Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC

The cblC disease is the most common inborn error of cobalamin metabolism. This is a monogenic disorder that affects all organs with patients presenting symptoms including cardiovascular, neurological, musculoskeletal, hematological and ocular problems. Oxidative stress and apoptosis have been implic...

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Detalles Bibliográficos
Autor principal: Tanimowo, Houston Segun
Otros Autores: Galleano, Mónica
Formato: Tesis de maestría acceptedVersion
Lenguaje:Inglés
Publicado: Facultad de Farmacia y Bioquímica 2019
Materias:
Efecto del hidrogéno peroxido
H2O2
CblC protein
Effect of hydrogen peroxide
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5940
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5940.dir/5940.PDF
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Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_5940
record_format dspace
spelling I28-R145-HWA_59402022-04-12 The cblC disease is the most common inborn error of cobalamin metabolism. This is a monogenic disorder that affects all organs with patients presenting symptoms including cardiovascular, neurological, musculoskeletal, hematological and ocular problems. Oxidative stress and apoptosis have been implicated in the pathogenesis of this disease and certain pathogenic variants of the CblC protein have been shown to produce reactive oxygen species.\nHydrogen peroxide (H2O2) is a biological oxidant and a signaling molecule that acts by modifying aminoacid residues in proteins. Cell biology studies have shown an increased formation of H2O2 in cells from cblC patients.\nThe aim of this Master Thesis is to study the effect of H2O2 on the catalytic activity of the wild type and pathogenic variants (R161Q, R161G and DelGln131) of the human CblC protein. The results showed that the oxidant H2O2 does not have any effect on the binding of substrate methylcobalamin (MeCbl) to wild type and pathogenic CblC protein. However, H2O2 reduced the observed rate of catalysis of both wild type and pathogenic CblC variants. Pathogenic variants R161Q and DelGln131 were the most sensitive to inhibition by H2O2. The substrates MeCbl and GSH protected the CblC protein from oxidative inactivation, suggesting that amino acid residues located at or near the active site could be the targets for H2O2. Examination of the X-ray crystal structure of CblC, as well as experiments with the thiol blocker iodoacetamide, suggest that Cys149 located in the cobalamin binding site could be important for catalysis and a target of oxidative inactivation. Finally, we tested two new cobalamins designed to recuperate loss of CblC activity in patients. The results show that the two cobalamins were effective in recuperating enzymatic activity in pathogenic variants of CblC, and also, in protecting wild type and CblC mutants from H2O2-mediated damage. Further studies are currently underway (beyond the scope of this thesis) to unequivocally identify the amino acid residue(s) targeted by H2O2 in CblC using redox proteomics and site-directed mutagenesis. Fil: Tanimowo, Houston Segun. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Galleano, Mónica Facultad de Farmacia y Bioquímica Hannibal, Luciana Tanimowo, Houston Segun 2019-03-06 application/pdf Valdez, Laura Frances, Daniel Borner, Christoph Efecto del hidrogéno peroxido H2O2 CblC protein Effect of hydrogen peroxide eng Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5940 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5940.dir/5940.PDF
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Inglés
orig_language_str_mv eng
topic Efecto del hidrogéno peroxido
H2O2
CblC protein
Effect of hydrogen peroxide
Ciencias de la vida
spellingShingle Efecto del hidrogéno peroxido
H2O2
CblC protein
Effect of hydrogen peroxide
Ciencias de la vida
Tanimowo, Houston Segun
Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
topic_facet Efecto del hidrogéno peroxido
H2O2
CblC protein
Effect of hydrogen peroxide
Ciencias de la vida
description The cblC disease is the most common inborn error of cobalamin metabolism. This is a monogenic disorder that affects all organs with patients presenting symptoms including cardiovascular, neurological, musculoskeletal, hematological and ocular problems. Oxidative stress and apoptosis have been implicated in the pathogenesis of this disease and certain pathogenic variants of the CblC protein have been shown to produce reactive oxygen species.\nHydrogen peroxide (H2O2) is a biological oxidant and a signaling molecule that acts by modifying aminoacid residues in proteins. Cell biology studies have shown an increased formation of H2O2 in cells from cblC patients.\nThe aim of this Master Thesis is to study the effect of H2O2 on the catalytic activity of the wild type and pathogenic variants (R161Q, R161G and DelGln131) of the human CblC protein. The results showed that the oxidant H2O2 does not have any effect on the binding of substrate methylcobalamin (MeCbl) to wild type and pathogenic CblC protein. However, H2O2 reduced the observed rate of catalysis of both wild type and pathogenic CblC variants. Pathogenic variants R161Q and DelGln131 were the most sensitive to inhibition by H2O2. The substrates MeCbl and GSH protected the CblC protein from oxidative inactivation, suggesting that amino acid residues located at or near the active site could be the targets for H2O2. Examination of the X-ray crystal structure of CblC, as well as experiments with the thiol blocker iodoacetamide, suggest that Cys149 located in the cobalamin binding site could be important for catalysis and a target of oxidative inactivation. Finally, we tested two new cobalamins designed to recuperate loss of CblC activity in patients. The results show that the two cobalamins were effective in recuperating enzymatic activity in pathogenic variants of CblC, and also, in protecting wild type and CblC mutants from H2O2-mediated damage. Further studies are currently underway (beyond the scope of this thesis) to unequivocally identify the amino acid residue(s) targeted by H2O2 in CblC using redox proteomics and site-directed mutagenesis.
author2 Galleano, Mónica
author_facet Galleano, Mónica
Tanimowo, Houston Segun
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Tanimowo, Houston Segun
author_sort Tanimowo, Houston Segun
title Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
title_short Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
title_full Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
title_fullStr Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
title_full_unstemmed Oxidation and repair of oxidative damage in the GSH-dependent enzyme CblC
title_sort oxidation and repair of oxidative damage in the gsh-dependent enzyme cblc
publisher Facultad de Farmacia y Bioquímica
publishDate 2019
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_5940
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_5940.dir/5940.PDF
work_keys_str_mv AT tanimowohoustonsegun oxidationandrepairofoxidativedamageinthegshdependentenzymecblc
_version_ 1766017555142541312

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