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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

STRUCTURAL AND FUNCTIONAL STUDIES OF SYNAPTIC ENZYMES

Lim, Eun Jeong 01 January 2006 (has links)
Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zincdependent metallopeptidases that metabolize small bioactive peptides. The two enzymes share60 % sequence identity and their crystal structures demonstrate that they adopt nearly identicalfolds. They generally cleave at the same sites, but they recognize different positions on somepeptides, including neurotensin, a 13-residue peptide involved in modulation of dopaminergiccircuits, pain perception, and thermoregulation.On the basis of crystal structures and previous mapping studies, four residues(E469/R470, M490/R491, H495/N496, and R498/T499, TOP residues listed first) in thesubstrate-binding channel appear positioned to account for differences in specificity. TOPmutated to the neurolysin residues at all four position cleaves neurotensin at the neurolysin siteand neurolysin mutated to the TOP residues at all four sites cleaves at the TOP position. Using aseries of constructs mutated at only three sites, it was determined that only two of the mutations,E469/R470 and R498/T499, are required to swap the specificity of TOP and neurolysin. Theseresults were confirmed by testing the two mutation constructs, and either single mutant of TOPshown an intermediate specificity, cleaving at both sites.Crystal structures of the two mutation constructs of TOP and neurolysin unligandedforms, the mutations do not perturb local structure, but side chain conformations at theR498/T499 position differ from those of the mimicked enzyme. A model for differentialrecognition of neurotensin based on differences in surface charge distribution in the substratebinding sites is proposed. The model is supported by finding that reducing the positive charge onthe peptide results in cleavage at both hydrolysis sites.This dissertation also includes a description of the production and crystallization trials ofhuman neprilysin (E.C. 3.4.24.11), which will be used as another model system for studyingspecificity in metallopeptidases. In addition, the production and crystallization, and crystalcharacterization of human choline acetyltransferase (EC 2.3.1.6) is described.
2

Caracterização fenotípica de camundongos knockout para neurolisina. / Phenotype characterization of neurolysin knockout mice.

Cavalcanti, Diogo Manuel Lopes de Paiva 22 May 2014 (has links)
A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.
3

Caracterização fenotípica de camundongos knockout para neurolisina. / Phenotype characterization of neurolysin knockout mice.

Diogo Manuel Lopes de Paiva Cavalcanti 22 May 2014 (has links)
A oligopeptidase neurolina (E.C.3.4.24.16; nln ) foi identificado pela primeira vez em membranas sinápticas de cérebro de ratos como sendo capaz de participar no metabolismo de peptídeos bioativos, como neurotensina e bradicinina. Recentemente, foi sugerido que a ausência de Nln pode melhorar a sensibilidade a insulina. Aqui, nós mostrado que camundongos knockout para Nln (KO) são mais tolrerantes à glicose, sensíveis à insulina e apresentam maior gliconeogênese. Os animais KO apresentou um aumento na expressão de mRNA de vários genes relacionados com a gliconeogênese no fígado. A semiquantificação de peptídeos intracelulares revelou um aumento em peptídeos intracelulares específicos no gastrocnêmio e tecido adiposo epididimal, que estão envolvidos com o aumento da tolerância a glicose e maior sensibilidade à insulina nos animais KO. Esses resultados sugerem fortemente a nova possibilidade de que Nln é uma enzima chave no metabolismo energético e pode ser um novo alvo terapêutico para melhorar a captação de glicose e sensibilidade a insulina. / The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln knockout mice (KO) have increased glucose tolerance, insulin sensitivity and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semi-quantitative peptidomic analysis suggests increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

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