Conformational and functional characterization of AAA domain proteins through the investigation of human ANKCLP and Rvbs from Leishmania major and Saccharomyces cerevisiae

Author(s)

Josielle Abrahão

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-01-26

Examining board members

Carlos Henrique Inacio Ramos; Maria Celia Bertolini; Mário Tyago Murakami; André Ricardo de Lima Damásio; Juliana Ferreira de Oliveira

• Advisor: Carlos Henrique Inacio Ramos

Abstract

AAA+ proteins family are ATPase enzymes associated with diverse cellular activities. Mainly characterized by the presence of the nucleotide binding domain, known as NBD (Nucleotide Binding Domain), the AAA proteins can act of a ranging of functions from DNA replication to protein degradation. Thus, this thesis describes the results obtained with some AAA+ proteins: human ANKCLP and Rvbs 1 and 2 from Leishmania major. The human ANKCLP protein has a NBD domain which is very similar to the NBD domais from chaperone disaggregases. Therefore, using the yeast model, we showed that the ANKCLP protein didn¿t complement the yeast cell survival in the absence of Hsp104, chaperone disaggregate with two AAA+ domains and well characterized in yeast. In addition, the ANKCLP was not able to remodel amyloid aggregates in yeast cells which contain prion formation factors. Due to the similarity with ANKCLP, we were interested in the characteristics of the Hsp78 protein, chaperone disaggregate from yeast mitochondria. Thus, we have produced a mini-review on this protein and a critical view about the ANKCLP protein as a hypothetical chaperone disaggregase in animals. Also, is this work we studied the proteins Rvbs which also have one NBD domain and, in the first time, they have been described from Leishmania major, which causes leishmaniasis in mammals. Indeed, although these proteins are well described from human and yeast, they are not well studied when it comes from protozoa. Therefore, the Rvb1 and Rvb2 were successfully purified, and LmRvb1 was able to form a pentamer in solution, but did not have ATPase activity in vitro. LmRvb2 forms a large oligomer and has ATPase activity in vitro. The co-expression of Rvb1 and Rvb2 formed at least a dimer in solution. Stability and fluorescence experiments indicated that these proteins interact under these conditions and this dimer has ATPase activity in vitro, as well as an LmRvb2. Finally, with this work we hope to contribute to greater understanding of these AAA+ proteins (AU)

• FAPESP's process: 13/10939-2 - Characterization of human genes as potential chaperone with desagregase function
• Grantee: Josielle Abrahão
• Support Opportunities: Scholarships in Brazil - Doctorate