Intersection State Routes 92 & 100, Winterthur, New Castle County, DE

  17. An Informal Report on Collegiate Successes with "The Adams Chronicles."

    ERIC Educational Resources Information Center

    Goldsberry, Gary G.

    In the spring of 1976, "The Adams Chronicles", a bicentennial television course developed by Coast Community College District and the University of California at San Diego, was distributed to colleges nationwide at no charge with the understanding that each college would return information regarding promotion, enrollment, and form of…