Estudo biofísico e funcional do complexo PrPc/STI-1

SEBASTIÁN A. ROMANO; YRAIMA CORDEIRO; LUIS MAURÍCIO T. R. LIMA; DÉBORA FOGUEL; RAFAEL LINDEN

Águas de Lindoia

Congreso; XXI Reunião Anual da Federação de Sociedades de Biologia Experimental; 2006

Federação de Sociedades de Biologia Experimental

Objetivos: Prion diseases are spongiform encephalopathies related to de cellular prion protein (PrPc), abundantly expressed in the central nervous system. During the course of the disease, PrPc is transformed in a pathological isoform, PrPSc, leading to neurodegeneration through accumulation of this abnormally folded protein. Numerous studies have focused on the pathological properties of PrPc, however, its cellular function is still unclear. Lately, our group has identified and studied PrPc binding proteins, showing that PrPc participates in a multi-protein complex resident in the cellular surface that it is involved in cell-cell and cell-extracellular matrix interactions that modulate neuronal survival and differentiation (J. Neurosci. 25, 11330-11339, 2005). One of the binding proteins is a 66kDa protein, known as stress-inducible protein (STI-1), which induces trophic and neuroprotective signals through the recruitment of neuronal PrPc. The present work aims at studying the biophysical and biochemical properties of the PrPc/STI-1 complex. These experiments provide important information for understanding this signalling complex, shedding light on the molecular mechanisms that underlie the PrPc cellular function. Métodos e Resultados: Mouse PrPc and STI-1 recombinant proteins were expressed in Eschericha coli and purified. Fluorescence anisotropy measures of the fluorescein-tagged PrPc were carried out to study the PrPc/STI-1 complex formation. UV Circular Dichroism (CD) provided information on the secondary structure of the proteins. SAXS measures carried out at the Laboratorio Nacional de Luz Síncrotron (LNLS). Confirming previous in vivo data, the in vitro formation of the PrPc/STI-1 complex was evidenced by a gradual increment of the fluorescence anisotropy of the tagged-PrPc as the STI-1 concentration was increased. CD data indicated the correct folding of the proteins and showed that the complex suffers a small but significant loss of secondary structure content when compared to the sum of the monomers spectra. SAXS data allowed us to obtain information about the structure and biophysical properties of the complex. A low-resolution model of the complex was developed. Conclusão: The low-resolution structure obtained by SAXS indicated a significant increase of the radius of gyration of the complex, compared to the radii of the monomeric proteins. Interestingly, this work evidenced that the PrPc/STI-1 interaction led to significant modifications of their secondary structures. This observation suggests a stretching of helical structures and a possible exposure of residues that could be relevant for the signalling capabilities of the complex.