IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Modulation of the intrinsically disordered HPV E7 oncoprotein by calcium.
Autor/es:
NOVAL MARÍA GABRIELA; CHEMES LUCÍA BEATRIZ; SALVAY ANDRES; DE PRAT-GAY GONZALO
Lugar:
Buenos Aires
Reunión:
Workshop; 3rd ICGEB WORKSHOP ON HUMAN RNA VIRUSES; 2012
Resumen:
Intrinsically disordered proteins are functional proteins that lack any constant secondary and terciary structure under physiological conditions. However, these proteins have regions with local structural tendencies which provide the possibility of undergo unstructured-structured transitions upon binding with their cellular targets. Most proteins containing disordered regions are involved in signal transduction and regulation events that imply multiple partner interactions. In particular, viral proteins are rich in intrinsic disordered regions such as the measles virus N protein, the flaviviruses core proteins, E7-HPV16 oncoprotein and many others. Due to the widespread presence of these intrinsically disordered regions, it is of great interest to further describe and characterize its structural behavior. As regards this, the E7 oncoprotein, the major transforming protein of human papiloma viruses, is a small acidic protein, which consists of two domains: an intrinsically disordered, extended N-terminal domain (E7N) and a globular C-terminal domain (E7C). The intrinsically disordered domain contains two conserved regions, CR1 and CR2. The CR2 region includes the LxCxE motif, which mediates high-affinity binding to Rb and is also present in the AdE1A and SV40-LT proteins. It has been postulated that calcium could be essential for E7-mediated Rb proteosomal degradation, suggesting that E7 could interact with this cation. In order to analyze such interaction and if this interaction modulates the conformation of the intrinsically disordered domain, we studied the effect of calcium in the full length E7-oncoprotein and in E7N. For this study we used biophysical techniques, such as circular dicroism, size exclusion chromatography, fluorescence spectroscopy and analytical ultracentrifugation. We have found that concentrations between 100 μM and 1 mM of Ca2+ changed the conformation of the full length protein and also E7N in aqueous media. To identify which structure is induced by Ca2+ in E7N, we used TFE titrations and variable temperature measurements. We found that Ca2+ increased the tendency towards formation of alpha helix, and decreased the tendency of PII structure with changes in the hydrodynamic radii. For further analysis, we used synthetic peptides of the CR1 and CR2 regions. We found that the effect on the hydrodynamic radii is centered only in the CR2 region. Instead, the effect in the tendency to alpha helix formation is centered in both CR1 and CR2. Finally, we performed additional studies using E7N domains of the high risk HPV-18, low risk HPV-11 and the BPV-1 strains that present different grade of disorder in their domain. We observed a positive correlation between the disorder rate and the capacity of these domains to be modulated by calcium. In spite of the requirement of further studies for completely understand the effect of this cation in the domain and in the whole protein, we can conclude that the interaction with Ca2+ modulates the conformation of E7N IDP domain, suggesting that in a cellular context this interaction could play an important rol in the binding with their targets.