60ª Reunião Anual da SBPC

INTRODUÇÃO:

Fasciculation and elongation protein zeta-1(FEZ1) is a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein involved in the axonal outgrowth and fasciculation and promotes neurite extension of PC12 cells through interaction with protein kinase C zeta. Nerve growth factor (NGF) stimulation induces significant expression of endogenous FEZ1 in PC12 cells. Upon NGF stimulation FEZ1 localizes in both cytoplasm and neuritis, co-localizing with mitochondria. Silencing of FEZ1 by RNAi efficiently reduces NGF-induced neurite elongation and the anterograde motility of mitochondria in PC12 cells. How exactly FEZ1 interacts with cytoskeleton and vesicles/organeles/proteins as cargoes is still being elucidated. We employed the yeast two-hybrid system and identified among others several transcription regulatory proteins as FEZ1(221-392) interactors. The other identified proteins are functionally associated to neuronal cell development, intracellular transport processes and apoptosis (Assmann et. al, 2006). Here, we focused our efforts on studies with a FEZ1 interacting protein involved in transcriptional regulation called SAP30L (Sin3A Associated Protein 30-like – which is involved mainly in chromatin compaction) and in structural analysis of FEZ1 dimerization.

METODOLOGIA:

Cloning, expression and purification: to express FEZ1(92-194) and the full length of SAP30L fused to a His tag, the corresponding nucleotide sequences were amplified by PCR and inserted into bacterial expression vector pET28a (Novagen/EMD Biosciences, San Diego, CA) modified to a TEV cleavage site, in our laboratory. Soluble FEZ1(92-194), and SAP30L fused to the His tag was purified for in vitro analyses from 2 liter of culture of E. coli BL21 (DE3) cells that were induced for 4h (to FEZ1) or 1.5h (to SAP30L) to protein expression at 30^oC using 0.2mM (to FEZ1) or 0.5mM (to SAP30L) isopropyl 1-thio-β-D-galactopyranoside. The 6xHis-tagged proteins used in this study were purified using a HiTrap chelating column in an AKTA^TM FPLC^TM (GE Healthcare). Structural analysis: the protein FEZ1(92-194) fused to a His tag was characterized by dynamic light scattering at 1.2 mg/mL in PBS 1X, circular dichroism at 10 µM in PBS 1X and submitted to crystallization trials with 6 different kits. Establishing a Stable Cell Line: a stable cell line constitutively expressing GFP-SAP30L was selected from transfected HEK293 cells treated with geneticin (0.6 mg/mL).

RESULTADOS:

We are trying to understand the structural basis of the FEZ1 dimerization on its poly-Glu N-terminal region by structural determination through X-ray crystallography or nuclear magnetic ressonance (NMR). The monodispersity of the sample (6xHis-FEZ1[92-194]) was checked by DLS experiments before the crystallization assay. Circular dichroism evidenced a predominantly random-coil molecule, according to our previous data (Lanza et al., 2008) for 6xHis-FEZ1(1-227). We submitted 6xHis-FEZ1(92-194) to 6 initial crystallization kits at 6.0 mg/mL. The protein 6xHis-SAP30L was expressed in E. coli and purified, but it shows a high degree of degradation. HEK293 cells with stable expression of GFP-SAP30L were selected and we confirmed the nuclear localization of this protein. The FEZ1(92-194) structural analysis, currently in progress, will be important to understand FEZ1 dimerization. The functional data from yeast two-hybrid assays and sub-cellular co-localization with SAP30L and other interactors will be important to understand its possible function in the transcriptional regulation.

CONCLUSÕES:

The aims of FEZ1 poly-Glu N-terminal region structural analyses are to define if the dimerization occurs in dependent way of the concentration of protein FEZ1 and to determine the dimer structure in high resolution, by protein crystallography or NMR. The confirmation of the dimer structure of FEZ1 highlights the functional aspects of this structure. Until the present date, the FEZ1 sub-cellular localization is cytoplasmatic. The interaction of FEZ1 with diverse nuclear proteins, amongst these some factors involved in the gene transcription regulation (as the protein SAP30L) and cytoskeleton proteins opens new clues about it functions in the growth of neuritis and its involvement in diseases such as schizophrenia where the interaction of FEZ1 and DISC1 is disrupted. This is associated with intense alterations in the gene expression profile and functional changes of proteins associated to the cytoskeleton. The generation of dominant negative FEZ1 constructs and the use of FEZ1 iRNA techniques and analysis of FEZ1 co-localization with its interacting proteins will allow us to get new clues about the dimerization and its relevance in processes like neuronal cell development and schizophrenia.

Instituição de fomento: FAPESP, CNPq e LNLS

Palavras-chave: FEZ1 protein, neural cell development, yeast two-hybrid system

E-mail para contato: jkobarg@lnls.br