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Doctoral Thesis
DOI
10.11606/T.75.2012.tde-24072012-172313
Document
Author
Full name
Kelly Pereira da Silva
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2012
Supervisor
Committee
Borges, Júlio César (President)
Araujo, Ana Paula Ulian de
Cano, Maria Isabel Nogueira
Carrilho, Emanuel
Souza, Dulce Helena Ferreira de
Title in Portuguese
Estudos estruturais e funcionais da Hsp90 de Leishmania braziliensis e suas co-chaperonas p23
Keywords in Portuguese
Leishmania braziliensis
chaperonas moleculares
Hsp90
Abstract in Portuguese
As chaperonas moleculares são proteínas que auxiliam no enovelamento correto de outras proteínas, entre outras funções importantes para as células, motivo pelo qual elas têm sido alvo para o combate de várias doenças. As Hsp90 (82-96 kDa) são chaperonas abundantes que interagem com diversas proteínas-cliente. São constituídas por três domínios: N-terminal, intermediário ou central (M) e C-terminal, o qual é responsável pela dimerização da proteína. A atividade da Hsp90 está diretamente relacionada à sua atividade ATPásica. Durante o ciclo funcional, as Hsp90 podem interagir com inúmeras co-chaperonas. Uma delas é a co-chaperona p23 (18-22 kDa) que interage com o dímero da Hsp90 e algumas das suas funções são a inibição da atividade ATPásica e atividade chaperona. O objetivo do trabalho foi obter a proteína recombinante Hsp90 de Leishmania braziliensis e os domínios N e N+M, determinar fatores importantes que relacionam mudanças conformacionais e função da Hsp90 e as bases moleculares da inibição por GA. Também obter as co-chaperonas Lbp23A e Lbp23B e investigar a interação com a LbHsp90 e suas funções. As proteínas produzidas foram purificadas e caracterizadas por técnicas biofísicas. Em solução, a LbHsp90 foi caracterizada como dímero assimétrico e as demais proteínas como monômeros assimétricos.A interação da LbHsp90 e domínios com nucleotídeos foi analisada por fluorescência e as constantes de dissociação ficaram em torno de 150 µM. A afinidade por GA foi maior que a verificada para ATP e em ordem crescente para LbHsp90, LbHsp90_NM e LbHsp90_N. A LbHsp90 apresentou grande atividade chaperona em relação à citrato sintase, de maneira independente de ATP. A LbHsp90 mostrou baixa atividade ATPásica, a qual foi inibida pela GA com IC50 de 0,7 µM. Tanto a Lbp23A quanto a Lbp23B inibiram a atividade ATPásica da LbHsp90, porém a Lbp23A aproximou-se de 100% de inibição e a Lbp23B apenas 30%. A interação in vitro entre a LbHsp90 e a Lbp23B foi observada por pull-down na presença/ausência de nucleotídeos e essa técnica não se mostrou adequada para a Lbp23A.O pioneirismo do trabalho com a Hsp90/p23 de L. braziliensis oferece uma grande contribuição para futuros trabalhos que visam o entendimento das relações funcionais entre essas proteínas e o contexto das Hsp90 no desenvolvimento da leishmaniose.
Title in English
Structural and functional studies of Leishmania braziliensis Hsp90 and its p23 co-chaperones
Keywords in English
Leishmania braziliensis
Hsp90
molecular chaperones
Abstract in English
Molecular chaperones are proteins involved in proper folding of other proteins, and others important cellular functions, why they have been targeted for combating various diseases. The Hsp90 (82-96 kDa) are ubiquitous chaperones that interact with a wide range of client proteins. They are formed by three domains: N-terminal, central or middle (M), and C-terminal, which is responsible by its dimerization. The Hsp90 activity is related to its ATPase activity. During the Hsp90 functional cycle, diverse co-chaperones. One of them is the p23 (18 kDa), that interacts with one Hsp90 dimer, and some p23 functions are the inhibition of Hsp90 ATPase activity and chaperone activity. The aim of this work was obtain the Hsp90 recombinant Leishmania braziliensis Hsp90, the N and N+M domains, to determine the important factors related to conformational changes and Hsp90 function, and the molecular basis of GA inhibition. Also, to obtain the Lbp23A and Lbp23B co-chaperones in order to establish relevant aspects for LbHsp90 interaction and its co-chaperones functions. The recombinant proteins were produced, purified and characterized by biophysics techniques. The LbHsp90 was identified as an asymmetric dimer for whereas the others were identified as asymmetric monomers. The interactions between LbHsp90 and domains with nucleotides were determined by fluorescence and the dissociation constants were about 150 µM. The GA-affinity was greater than ATP one, in increasing order for LbHsp90, LbHsp90_NM, and LbHsp90_N. The LbHsp90 showed large chaperone activity related to citrate synthase independently of ATP. The LbHsp90 presented low ATPase activity, which was inhibited by GA with a IC50 of 0,7. The Lbp23A and Lbp23B inhibited the ATPase activity with different values, the Lbp23A inhibition was closed to 100% whereas the Lbp23B one was 30%. The in vitro interaction between the LbHsp90 and Lbp23B was observed by pull-down, in the absence or presence of nucleotides, and for Lbp23A this technique was not appropriated. The pioneering work with Hsp90/p23 from L. braziliensis offers an important contribution to future studies aimed at understanding the functional relationships between these proteins and the context of Hsp90 in the development of leishmaniasis.
 
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Publishing Date
2012-10-23
 
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