60ª Reunião Anual da SBPC

INTRODUÇÃO:

The NIMA-related kinases (Neks) represent an evolutionarily conserved family of 11 serine/threonine kinases, containing 40-45% identity to the Aspergillus nidulans mitotic regulator NIMA within their N-terminal catalytic domain. NIMA kinase is involved in multiple aspects of mitotic progression including mitotic entry, chromatin condensation, spindle organization and cytokinesis. Both Nek1 and Nek8 were implicated in cilia function since mutation in mouse models of polycystic kidney disease (PKD) mapped to their genes. Human Nek8 is overexpressed in multiple breast tumors and hNek6 was also recently found to be linked to hepatic cell carcinomas. In previous two-hybrid studies our group identified hNek1 interacting proteins involved in the G2/M DNA damage checkpoint, neural cell development and PKD. These facts highlight Neks as potencial chemotherapeutic targets. Considering the relevance of a drug design approach, as long as a better understanding about their roles in the cell environment, we attempt to express and purify these proteins for structural and functional studies. In this work we present the new constructions of human Neks 1, 6 and 8, as long as the expression, purification and initial crystallization trials for the mutant hNek6(S206A).

METODOLOGIA:

Cloning: we PCR amplified cDNAs encoding full length proteins, kinase and regulatory domains of hNeks 1, 6 and 8 and sub-cloned into pET28a-TEV vectors for expression in E. coli and/or into pFastBacHT vectors for the expression in the baculo virus system. The PCR products were also cloned into mammalian expression vectors for subcellular localization analysis and/or into pBTM116KQ for yeast two-hybrid studies. Site-directed Mutagenesis: mutations were introduced by PCR-based mutagenesis of the mentioned plasmid constructs using QuickChange Site-Directed Mutagenesis kit. Expression and Purification: the expression of the recombinant proteins hNek6 and hNek6(S206A) fused to a His-tag were optimized in E. coli (BL21 pRARE) with 1mM IPTG, 18^oC for 4h and 0,5mM IPTG, 37^oC for 4h, respectively. The proteins were purified by Ni-affinitty chromatography using a Hitrap Chelating 1mL column and eluted with Buffer B (50mM HEPES pH 7,5, 5mM Sodium Phosphate, 300mM NaCl, 300mM Imidazole and 5% Glycerol). The expression and purification of hNek1KD(T162A) is currently being optimized. Structural Analysis: the mutant protein hNek6(S206A) was characterized by dynamic light scattering at different concentrations in Buffer B and submitted to crystallization trials with 6 different kits.

RESULTADOS:

Several constructions involving hNeks 1, 6 and 8 were tested but only the recombinant protein 6xHis-TEV-hNek6, the mutant version 6xHis-hNek6(S206A) and the mutated kinase domain of hNek1 – 6xHis-TEV-Nek1KD(T162A) – expressed in E. coli. Although the induction and purification of higher yields of 6xHis-hNek6(S206A) has been successfully achieved in comparison to the non-mutaded 6x-His-hNek6, the protein is not very stable and aggregates in certain conditions. In that way, buffer conditions are being optimized for structural studies in solution (Circular Dichroism, SAXS and Fluorescence).

CONCLUSÕES:

In this work we have successfully expressed some Nek recombinant proteins, in particular the mutant hNek6(S206A), for which the structural studies are in advance. These studies may provide useful information for the development of new chemotherapeutic strategies in the future with the development of more potent inhibitors. Although some of the tested Nek kinase domains and mutant kinase domains show significant expression in the bacterial systems, our results suggest that for the expression of the full length proteins Nek1 and Nek8 we need to employ the baculo virus expression system.

Instituição de fomento: FAPESP, LNLS and CNPq

Palavras-chave: kinase, cancer, drug design

E-mail para contato: gmeirelles@lnls.br, jkobarg@lnls.br