C. Ciências Biológicas - 3. Bioquímica - 1. Biologia Molecular
FEZ1 IS AN NATIVELY UNFOLDED PROTEIN AND ITS OVER-EXPRESSION CAUSES A“FLOWER LIKE” PHENOTYPE IN HUMAN CELLS.
Daniel Carlos Ferreira Lanza1, 2
Júlio César da Silva1, 3
Daniel Maragno Trindade1, 2
Iris Concepcion Linares de Torriani1, 3
Jörg Kobarg1, 2
1. Laboratório Nacional de Luz Síncrotron
2. Instituto de Biologia, Universidade Estadual de Campinas
3. Instituto de Física “Gleb Wataghin”, Universidade Estadual de Campinas
INTRODUÇÃO:The protein FEZ1 (Fasciculation and Elongation protein Zeta-1) was initially identified as an orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for the formation and extension of the worms axons. Human FEZ1 interacts with Protein Kinase C (PKC) and several regulatory proteins involved in functions ranging from microtubule associated transport to transcriptional regulation. FEZ1 can be also classified as a hub protein, since it was reported to interact with over 40 proteins.METODOLOGIA:We performed experiments of circular dichroism, fluorescence spectroscopy, limited proteolysis and SAXS. Furthermore, in vitro phosphorylation and pull down assays were performed. Over-expression of FEZ1 constructs in human HEK293 and HeLa cells and subsequent analysis by FISH, FACS and confocal microscopy were utilized for cellular studies. Endogenous localization of FEZ1 was also observed by fluorescence microscopy.RESULTADOS:Our experiments of circular dichroism, fluorescence spectroscopy and proteolysis suggest that FEZ1 contains disordered regions, especially in its N-terminal. Pull down and SAXS experiments confirmed that FEZ1 dimerizes at its N-terminus. Ab initio modeling of the FEZ1 envelope showed a dimer of elongated shape. We performed in vitro phosphorylation assays of FEZ1 and phosphorylation occurred mainly in its C-terminal region and inhibited FEZ1 interaction with CLASP2 in vitro. Furthermore, we over-expressed GFP-FEZ1 in human cells and observed that over 40% of transiently transfected cells develop flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to cytoplasm and nuclei, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with alpha and gamma tubulin, which localizes as a centrosome like structure at the center of the multiple lobules.CONCLUSÕES:Concluding, our data suggest that FEZ1 is a natively unfolded protein, its binding and transport functions may be subject to regulation by phosphorylation and that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.
Instituição de fomento: We thank FAPESP and CNPq for financial support
Palavras-chave: microtubules, leukemia, PKC
E-mail para contato: danielcarlosf@yahoo.com.br
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