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Functional characterization of naturally occurring mutant transglutaminase 2Salter, Neil William 01 September 2011 (has links)
Transglutaminase 2 (TG2) is a functionally and structurally complex ubiquitous protein. TG2 has Ca2+-dependent transamidating activity, can bind and hydrolyze ATP/GTP, function as a G-protein in intracellular signaling, and has reported kinase activity. TG2 knockout mice are observed to have impaired glucose-stimulated insulin secretion (GSIS). Three naturally occurring mutations, including Met330Arg, Ile331Asn, and Asn333Ser, have been reported in the TG2 protein and observed to be related with maturity onset diabetes of the young (MODY). Overexpression of the naturally occurring Myc-tag mutants in INS-1E cells generated a loss in GSIS compared to wild type-TG2 overexpression. Each mutant was shown to have diminished transamidation and kinase activities, along with altered GTP-binding which was responsive to glucose stimulation. Naturally occurring mutations in TG2 impact the transamidation, kinase, and GTP-binding functions of TG2. The GTP-binding function of TG2 has a significant impact on GSIS from pancreatic beta cells in addition to its transamidating activity.
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Functional characterization of naturally occurring mutant transglutaminase 2Salter, Neil William 01 September 2011 (has links)
Transglutaminase 2 (TG2) is a functionally and structurally complex ubiquitous protein. TG2 has Ca2+-dependent transamidating activity, can bind and hydrolyze ATP/GTP, function as a G-protein in intracellular signaling, and has reported kinase activity. TG2 knockout mice are observed to have impaired glucose-stimulated insulin secretion (GSIS). Three naturally occurring mutations, including Met330Arg, Ile331Asn, and Asn333Ser, have been reported in the TG2 protein and observed to be related with maturity onset diabetes of the young (MODY). Overexpression of the naturally occurring Myc-tag mutants in INS-1E cells generated a loss in GSIS compared to wild type-TG2 overexpression. Each mutant was shown to have diminished transamidation and kinase activities, along with altered GTP-binding which was responsive to glucose stimulation. Naturally occurring mutations in TG2 impact the transamidation, kinase, and GTP-binding functions of TG2. The GTP-binding function of TG2 has a significant impact on GSIS from pancreatic beta cells in addition to its transamidating activity.
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Design, synthesis and biological evaluation of TG2 transglutaminase inhibitors / Conception, synthèse et évaluation biologique des inhibiteurs de la transglutaminase TG2Fidalgo Lopez, Javier 23 November 2016 (has links)
La transglutaminase tissulaire (TG2) est une enzyme de la famille des transglutaminases (EC 2.3.2.13) qui est exprimée de manière ubiquitaire chez les mammifères. Cette enzyme catalyse la formation d'une liaison amide intra- ou intermoléculaire entre un résidu glutamine et un résidu lysine. Ce processus biologique conduit à la modification post-traductionnelle des protéines. Un nombre croissant de publications associe la surexpression de cette enzyme et la déréglementation de son activité, avec un certain nombre de pathologiques humaines telles que les maladies neurodégénératives (maladie d’Alzheimer, maladie de Huntington, maladie de Parkinson), la fibrose tissulaire, certains cancers et la maladie cœliaque. Le développement d'inhibiteurs puissants et sélectifs de la TG2 est primordial pour identifier soit des outils pharmacologiques pour comprendre les processus biologiques dépendant de cette enzyme ou soit des candidats médicaments pour traiter les pathologies liées à la surexpression de la TG2. La majorité des composés inhibiteurs synthétisés jusqu'à présent agissent en bloquant de manière irréversible la réaction de transamidification de la TG2 en ciblant spécifiquement la cystéine 277 présente dans le site actif de la TG2.L’objectif de ce travail a été d’identifier et de sélectionner des molécules de faible poids moléculaire inhibant de façon sélective et puissante l’activité de transamidification de la TG2. Nous présenterons l’optimisation de deux séries originales de composés (synthèse, études de relation de structure-activité) comportant un noyau aromatique central de type naphtalénique ou indolique et une fonction acrylamide comme accepteur de Michael pour piéger la fonction thiol de la cystéine 277. Un certain nombre de composés synthétisés montre une inhibition nanomolaire de la TG2 (IC50 = 1.7-6 nM) avec un excellent profil de sélectivité vis-à-vis de TG1, TG6 et FXIIIa (IC50 > 10 µM). Ces inhibiteurs inhibent efficacement la TG2 dans des extraits de tissus et de cellules. Aucune toxicité apparente n’a été observée pour des concentrations inférieures à 10 µM d’inhibiteur sur les lignées vSMCs et SH-SY5Y. Les valeurs de KI, kinact et kinact/KI ont été également déterminés sur deux inhibiteurs sélectionnés (23b et 78f) pour leurs activités biologiques. La formation d’une liaison covalente entre la cystéine 277 de la TG2 et ces deux inhibiteurs a été prouvée par digestion trypsique suivie d’une analyse LC-MS/MS / Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme of the mammalian transglutaminase (TG) family which catalyzes the formation of an intra- or inter-molecular isopeptide bond between a glutamine and a lysine, leading to the post-translational modification of proteins. An increasing number of literature has associated the over-expression of this enzyme, and the deregulation of its activity, with a number of human physio-pathological states like neurodegenerative disorders (Alzheimer’s disease, Huntington’s disease, Parkinson’s disease), tissue fibrosis, certain cancers, and celiac disease. The development of potent and selective TG2 inhibitors has become primordial to reach either a pharmacological probe, to understand the biological processes that depend on this enzyme, or a drug candidate, to treat the pathologies related with its overexpression. The majority of the inhibitory compounds synthesized so far act by irreversibly blocking the transamidation reaction of TG2. These TG2 inhibitors specifically target the cysteine 277 present in the TG2 active site. The aim of this work was the identification and selection of new potent and selective small molecules to inhibit the TG2 transamidation activity. We present the optimization of two new series of compounds (synthesis, structure-activity relationship studies) bearing naphthalene or indole aromatic rings as the central backbone structure. Both series present an acrylamide group as the Michael acceptor in order to react with the thiol group of cysteine 277. Several of the synthesized compounds showed a nanomolar inhibition over TG2 (1.7-6 nM) with an excellent selectivity profile over TG1, TG6 and FXIIIa (IC50 > 10 µM). These inhibitors showed high specificity on inhibiting TG2 in tissue and cell extracts. No apparent toxicity up to 10 µM was observed in vSMCs and SH-SY5Y cell lines. Their KI, kinact et kinact/KI were also determined on two selected inhibitors (23b and 78f) for their biological activities. The formation of a covalent bond between the cysteine 277 of TG2 and these two inhibitors was proven by tryptic digestion followed by LC-MS/MS analysis
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