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Master's Dissertation
DOI
https://doi.org/10.11606/D.75.2020.tde-17022020-141644
Document
Author
Full name
Arthur Alexandre Pereira
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2019
Supervisor
Committee
Borges, Júlio César (President)
Bertolini, Maria Celia
Melo, Fernando Alves de
Souza, Dulce Helena Ferreira de
Title in Portuguese
Obtenção da co-chaperona mitocondrial hDjC20 humana
Keywords in Portuguese
chaperona molecular
DjC20
Hsp70
J-protein
Abstract in Portuguese

A estrutura nativa de uma proteína é essencial para o correto funcionamento e em ambiente celular, essa estrutura é adquirida durante um processo denominado de enovelamento proteico. Contudo, falhas durante o enovelamento da proteína podem ocorrer, levando a formação de agregados, que apresentam relação com o surgimento de algumas doenças. Para contornar esse problema a evolução desenvolveu uma maquinaria celular para auxiliar as proteínas nascentes ou mal enoveladas a adquirirem a estrutura nativa. As chaperonas moleculares são um grupo de proteínas que estão envolvidas em diversos processos, como no enovelamento de proteínas nascentes ou mal enoveladas, na prevenção da agregação proteica, no encaminhamento de proteínas agregadas para o processo de degradação, entre outros. As Hsp70, sendo uma das principais famílias de chaperonas moleculares, têm participação em vários desses processos celulares e necessitam de auxílio de cofatores e co-chaperonas para a realização do seu ciclo funcional. A elucidação do mecanismo e a interação dos fatores presentes se fazem necessário para o entendimento do seu ciclo funcional. O objetivo deste trabalho foi a obtenção de uma co-chaperona das Hsp70, de origem humana, pertencente à família das J-proteins, conhecida por atuar na mitocôndria. A proteína recombinante hDjC20 humana (hDjC20) foi expressa sem o peptídeo sinal de encaminhamento para a mitocôndria, purificada e submetida a caracterização biofísica inicial utilizando as técnicas de dicroísmo circular (CD), fluorescência intrínseca do triptofano, cromatografia de exclusão por tamanho analítica (aSEC) e calorimetria de varredura diferencial (DSC). Os resultados indicam que a hDjC20 recombinante foi obtida com um alto grau de pureza, enovelada e monomérica em solução. Testes de desnaturação química e térmica indicam que a hDjC20, que tem um domínio zinc-finger, se apresenta provavelmente ligada ao zinco que contribui para a estabilidade estrutural da proteína.

Title in English
Obtaining human hDjC20 mitochondrial co-chaperone
Keywords in English
DjC20
Hsp70
molecular chaperone
protein J
Abstract in English

The native structure of a protein is essential for proper cellular functioning, this structure is acquired during a process known folding. However, failure during protein folding may occur, leading to the formation of protein aggregates, which are related to the onset of some diseases. To order to overcome this problem, the cellular machinery has developed a way to lead nascent or badly folded proteins to acquire their native structure. Molecular chaperones are a group of proteins that are involved in several processes, such as in the folding of nascent or badly folded proteins, in the prevention of protein aggregation, in the routing of aggregated proteins to the degradation process, among others. Hsp70, being one of the main families of molecular chaperones, participate in several of these cellular processes and require the help of cofactors and co-chaperones to perform their functional cycle. The elucidation of the mechanism and the interaction of the present factors are necessary for the understanding of its functional cycle. The objective of this study was to obtain a human-origin Hsp70 co-chaperone from the J-protein family, known to act in mitochondria. The recombinant human protein hDjC20 was expressed without the mitochondrial signaling peptide, purified and subjected to initial biophysical characterization using circular dichroism (CD), tryptophan intrinsic fluorescence, size exclusion chromatography (aSEC) and differential scanning calorimetry (DSC). The results indicate that recombinant hDjC20 was obtained with a high purity, folded and monomeric solution. Chemical and thermal denaturation tests indicate that hDjC20, which has a zinc-finger domain, is probably linked to zinc which contributes to the structural stability of the protein.

 
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Publishing Date
2020-03-05
 
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