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2. Structure-Based Design of Xanthine-Imidazopyridines and -Imidazothiazoles as Highly Potent and In Vivo Efficacious Tryptophan Hydroxylase Inhibitors

Author

Specker, Edgar, primary, Wesolowski, Radoslaw, additional, Schütz, Anja, additional, Matthes, Susann, additional, Mallow, Keven, additional, Wasinska-Kalwa, Malgorzata, additional, Winkler, Lars, additional, Oder, Andreas, additional, Alenina, Natalia, additional, Pleimes, Dirk, additional, von Kries, Jens Peter, additional, Heinemann, Udo, additional, Bader, Michael, additional, and Nazaré, Marc, additional

Published
2023
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5. cCPE Fusion Proteins as Molecular Probes to Detect Claudins and Tight Junction Dysregulation in Gastrointestinal Cell Lines, Tissue Explants and Patient-Derived Organoids

Author

Waldow, Ayk, primary, Beier, Laura-Sophie, additional, Arndt, Janine, additional, Schallenberg, Simon, additional, Vollbrecht, Claudia, additional, Bischoff, Philip, additional, Farrera-Sal, Martí, additional, Loch, Florian N., additional, Bojarski, Christian, additional, Schumann, Michael, additional, Winkler, Lars, additional, Kamphues, Carsten, additional, Ehlen, Lukas, additional, and Piontek, Jörg, additional

Published
2023
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7. Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures

Author

Alcaniz, Joshua, primary, Winkler, Lars, additional, Dahlmann, Mathias, additional, Becker, Michael, additional, Orthmann, Andrea, additional, Haybaeck, Johannes, additional, Krassnig, Stefanie, additional, Skofler, Christina, additional, Kratzsch, Tobias, additional, Kuhn, Susanne A., additional, Jödicke, Andreas, additional, Linnebacher, Michael, additional, Fichtner, Iduna, additional, Walther, Wolfgang, additional, and Hoffmann, Jens, additional

Published
2023
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9. Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures

Author

Alcaniz, Joshua, Winkler, Lars, Dahlmann, Mathias, Becker, Michael, Orthmann, Andrea, Haybaeck, Johannes, Krassnig, Stefanie, Skofler, Christina, Kratzsch, Tobias, Kuhn, Susanne A., Jödicke, Andreas, Linnebacher, Michael, Fichtner, Iduna, Walther, Wolfgang, and Hoffmann, Jens

Subjects
Cancer Research, Oncology
Abstract

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

Published
2023

12. JCB904687 Supplemental Material2 - Supplemental material for Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

Author

Winkler, Lars, Blasig, Rosel, Breitkreuz-Korff, Olga, Berndt, Philipp, Dithmer, Sophie, Helms, Hans C, Puchkov, Dmytro, Kavi Devraj, Kaya, Mehmet, Zhihai Qin, Liebner, Stefan, Wolburg, Hartwig, Anuska V Andjelkovic, Rex, Andre, Blasig, Ingolf E, and Haseloff, Reiner F

Subjects
110320 Radiology and Organ Imaging, FOS: Clinical medicine, FOS: Biological sciences, Medicine, Cell Biology, 110305 Emergency Medicine, 110306 Endocrinology, Biochemistry, 69999 Biological Sciences not elsewhere classified, 110904 Neurology and Neuromuscular Diseases, Neuroscience
Abstract

Supplemental material, JCB904687 Supplemental Material2 for Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins by Lars Winkler, Rosel Blasig, Olga Breitkreuz-Korff, Philipp Berndt, Sophie Dithmer, Hans C Helms, Dmytro Puchkov, Kavi Devraj, Mehmet Kaya, Zhihai Qin, Stefan Liebner, Hartwig Wolburg, Anuska V Andjelkovic, Andre Rex, Ingolf E Blasig and Reiner F Haseloff in Journal of Cerebral Blood Flow & Metabolism

Published
2020
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14. Tight junctions in the blood–brain barrier promote edema formation and infarct size in stroke – Ambivalent effects of sealing proteins

Author

Winkler, Lars, primary, Blasig, Rosel, additional, Breitkreuz-Korff, Olga, additional, Berndt, Philipp, additional, Dithmer, Sophie, additional, Helms, Hans C, additional, Puchkov, Dmytro, additional, Devraj, Kavi, additional, Kaya, Mehmet, additional, Qin, Zhihai, additional, Liebner, Stefan, additional, Wolburg, Hartwig, additional, Andjelkovic, Anuska V, additional, Rex, Andre, additional, Blasig, Ingolf E, additional, and Haseloff, Reiner F, additional

Published
2020
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17. Claudin-12 deficiency causes nerve barrier breakdown, mechanical hypersensitivity and painfulness in polyneuropathy

Author

Chen, Jeremy Tsung-Chieh, primary, Hu, Xiawei, additional, Doppler, Kathrin, additional, Breitkreuz-Korff, Olga, additional, Otto, Isabel U. C., additional, Schwabe, Joachim, additional, Reinhold, Ann-Kristin, additional, Günzel, Dorothee, additional, Dithmer, Sophie, additional, Hankir, Mohammed K., additional, Fallier-Becker, Petra, additional, Winkler, Lars, additional, Blasig, Rosel, additional, Sommer, Claudia, additional, Brack, Alexander, additional, Blasig, Ingolf E., additional, and Rittner, Heike L., additional

Published
2019
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18. Abstracts from the 20th International Symposium on Signal Transduction at the Blood-Brain Barriers

Author

Małecki, Andrzej, primary, Skipor-Lahuta, Janina, additional, Toborek, Michal, additional, Abbott, N. Joan, additional, Antonetti, David A., additional, Su, Enming Joe, additional, Lawrence, Daniel A., additional, Atış, Müge, additional, Akcan, Uğur, additional, Yılmaz, Canan Uğur, additional, Orhan, Nurcan, additional, Düzgün, Poyraz, additional, Ceylan, Umut Deniz, additional, Arıcan, Nadir, additional, Karahüseyinoğlu, Serçin, additional, Şahin, Gizem Nur, additional, Ahıshalı, Bülent, additional, Kaya, Mehmet, additional, Aydin, Sidar, additional, Klopstein, Armelle, additional, Engelhardt, Britta, additional, Baumann, Julia, additional, Tsao, Chih-Chieh, additional, Huang, Sheng-Fu, additional, Ogunshola, Omolara, additional, Boytsova, Elizaveta B., additional, Morgun, Andrey V., additional, Khilazheva, Elena D., additional, Pozhilenkova, Elena A., additional, Gorina, Yana V., additional, Martynova, Galina P., additional, Salmina, Alla B., additional, Bueno, David, additional, Garcia-Fernàndez, Jordi, additional, Castro, Victor, additional, Skowronska, Marta, additional, Chupel, Matheus Uba, additional, Minuzzi, Luciele Guerra, additional, Filaire, Edith, additional, Teixeira, Ana Maria, additional, Corsi, Mariangela, additional, Versele, Romain, additional, Fuso, Andrea, additional, Sevin, Emmanuel, additional, Di Lorenzo, Cherubino, additional, Businaro, Rita, additional, Fenart, Laurence, additional, Gosselet, Fabien, additional, Candela, Pietra, additional, Deli, Mária A., additional, Delaney, Conor, additional, O’Keefe, Eoin, additional, Farrell, Michael, additional, Doyle, Sarah, additional, Campbell, Matthew, additional, Drewes, Lester R., additional, Appelt-Menzel, A., additional, Cubukova, A., additional, Metzger, M., additional, Fischer, R., additional, Francisco, David M. F., additional, Bruggmann, Rémy, additional, Fries, Alexa, additional, Blecharz, Kinga G., additional, Wagner, Josephin, additional, Winkler, Lars, additional, Schneider, Ulf, additional, Vajkoczy, Peter, additional, Furuse, Mikio, additional, Gabbert, Lydia, additional, Dilling, Christina, additional, Sisario, Dmitri, additional, Soukhoroukov, Vladimir, additional, Burek, Malgorzata, additional, Guérit, S., additional, Fidan, E., additional, Devraj, K., additional, Czupalla, C. J., additional, Macas, J., additional, Thom, S., additional, Plate, K. H., additional, Gerhardt, H., additional, Liebner, S., additional, Harazin, András, additional, Bocsik, Alexandra, additional, Váradi, Judit, additional, Fenyvesi, Ferenc, additional, Tubak, Vilmos, additional, Vecsernyés, Miklós, additional, Helms, Hans Christian, additional, Waagepetersen, Helle Sønderby, additional, Nielsen, Carsten Uhd, additional, Brodin, Birger, additional, Hoyk, Zsófia, additional, Tóth, Melinda E., additional, Lénárt, Nikolett, additional, Dukay, Brigitta, additional, Kittel, Ágnes, additional, Vígh, Judit, additional, Veszelka, Szilvia, additional, Walter, Fruzsina, additional, Zvara, Ágnes, additional, Puskás, László, additional, Sántha, Miklós, additional, Engelhardt, Sabrina, additional, Ogunshola, Omolara O., additional, Huber, Anna, additional, Reitner, Alexander, additional, Osmen, Samar, additional, Hahn, Kathrin, additional, Bounzina, Neli, additional, Gerhartl, Anna, additional, Schönegger, Anna, additional, Steinkellner, Hannes, additional, Laccone, Franco, additional, Neuhaus, Winfried, additional, Hudson, Natalie, additional, Celkova, Lucia, additional, Iltzsche, Anne, additional, Drndarski, Svetlana, additional, Begley, David J, additional, Janiurek, Mette Mathiesen, additional, Kucharz, Krzysztof, additional, Christoffersen, Christina, additional, Nielsen, Lars Bo, additional, Lauritzen, Martin, additional, Johnson, Rebecca H, additional, Kho, Dan T, additional, O’Carroll, Simon J, additional, Angel, Catherine E, additional, Graham, E. Scott, additional, Pereira, Jennifer, additional, Karali, Christina Simoglou, additional, Cheng, Vinton, additional, Zarghami, Niloufar, additional, Soto, Manuel Sarmiento, additional, Couch, Yvonne, additional, Anthony, Daniel C., additional, Sibson, Nicola R., additional, Kealy, John, additional, Keep, Richard F., additional, Routhe, Lisa J., additional, Xiang, Jianming, additional, Ye, Hong, additional, Hua, Ya, additional, Moos, Torben, additional, Xi, Guohua, additional, Kristensen, M., additional, Bach, A., additional, Strømgaard, K., additional, Kutuzov, Nikolay, additional, Lopes-Pinheiro, Melissa A., additional, Lim, Jamie, additional, Kamermans, Alwin, additional, van Horssen, Jack, additional, Unger, Wendy W.J., additional, Fontijn, Ruud, additional, de Vries, Helga E., additional, Majerova, Petra, additional, Garruto, Ralph M., additional, Marchetti, Luca, additional, Francisco, David, additional, Gruber, Isabelle, additional, Lyck, Ruth, additional, Mészáros, Mária, additional, Porkoláb, Gergő, additional, Kiss, Lóránd, additional, Pilbat, Ana-Maria, additional, Török, Zsolt, additional, Bozsó, Zsolt, additional, Fülöp, Lívia, additional, Michalicova, Alena, additional, Galba, Jaroslav, additional, Mihaljevic, Sandra, additional, Novak, Michal, additional, Kovac, Andrej, additional, Morofuji, Yoichi, additional, Fujimoto, Takashi, additional, Watanabe, Daisuke, additional, Nakagawa, Shinsuke, additional, Ujifuku, Kenta, additional, Horie, Nobutaka, additional, Izumo, Tsuyoshi, additional, Anda, Takeo, additional, Matsuo, Takayuki, additional, Niu, Fang, additional, Buch, Shilpa, additional, Nyúl-Tóth, Ádám, additional, Kozma, Mihály, additional, Nagyőszi, Péter, additional, Nagy, Krisztina, additional, Fazakas, Csilla, additional, Haskó, János, additional, Molnár, Kinga, additional, Farkas, Attila E., additional, Galajda, Péter, additional, Wilhelm, Imola, additional, Krizbai, István A., additional, Kelly, Eoin, additional, Wallace, Eugene, additional, Greene, Chris, additional, Hughes, Stephanie, additional, Doyle, Niamh, additional, Humphries, Marian M., additional, Grant, Gerald A., additional, Friedman, Alon, additional, Veksler, Ronel, additional, Molloy, Michael G., additional, Meaney, James F., additional, Pender, Niall, additional, Doherty, Colin P., additional, Park, Minseon, additional, Liskiewicz, Arkadiusz, additional, Przybyla, Marta, additional, Kasprowska-Liśkiewicz, Daniela, additional, Nowacka-Chmielewska, Marta, additional, Malecki, Andrzej, additional, Pombero, Ana, additional, Garcia-Lopez, Raquel, additional, Martinez-Morga, Marta, additional, Martinez, Salvador, additional, Prager, Ofer, additional, Solomon-Kamintsky, Lyna, additional, Schoknecht, Karl, additional, Bar-Klein, Guy, additional, Milikovsky, Dan, additional, Vazana, Udi, additional, Rosenbach, Dror, additional, Kovács, Richard, additional, Radak, Zsolt, additional, Rodríguez-Lorenzo, Sabela, additional, Bruggmann, Remy, additional, Kooij, Gijs, additional, de Vries, Helga E, additional, Oxana, Semyachkina-Glushkovskaya, additional, Denis, Bragin, additional, Elena, Vodovozova, additional, Anna, Alekseeva, additional, Alla, Salmina, additional, Vladimir, Salmin, additional, Andrey, Morgun, additional, Nataliya, Malinovskaya, additional, Elena, Khilazheva, additional, Elizaveta, Boytsova, additional, Alexander, Shirokov, additional, Nikita, Navolokin, additional, Alla, Bucharskaya, additional, Yirong, Yang, additional, Arkady, Abdurashitov, additional, Artem, Gekalyuk, additional, Mariya, Ulanova, additional, Anastasia, Shushunova, additional, Madina, Bodrova, additional, Artem, Sagatova, additional, Alexander, Khorovodov, additional, Esmat, Shareef Ali, additional, Valery, Pavlov, additional, Artem, Tuchin, additional, Jürgen, Kurths, additional, de Abreu, Marcelle Silva, additional, Calpena, Ana C., additional, Espina, Marta, additional, García, Maria Luisa, additional, Romero, Ignacio A., additional, Male, David, additional, Storck, Steffen, additional, Hartz, Anika, additional, Pahnke, Jens, additional, Surma, Claus U., additional, Surma, M., additional, Giżejewski, Z., additional, Zieliński, H., additional, Szczepkowska, Aleksandra, additional, Kowalewska, Marta, additional, Krawczynska, Agata, additional, Herman, Andrzej P., additional, Skipor, Janina, additional, Kachappilly, Nicole, additional, Veenstra, Mike, additional, Rivera, Rosiris Leon, additional, Williams, Dionna W., additional, Morgello, Susan, additional, Berman, Joan W., additional, Wyneken, Ursula, additional, Batiz, Luis Federico, additional, Temizyürek, Arzu, additional, Khodadust, Rouhollah, additional, Küçük, Mutlu, additional, Gürses, Candan, additional, Emik, Serkan, additional, Zielińska, Magdalena, additional, Obara-Michlewska, Marta, additional, Milewski, Krzysztof, additional, Skonieczna, Edyta, additional, Fręśko, Inez, additional, Neuwelt, Edward A., additional, Maria, Ana Raquel Santa, additional, Bras, Ana Rita, additional, Lipka, Dóra, additional, Valkai, Sándor, additional, Kincses, András, additional, Dér, András, additional, and Deli, Maria A., additional

Published
2017
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19. Role of interleukin-6 in the brain endothelial dysfunction after SAH

Author

Wagner, Josephin, Blecharz, Kinga G., Schneider, Ulf, Winkler, Lars, and Vajkoczy, Peter

Subjects
Blood- Brain Barrier, ddc: 610, Interleukin- 6, Subarachnoid hemorrhage, cardiovascular diseases, 610 Medical sciences, Medicine, nervous system diseases
Abstract

Objective: Until date, mechanisms underlying the intravascular inflammatory response post subarachnoid hemorrhage (SAH) remain unclear. Interleukin-6 (IL-6) has been implicated to play a crucial role in the inflammatory response post SAH. Here, we aimed to show the time-lapse of the expression and localization[for full text, please go to the a.m. URL], 66. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Published
2015

20. Continuity of the blood-brain barrier during subarachnoid hemorrhage in mice

Author

Blecharz, Kinga G., Wagner, Josephin, Schneider, Ulf, Winkler, Lars, and Vajkoczy, Peter

Subjects
subarachniod hemorrhage, endothelium, ddc: 610, cardiovascular diseases, 610 Medical sciences, Medicine, blood-brain barrier, nervous system diseases
Abstract

Objective: Despite decades of research, processes leading to the intravascular inflammatory response accompanying subarachnoid hemorrhage (SAH) remain elusive. The herein presented study focusses on investigating the extent and the progression of blood-brain barrier (BBB) disruption through the loss[for full text, please go to the a.m. URL], 65. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)

Published
2014

21. Redox Regulation of Cell Contacts by Tricellulin and Occludin: Redox-Sensitive Cysteine Sites in Tricellulin Regulate Both Tri- and Bicellular Junctions in Tissue Barriers as Shown in Hypoxia and Ischemia

Author

Cording, Jimmi, primary, Günther, Ramona, additional, Vigolo, Emilia, additional, Tscheik, Christian, additional, Winkler, Lars, additional, Schlattner, Isabella, additional, Lorenz, Dorothea, additional, Haseloff, Reiner F., additional, Schmidt-Ott, Kai M., additional, Wolburg, Hartwig, additional, and Blasig, Ingolf E., additional

Published
2015
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23. Die Bedeutung der zweiten extrazellulären Schleife von Claudin-3 und Claudin-5 für Claudin-Interaktionen und Tight junction-Strangbildung

Author

Winkler, Lars

Subjects
protein-protein interaction, tight junction, Clostridium perfringens enterotoxin, FRET, 500 Naturwissenschaften und Mathematik::570 Biowissenschaften, Biologie::570 Biowissenschaften, Biologie, Claudin
Abstract

Titel und Inhaltsverzeichnis 1. Einleitung 1 2. Material und Methode 21 3. Ergebnisse 50 4. Diskussion 92 5. Zusammenfassung 110 6. Summary 112 7. Literaturverzeichnis 114 8. Anhang 129, Epithelien und Endothelien bilden in multizellulären Organismen Barrieren, die für die Funktion dieser Organe essentiell sind, jedoch die Wirkstoffpermeation einschränken können. Um die freie Diffusion wasserlöslicher Moleküle durch die Zwischenräume von Epithel- und Endothelzellen zu begrenzen, ist der parazelluläre Spalt zwischen diesen Zellen durch Tight junctions (TJ) abgedichtet. TJs erscheinen in der Gefrierbruch-Elektronenmikroskopie als intramembranöse Netzwerke aus Strängen im apikalen Bereich der lateralen Plasmamembran. Strukturelles Rückgrat der TJs der besonders dichten Blut- Hirnschranke sind die Claudine (Cld) 3 und 5. Cld5 dichtet die TJs der Blut- Hirnschranke für Moleküle kleiner als 800 Da ab. Hypothese dieser Arbeit ist, dass die Strangbildung und der Verschluss des parazellulären Spalts auf homo- und heterophile cis\- und trans-Interaktionen der Claudine zurückzuführen ist, deren molekularer Interaktionsmechanismus bisher ungeklärt gewesen ist. Mit dieser Arbeit wurde eine direkte homophile Selbstassoziation der zweiten extrazellulären Schleife (2.EZS) der Claudine 3 und 5 mit Größenausschlusschromatographie nachgewiesen. Eine Beteiligung der 2.EZS an der trans-Interaktion, nicht jedoch an der cis-Interaktion, wurde durch systematische Aminosäuresubstitutionen der 2.EZS von Cld5 bewiesen. Zudem wurden molekulare Determinanten identifiziert, die in die trans-Interaktion involviert sind. Dazu wurden TJ-lose HEK-293-Zellen verwendet, in denen die homophile cis\- und trans-Interaktion sowie die Strangbildung von Cld5 frei vom Einfluss endogener Claudine untersucht wurde. Die Analyse der trans- Interaktionsfähigkeit mittels konfokaler Mikroskopie sowie die Ermittlung der subzellulären Lokalisation der Cld-Mutanten durch Zelloberflächen- Biotinylierung und konfokale Mikroskopie führten zu strukturellen Modellen eines Cld5-Monomers und eines Cld5-trans-Dimers. Dazu wurde auf die 2.EZS von Cld5 die sequenzhomologe Teilstruktur eines hypothetischen, bakteriellen Proteins (BB2244) übertragen. Die 2.EZS von Cld5 ist demnach eine Helix-turn- Helix-Struktur, deren turn-Konformation durch ein Netzwerk aus Wasserstoffbrücken, die Proline P150 und P153 sowie durch hydrophobe Seitenketteninteraktionen zwischen V152 und L160 stabilisiert wird. Nach dem Dimer-Modell trans-dimerisiert die 2.EZS durch eine Wechselwirkung von drei aromatischen Seitenketten (F147, Y148, Y158) zweier Schleifen miteinander. Eine potentielle Bindungsstelle der ebenfalls an der trans-Interaktion beteiligten Aminosäuren Q156 und E159 konnten bisher nicht identifiziert werden. Die Strukturmodelle lassen sich auf Claudine mit hoher Sequenzhomologie übertragen, die dadurch erstmals als klassische Claudine 1-10, 14, 15, 17 und 19 definiert werden. Weiterhin wurde das Zusammenspiel von Claudinen bei der Strangbildung hinsichtlich cis\- und trans-Interaktionen untersucht. Mutanten mit gestörter trans-Interaktion zeigten in der Gefrierbruch-Elektronenmikrospkopie eine stark verringerte Zahl intramembranöser Stränge, obwohl die cis-Interaktion unbeeinträchtigt blieb. Es wird ein Schema postuliert, das die Morphologie von Claudin-5-gebildeten diskontinuierlichen Strängen erklärt. Die Partikelkomplexe der diskontinuierlichen Stränge von Cld5 müssen demnach mit den Partikelkomplexen einer benachbarten Zelletrans-interagieren, um TJ- Stränge zu bilden. Bei den Partikelkomplexen handelt es sich möglicherweise um Cld5-Hexamere. Ein weiterer Aspekt der Arbeit ist die Analyse der Wechselwirkungen zwischen der 2.EZS von Claudinen und Clostridium perfringens Enterotoxin (CPE). Durch die Analyse von membrangebundene Peptidbibliotheken wurde die Interaktion von CPE mit Peptiden der 2.EZS der Claudine 3, 6, 7, 9 und 14, nicht jedoch der Claudine 1, 2, 4, 5, 6, 8, 10-13, 15, 18, 19, 20 und 22 aufgezeigt. Substitutionsanalysen mit membrangebundenen Peptidbibliotheken identifizierten das turn-Motiv NPLVP als essentiell für die Interaktion zwischen CPE und den isolierten 2.EZS von Claudinen. Im Gegensatz dazu scheint das turn-Motiv für die Interaktion von CPE mit nativen Cld-Molekülen auf der Zelloberfläche von Cld-exprimierenden Zellen ohne Bedeutung zu sein. Das deutet darauf hin, dass die bindungsrelevante Struktur der 2.EZS von Cld5 stark von der Membrantopologie der Claudine abhängt. Durch CPE-Bindungsstudien an transfizierten HEK-293-Zellen wurden zwei bindungsrelevante Aminosäuresubstitutionen, T151A und Q156E, in der 2.EZS von Cld5 identifiziert. Weiterhin wurde gezeigt, dass CPE an Cld-Moleküle außerhalb der TJs von Cld3- bzw. Cld5-exprimierenden HEK-293-Zellen bindet. Die CPE- induzierte Öffnung der TJs durch eine Störung des Polymerisations -/Depolymerisations-Gleichgewichts der Cld-Stränge ist daher wahrscheinlich. Insgesamt tragen die durchgeführten Untersuchungen zu einem besseren Verständnis der TJ-Struktur bei. Zudem ermöglicht die Charakterisierung der CPE-Cld- bzw. Cld-Cld-Interaktion die Entwicklung von Peptiden oder niedermolekularen Substanzen, die gezielt TJs modulieren, um so z. B. die Wirkstoffzufuhr ins Gehirn zu verbessern., In multicellular organisms, epithelia and endothelia form barriers to maintain the function of organs, but also to prevent the passage of drugs. To prevent diffusion of water soluble molecules through the paracellular pathway, the intercellular space between adjacent epithelial or endothelial cells is sealed by tight junctions (TJ). TJs appear in freeze-fracture replicas as intramembranous networks of strands on the apical end of the lateral surface of the cells. Structural backbone of the TJs in the specifically tight blood- brain barrier are the claudins (cld) 3 and 5. Cld5 is sealing the TJs of the blood-brain barrier for molecules

Published
2007

26. Sodium caprate transiently opens claudin-5-containing barriers at tight junctions of epithelial and endothelial cells

Author

Del Vecchio, Giovanna, Tscheik, Christian, Tenz, Kareen, Helms, Hans Christian, Winkler, Lars, Blasig, Rosel, Blasig, Ingolf, Del Vecchio, Giovanna, Tscheik, Christian, Tenz, Kareen, Helms, Hans Christian, Winkler, Lars, Blasig, Rosel, and Blasig, Ingolf

Abstract

Claudin-5 is a tight junction (TJ) protein which limits the diffusion of small hydrophilic molecules. Thus, it represents a potential pharmacological target to improve drug delivery to the tissues protected by claudin-5-dependent barriers. Sodium caprate is known as an absorption enhancer which opens the paracellular space acting on TJ proteins and actin cytoskeleton. Its action on claudin-5 is not understood so far. Epithelial and endothelial systems were used to evaluate the effect of caprate on claudin-5 in TJ-free cells and on claudin-5 fully integrated in TJ. To this aim, confocal microscopy on live and fixed cells and isolated mouse brain capillaries, Western blotting and permeability assays were employed. Caprate reversibly reduced claudin-5 trans-interactions in TJ-free human embryonic kidney-293 cells expressing claudin-5-YFP. It decreased the membranous claudin-5 and the F-actin content in Madin–Darby canine kidney-II cells expressing Flag-claudin-5, thereby increasing the permeability to the small molecule lucifer yellow. Interestingly, zonula occludens protein 1 (ZO-1), which links transmembranous TJ proteins to the actin cytoskeleton, was not affected by caprate treatment. Similarly, endogenous claudin-5 in the membrane of brain endothelia was displaced together with F-actin, whereas ZO-1 remained unaffected. Caprate transiently opens the paracellular space, reducing the intercellular claudin-5/claudin-5 interactions and the polymerized actin at the perijunctional region of endothelial and epithelial cells. In conclusion, the study further elucidates the cellular effects of caprate at the tight junctions.

Published
2012

30. Structure and function of claudins

Author

Krause, Gerd, primary, Winkler, Lars, additional, Mueller, Sebastian L., additional, Haseloff, Reiner F., additional, Piontek, Jörg, additional, and Blasig, Ingolf E., additional

Published
2008
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34. Visualization of a Covalent Intermediate between Microsomal Epoxide Hydrolase, but not Cholesterol Epoxide Hydrolase, and their Substrates

Author

Muller, Frank, primary, Arand, Michael, additional, Frank, Heinz, additional, Seidel, Albrecht, additional, Hinz, Willy, additional, Winkler, Lars, additional, Hanel, Karen, additional, Blee, Elizabeth, additional, Beetham, Jeffrey K., additional, Hammock, Bruce D., additional, and Oesch, Franz, additional

Published
1997
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36. Formation of tight junction: determinants of homophilic interaction between classic claudins.

Author

Piontek, Jög, Winkler, Lars, Wolburg, Hartwig, Müller, Sebastian L., Zuleger, Nikolaj, Piehl, Christian, Wiesner, Burkhard, Krause, Gerd, and Blasig, Ingolf E.

Subjects
*TIGHT junctions, *MEMBRANE proteins, *PROTEIN-protein interactions, *GENETIC mutation, *POLYMERIZATION, *CELL membranes, *HOMOLOGY (Biology)
Abstract

Claudins are the critical transmembrane proteins in tight junctions. Claudin-5, for instance, prevents paracellular permeation of small molecules. However, the molecular interaction mechanism is unknown. Hence, the claudin-claudin interaction and tight junction strand formation were investigated using systematic single mutations. Claudin-5 mutants transfected into tight junction-free cells demonstrated that the extracellular loop 2 is involved in strand formation via trans-interaction, but not via polymerization, along the plasma membrane of one cell. Three phenotypes were obtained: the tight junction type (wild-type-like trans- and cis-interaction; the disjunction type (blocked trans-interaction); the intracellular type (disturbed folding). Combining site-directed mutagenesis, live-cell imaging-, electron microscopy-, and molecular modeling data led to an antiparallel homodimer homology model of the loop. These data for the first time explain how two claudins hold onto each other and constrict the paracellular space. The intermolecular interface includes aromatic (F147, Y148, Y158) and hydrophilic (Q156, E159) residues. The aromatic residues form a strong binding core between two loops from opposing cells. Since nearly all these residues are conserved in most claudins, our findings are of general relevance for all classical claudins. On the basis of the data we have established a novel molecular concept for tight junction formation. [ABSTRACT FROM AUTHOR]

Published
2008
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