In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)

Detalhes bibliográficos
Autor(a) principal: Ferreira, Cátia Alexandra Marques Bonito
Data de Publicação: 2015
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10451/18402
Resumo: Tese de mestrado, Ciências Biofarmaêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015
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spelling In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)Mitochondrial fatty acid beta-oxidationMCADp.K304EMolecular dynamic simulationsChaperonesTeses de mestrado - 2015Tese de mestrado, Ciências Biofarmaêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015The medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid β-oxidation (mFAO) pathway, in particular the first dehydrogenation reaction of C4-C14 fatty acyl substrates. The mature MCAD enzyme is a homotetramer with Flavin Adenine Dinucleotide (FAD) as its natural cofactor. The most common mutation found in MCADD patients (c.985A>G in ACADM gene) is translated in the substitution of lysine by a glutamic acid residue at position 304 (p.K304E) of the mature protein. Accordingly, this substitution has been widely related with protein misfolding and tetramer instability. To better understand the molecular basis of the pathogenicity of the p.K304E variant in MCADD, in vitro and in silico approaches were undertaken. Experimental in vitro studies comprised the heterologous expression of human MCAD wild-type (hMCADwt) and p.K304E variant proteins, followed by the recombinant proteins’ purification and functional and structural characterization. The results obtained show that, when compared to hMCADwt, the p.K304E variant is structurally unstable, presenting an alteration in its oligomeric profile, thermal instability and a higher susceptibility to trypsin limited proteolysis, the latter pointing to a higher conformational flexibility of the variant protein. The addition of FAD in the initial step of the purification of the recombinant proteins seems to increase the tetrameric fraction of the variant protein and recover its deficient enzymatic activity. Moreover, FAD also incremented the thermal stability of both hMCADwt and p.K304E proteins, as expected being FAD the MCAD’s cofactor. Promising results were also obtained with glycerol and TMAO tested, among other compounds, as chemical chaperones for the p.K304E variant. To complement the in vitro data, molecular dynamics (MD) simulations of the MCAD enzyme were also performed and both porcine and human structures were studied. The initial coordinates were obtained, respectively, from the crystallographic structure of the porcine MCAD protein (PDB: 1UDY; pMCADwt), and by reversing the crystallographic structure of the E376G/T255E mutant of the human MCAD protein (PDB: 1EGC; hMCADwt). In a following step, both pMCADwt and hMCADwt proteins were used to generate the corresponding p.K304E variants. The results obtained showed that both wild-type proteins were stable during the simulation time. The FAD seems to have a structural role in the catalytic pockets and in the tetramer stability. The catalytic pockets are highly flexible and dynamic to better accommodate the substrate. The p.K304E variant seems to induce a different behavior over MD simulations regarding both pMCADwt and hMCADwt. The p.K304E mutation seems also to induce structural modifications in the catalytic pockets, ultimately affecting their volume. Finally, the mutation seems to asymmetrically affect the FAD and substrate binding affinities in both pMCAD and hMCAD variants, which may explain the decrease of the enzymatic activity and catalytic efficiency observed in the p.K304E/hMCAD variant (~50% residual activity in respect to hMCADwt). Altogether, the in vitro and in silico data obtained contribute to better clarify the structural and functional abnormalities of the p.K304E variant of hMCAD thereby opening perspectives for the search for small molecules as protein stabilizers towards the development of pharmacological strategies for the treatment of MCADD.Ventura, Fátima V.Guedes, Rita C.Repositório da Universidade de LisboaFerreira, Cátia Alexandra Marques Bonito2017-03-31T00:30:16Z201520152015-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfapplication/pdfhttp://hdl.handle.net/10451/18402TID:201251329enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-11-08T16:04:43Zoai:repositorio.ul.pt:10451/18402Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:37:58.986125Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
title In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
spellingShingle In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
Ferreira, Cátia Alexandra Marques Bonito
Mitochondrial fatty acid beta-oxidation
MCAD
p.K304E
Molecular dynamic simulations
Chaperones
Teses de mestrado - 2015
title_short In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
title_full In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
title_fullStr In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
title_full_unstemmed In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
title_sort In vitro and in silico : two complementary approaches to elucidate the molecular mechanisms of Medium-Chain Acyl-CoA dehydrogenase deficiency (MCADD)
author Ferreira, Cátia Alexandra Marques Bonito
author_facet Ferreira, Cátia Alexandra Marques Bonito
author_role author
dc.contributor.none.fl_str_mv Ventura, Fátima V.
Guedes, Rita C.
Repositório da Universidade de Lisboa
dc.contributor.author.fl_str_mv Ferreira, Cátia Alexandra Marques Bonito
dc.subject.por.fl_str_mv Mitochondrial fatty acid beta-oxidation
MCAD
p.K304E
Molecular dynamic simulations
Chaperones
Teses de mestrado - 2015
topic Mitochondrial fatty acid beta-oxidation
MCAD
p.K304E
Molecular dynamic simulations
Chaperones
Teses de mestrado - 2015
description Tese de mestrado, Ciências Biofarmaêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2015
publishDate 2015
dc.date.none.fl_str_mv 2015
2015
2015-01-01T00:00:00Z
2017-03-31T00:30:16Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10451/18402
TID:201251329
url http://hdl.handle.net/10451/18402
identifier_str_mv TID:201251329
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/embargoedAccess
eu_rights_str_mv embargoedAccess
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
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