Nutzung von Nukleinsäure-Protein-Wechselwirkungen für die Wirkanalytik von reaktiven Species

Reaktive Species wie Superoxid, Stickstoffmonoxid oder Peroxynitrit sind in eine Vielzahl pathophysiologischer Situationen wie z.B. das Reperfusionssyndrom involviert. Besonders hohe Konzentrationen treten auf, wenn das antioxidative Abwehrsystem nicht mehr in der Lage ist, den Radikalanstieg abzufangen. Die biomedizinische Forschung hat sich auf die Mechanismen der Freisetzung dieser Species unter unterschiedlichen Streßbedingungen fokussiert. Aus diesem Grund sind verschiedene analytische Meßmethoden für den Nachweis entwickelt worden. Sensorische Methoden bieten den Vorteil einer räumlich und zeitlich aufgelösten Analyse der Einzelteilchen. Jedoch gibt es beträchtliche Wechselwirkungen zwischen den verschiedenen reaktiven Species, was die Aussagefähigkeit von Einzelmessungen in komplexen Situationen einschränkt. Hier erscheint ein Summenparameter für die Radikalwirkung im Sinne einer Wirkanalytik aussagekräftiger. Das ‘iron regulatory protein 1’ (IRP1) kann als ein solches Markerprotein betrachtet werden, dessen Konzentration Aufschluß über den zellulären Streßlevel gibt. Das Protein wird unter der Wirkung von oxidativem Streß aus dem 4Fe-4S-Enzym cytosolische Aconitase gebildet. Das entstandene IRP1 zeigt im Gegensatz zur Aconitase eine ausgeprägte Bindungsaffinität zu spezifischen m-RNA-Strukturen - den sogenannten ‘iron responsive elements’ - IREs. Dieses Verhalten kann als Grundlage für die sensorische Detektion des Markerproteins genutzt werden. In dem hier vorzustellenden experimentellen Ansatz wurde die Proteinbindung mit Hilfe der Oberflächenplasmonresonanz detektiert. Hierzu wurde eine IRE-Konsensus-Sequenz in vitro transkribiert und anschließend auf einem Carboxydextran–modifizierten Biacore-Chip immobilisiert. Eine relativ hohe Oberflächenbelegung (2000 RU /mm2) wurde sichergestellt

Post-Translational Modifications Modulate Ligand Recognition by the Third PDZ Domain of the MAGUK Protein PSD-95

The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2–β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.This research was supported by grants CVI-05915, from the Andalusian Regional Government (http://www.juntadeandalucia.es), BIO2009-13261-C02 and BIO2012-39922-C02, from the Spanish Ministry of Science and Innovation (http://www.idi.mineco.gob.es/portal/site​/MICINN/) and FEDER. JMC received a postdoctoral contract from the Spanish Ministry of Science and Innovation. CCV was a recipient of a Formación de Personal Investigador fellowship from the Spanish Ministry of Science and Innovation

ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton

ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin

Формирование хранилища и анализ больших данных передвижений в городе

В статье рассмотрена концепция построения хранилища данных путей передвижения для интеллектуальной транспортной системы города. Приведен математический аппарат формализованного описания транспортной системы на основе гиперсети, дуги которой являются путями передвижения. Рассмотрен рекурсивный алгоритм получения данных о путях передвижения, а также механизм использования матриц смежности по маршрутным связям. Исследования проводятся в рамках проекта АР05133699 «Исследование и разработка инновационно-телекоммуникационных технологий с использованием современных кибертехнических средств для интеллектуальной транспортной системы города». The article discusses the concept of constructing a repository of data on travel routes for the intellectual transport system of the city. The mathematical apparatus of a formalized description of a transport system based on a hypernet, whose arcs are paths of movement, is given. A recursive algorithm for obtaining data on paths of movement, as well as a mechanism for using adjacency matrices for route connections, is considered. Research is carried out as part of the project AR05133699 “Research and development of innovative telecommunication technologies using modern cyber-technical means for the intellectual transport system of the city”

Tissue plasminogen activator enhances the hypoxia/reoxygenation-induced impairment of the blood-brain barrier in a primary culture of rat brain endothelial cells.

Hemorrhagic transformation is a major complication associated with tissue plasminogen activator (tPA) therapy for ischemic stroke. We studied the effect of tPA on the blood-brain barrier (BBB) function with our in vitro monolayer model generated using rat brain microvascular endothelial cells subjected either to normoxia or to hypoxia/reoxygenation (H/R) with or without the administration of tPA. The barrier function was evaluated by the transendothelial electrical resistance (TEER), the permeability of sodium fluorescein and Evans\u27 blue-albumin (EBA), and the uptake of lucifer yellow (LY). The permeability of sodium fluorescein and EBA was used as an index of paracellular and transcellular transport, respectively. The administration of tPA increased the permeability of EBA and the uptake of LY under normoxia. It enhanced the increase in the permeability of both sodium fluorescein and EBA, the decrease in the TEER, and the disruption in the expression of ZO-1 under H/R conditions. Administration of tPA could cause an increase in the transcellular transport under normoxia, and both the transcellular and paracellular transport of the BBB under H/R conditions in vitro. Even in humans, tPA may lead to an opening of the BBB under non-ischemic conditions and have an additional effect on the ischemia-induced BBB disruption.The original publication is available at www.springerlink.co

PDZ domains and their binding partners: structure, specificity, and modification

PDZ domains are abundant protein interaction modules that often recognize short amino acid motifs at the C-termini of target proteins. They regulate multiple biological processes such as transport, ion channel signaling, and other signal transduction systems. This review discusses the structural characterization of PDZ domains and the use of recently emerging technologies such as proteomic arrays and peptide libraries to study the binding properties of PDZ-mediated interactions. Regulatory mechanisms responsible for PDZ-mediated interactions, such as phosphorylation in the PDZ ligands or PDZ domains, are also discussed. A better understanding of PDZ protein-protein interaction networks and regulatory mechanisms will improve our knowledge of many cellular and biological processes

NO and oxyradical metabolism in new cell lines of rat brain capillary endothelial cells forming the blood–brain barrier

To investigate the relevance of •NO and oxyradicals in the blood–brain barrier (BBB), differentiated and well-proliferating brain capillary endothelial cells (BCEC) are required. Therefore, rat BCEC (rBCEC) were transfected with immortalizing genes. The resulting lines exhibited endothelial characteristics (factor VIII, angiotensin-converting enzyme, high prostacyclin/thromboxane release rates) and BBB markers (γ-glutamyl transpeptidase, alkaline phosphatase). The control line rBCEC2 (mock transfected) revealed fibroblastoid morphology, less factor VIII, reduced γ-glutamyl transpeptidase, weak radical defence, low prostanoid metabolism, and limited proliferation. Lines transfected with immortalizing genes (especially rBCEC4, polyoma virus large T antigen) conserved primary properties: epitheloid morphology, subcultivation with high proliferation rate under pure culture conditions, and powerful defence against reactive oxygen species (Mn–, Cu/Zn–superoxide dismutase, catalase, glutathione peroxidase, glutathione) effectively controlling radical metabolism. Only 100 μM H2O2 overcame this defence and stimulated the formation of eicosanoids similarly as in primary cells. Some BBB markers were expressed to a lower degree; however, cocultivation with astrocytes intensified these markers (e.g., alkaline phosphatase) and paraendothelial tightness, indicating induction of BBB properties. Inducible NO synthase was induced by a cytokine plus lipopolysaccharide mixture in all lines and primary cells, resulting in •NO release. Comparing the cell lines obtained, rBCEC4 are stable immortalized and reveal the best conservation of properties from primary cells, including enzymes producing or decomposing reactive species. These cells can be subcultivated in large amounts and, hence, they are suitable to study the role of radical metabolism in the BBB and in the cerebral microvasculature

Dynamic control of RSK complexes by phosphoswitch-based regulation

Assembly and disassembly of protein–protein complexes needs to be dynamically controlled and phosphoswitches based on linear motifs are crucial in this process. Extracellular signal–regulated kinase 2 (ERK2) recognizes a linear-binding motif at the C-terminal tail (CTT) of ribosomal S6 kinase 1 (RSK1), leading to phosphorylation and subsequent activation of RSK1. The CTT also contains a classical PDZ domain-binding motif which binds RSK substrates (e.g. MAGI-1). We show that autophosphorylation of the disordered CTT promotes the formation of an intramolecular charge clamp, which efficiently masks critical residues and indirectly hinders ERK binding. Thus, RSK1 CTT operates as an autoregulated phosphoswitch: its phosphorylation at specific sites affects its protein-binding capacity and its conformational dynamics. These biochemical feedbacks, which form the structural basis for the rapid dissociation of ERK2-RSK1 and RSK1-PDZ substrate complexes under sustained epidermal growth factor (EGF) stimulation, were structurally characterized and validated in living cells. Overall, conformational changes induced by phosphorylation in disordered regions of protein kinases, coupled to allosteric events occurring in the kinase domain cores, may provide mechanisms that contribute to the emergence of complex signaling activities. In addition, we show that phosphoswitches based on linear motifs can be functionally classified as ON and OFF protein–protein interaction switches or dimmers, depending on the specific positioning of phosphorylation target sites in relation to functional linear-binding motifs. Moreover, interaction of phosphorylated residues with positively charged residues in disordered regions is likely to be a common mechanism of phosphoregulation

The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties

The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins