transmembrane-domain-oligomerization-propensity-determined-toxr

Transmembrane Domain Oligomerization Propensity determined by ToxR Assay

The oversimplified view of protein transmembrane domains as merely anchors in phospholipid bilayers has long since been disproven.  In many cases ...

The bisindolylmaleimides are selective protein kinase inhibitors that can adopt two limiting diastereomeric (syn and anti) conformations. The configurational stability of a range of substituted and macrocyclic bisindolylmaleimides was investigated by using appropriate techniques. With unconstrained bisindolylmaleimides, the size of the 2-indolyl substituents was found to affect configurational stability, though not sufficiently to allow atropisomeric bisindolylmaleimides to be obtained. However, with a tether between the two indole nitrogen atoms in place, the steric effect of 2-indolyl substituents was greatly exaggerated, leading to large differences in configurational stability. The rate of interconversion of the syn and anti conformers varied by over twenty orders of magnitude through substitution of a bisindolylmaleimide ring system, which was constrained within a macrocyclic ring. Indeed, the first examples of configurationally stable atropisomeric bisindolylmaleimides are reported; the half-life for epimerisation of these compounds at room temperature was estimated to be >10(7) years.

Configurational stability of bisindolylmaleimide cyclophanes: from conformers to the first configurationally stable, atropisomeric bisindolylmaleimides.

Konfigurative Stabilit&#xE4;t von Bisindolylmaleimid Cyclophane: von Konformeren zu den ersten konfigurativ stabil, atropisomere bisindolylmaleimides.

The efficient synthesis of a range of stable SAM mimetics, and their ability to promote the binding of the E. coli methionine repressor (MetJ) to its operator DNA, is described.

Identification of stable S-adenosylmethionine (SAM) analogues derivatised with bioorthogonal tags: effect of ligands on the affinity of the E. coli methionine repressor, MetJ, for its operator DNA.

Die effiziente Synthese von eine Reihe von stabilen SAM Mimetics und ihre F&auml;higkeit zur F&ouml;rderung der Bindung von E. Coli Methionin Verdr&auml;nger (MetJ) zu seinem Betreiber DNA, wird beschrieben.

Identifizierung von stabilen S-Adenosylmethionine (SAM)-Analoga derivatisierte mit Bioorthogonal Tags: Wirkung der Liganden auf die Affinit&auml;t der E. Coli-Methionin Verdr&auml;nger, MetJ, f&uuml;r seine DNA-Operator.

Steigende Zahl von Zielstrukturen f&uuml;r computational Studien Protein macht das Struktur-basiertes Screening-Paradigma f&uuml;r ersten hit Identifizierung attraktiver. Haben wir entwickelt, einen Roman in Silico screening-Methodik zur molekularen Mechanik (MM) / implizite L&ouml;sungsmittel Methoden auswerten Bindung freie Energien und diese Technologie zur Identifizierung von Inhibitoren der Interaktion TLR4/MD-2 angewendet.

Anwendung eines Romans in Silico Hochdurchsatz Schirm selektive Inhibitoren f&uuml;r Protein-Protein Interaktionen zu identifizieren.

Increasing numbers of target protein structures available for computational studies makes the structure-based screening paradigm more attractive for initial hit indentification. We have developed a novel in silico screening methodology incorporating Molecular Mechanics (MM)/implicit solvent methods to evaluate binding free energies and applied this technology to the identification of inhibitors of the TLR4/MD-2 interaction.

Application of a novel in silico high-throughput screen to identify selective inhibitors for protein-protein interactions.

Epstein-Barr virus (EBV), a human Î³-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFÎºB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.

Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1.

Epstein - Barr-Virus (EBV), eine menschliche &gamma;-Herpesvirus, schafft lebenslange Infektion durch gezielte das adaptive Immunsystem des Wirtes durch B-Ged&auml;chtniszellen. Obwohl normalerweise gutartig, tr&auml;gt EBV lymphoide Malignome und Lymphoproliferative Syndrome bei immungeschw&auml;chten Personen. Das virale OIT3 latentes Membranprotein 1 (LMP-1) ist f&uuml;r B-Lymphozyte Immortalization von EBV. Die konstitutive Signalling Aktivit&auml;t der LMP-1 ist abh&auml;ngig von Homo-Oligomerisierung von seiner sechs-spanning hydrophobe Transmembranegebiet (TMD). Jedoch ist der Mechanismus, der LMP-1 intermolekulare Interaktion fahren oft missverstanden. Hier zeigen wir, dass die f&uuml;nfte transmembrane-Helix (TM5) LMP-1 stark self-associates, bilden eine komplexe homotrimeres, vermittelt durch einen polar R&uuml;ckstand in der Membran, D150 eingebettet. Ersatz von diesem Asparagins&auml;ure R&uuml;ckstand mit Alanin st&ouml;rt TM5 Self-association in Waschmitteln Micellen und bakteriellen Zellmembranen. Eine in LMP-1-Variante beherbergen die D150A Substitution ist mangelhaft NF&kappa;B-Aktivierung, die Schl&uuml;sselrolle der f&uuml;nfte Transmembran-Helix in konstitutiven Aktivierung der Signalisierung durch dieses OIT3 unterst&uuml;tzen.

Transmembran-Peptide verwendet, um die Homo-Oligomere Schnittstelle und Bindung Hotspot latentes Membrane Protein 1 zu untersuchen.

ToxR-based transcriptional reporter assays allow the strength of transmembrane helix interactions in biological membranes to be measured. Previously, these assays have only been used to study single-pass transmembrane systems. To facilitate investigation of polytopic transmembrane domain (TMD) oligomerization, we applied the ToxR methodology to the study of multi-pass TMD oligomerization to give 'Multi-Tox'. Association propensities of the viral oncoprotein, latent membrane protein-1 (LMP-1), and the E. coli membrane-integral diacylglycerol kinase (DAGK) were studied by Multi-Tox, highlighting residues of particular mechanistic importance. Both homo- and hetero-oligomerizations were studied.

Multi-Tox: application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization.

Transkriptionelle Reporter ToxR basierende Assays erm&ouml;glichen die St&auml;rke der Transmembran-Helix-Wechselwirkungen in biologischen Membranen gemessen werden. Fr&uuml;her wurden diese Proben nur Untersuchung Einschritt-Transmembran-Systeme verwendet. Zur Erleichterung der Untersuchung von polytopischen Transmembranegebiet (TMD) Oligomerisierung angewendet wir die ToxR Methodik zum Studium der Multipass TMD Oligomerisierung, &#39;Multi-Tox&#39; zu geben. Verein Neigungen die virale OIT3, latentes Membrane Protein-1 (LMP-1) und das E. Coli membranst&auml;ndiger Diacylglycerol Kinase (DAGK) von Multi-Tox, Hervorhebung R&uuml;ckst&auml;nde von besonderer Bedeutung der mechanistischen studiert wurden. Beide Homo und Hetero-Oligomerizations wurden untersucht.

Multi-Tox: Anwendung des ToxR-transkriptionelle Reporter Assays zum Studium der Multipass Protein Transmembranegebiet Oligomerisierung.

In optimalen F&auml;llen k&ouml;nnen bivalenten Liganden mit au&szlig;ergew&ouml;hnlich hoher Affinit&auml;t zu ihren Zielen Protein binden. Entwerfen optimierte Verkn&uuml;pfungsprogramme, die die Bindung der beiden Gruppen perfekt zu orientieren, ist jedoch anspruchsvoll und doch entscheidend in beiden Fragment-basierte Ligand-Design und in der Entdeckung der Bisubstrate-Enzym-Hemmer. S-Adenosylmethionine (SAM)-Analoga wurden mit dem Ziel der Interaktion mit dem MetJ-Dimer in einem bivalenten Sinn (1:1 Ligand/MetJ Dimer) entwickelt, um unser Verst&auml;ndnis der Linker-Design, eine Reihe von neuartigen hergestellten weiter. Eine Reihe von Liganden wurde synthetisiert und F&auml;higkeit zur F&ouml;rderung der Bindung der Escherichia coli Methionin Verdr&auml;nger, MetJ, zu seinem Betreiber DNA analysiert. Von zweiwertigem SAM-Analoga zu den MetJ Homodimer in Anwesenheit von Operator binden DNA wurde durch Fluoreszenz Anisotropie und der Effekt der Linker ausgewertet und Struktur wurde untersucht. Die effektivste bivalenten Ligand identifiziert hatte einen flexiblen Linker und bef&ouml;rdert die DNA-Protein-Interaktion bei 21-mal niedriger Konzentration als das entsprechende monovalenten Steuerelement-Compound.

Design, Synthese und in-vitro-Auswertung von Roman bivalenten S-Adenosylmethionine-Analoga.

In optimal cases, bivalent ligands can bind with exceptionally high affinity to their protein targets. However, designing optimised linkers, that orient the two binding groups perfectly, is challenging, and yet crucial in both fragment-based ligand design and in the discovery of bisubstrate enzyme inhibitors. To further our understanding of linker design, a series of novel bivalent S-adenosylmethionine (SAM) analogues were designed with the aim of interacting with the MetJ dimer in a bivalent sense (1:1 ligand/MetJ dimer). A range of ligands was synthesised and analyzed for ability to promote binding of the Escherichia coli methionine repressor, MetJ, to its operator DNA. Binding of bivalent SAM analogues to the MetJ homodimer in the presence of operator DNA was evaluated by fluorescence anisotropy and the effect of linker length and structure was investigated. The most effective bivalent ligand identified had a flexible linker, and promoted the DNA-protein interaction at 21-times lower concentration than the corresponding monovalent control compound.

Article	Year
Configurational stability of bisindolylmaleimide cyclophanes: from conformers to the first configurationally stable, atropisomeric bisindolylmaleimides. Chemistry (Weinheim an der Bergstrasse, Germany)\| PubMed ID: 16075446	2005
Identification of stable S-adenosylmethionine (SAM) analogues derivatised with bioorthogonal tags: effect of ligands on the affinity of the E. coli methionine repressor, MetJ, for its operat... Organic & biomolecular chemistry\| PubMed ID: 19194573	2009
Application of a novel in silico high-throughput screen to identify selective inhibitors for protein-protein interactions. Bioorganic & medicinal chemistry letters\| PubMed ID: 20709548	2010
Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1. Biopolymers\| PubMed ID: 21560118	2011
Multi-Tox: application of the ToxR-transcriptional reporter assay to the study of multi-pass protein transmembrane domain oligomerization. Biochimica et biophysica acta\| PubMed ID: 21791200	2011
Design, synthesis and in vitro evaluation of novel bivalent S-adenosylmethionine analogues. Bioorganic & medicinal chemistry letters\| PubMed ID: 22137339	2012

Catherine Joce

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