The tumour suppressor p53 plays a crucial role in cell stress response and its anticancer activity is mainlydown-regulated by the oncoprotein Mdm2 that, upon binding to p53, blocks its transcriptional activity andpromotes its ubiquitin-dependent degradation. Targeting Mdm2–p53 interaction is believed to be the most directof all p53-activating strategies to treat tumours in which p53 has retained its wild-type function. The bacterialprotein Azurin has been shown to bind p53, inhibiting cancer cell proliferation likely through a post-translationalincreasing of the p53 level. This apparent antagonist action with respect to the Mdm2–p53 functional interactionsuggests that binding of Azurin to p53 might interfere with the Mdm2–p53 association and, thus, preventing p53from degradation. Toward this end, a detailed kinetic characterization of the binding interaction of these threeproteins has been performed by surface plasmon resonance. The occurrence of specific binary interactions of bothAzurin andMdm2 with p53, as investigated more appropriately in their full-length conformation, is ascertained andthe corresponding association and dissociation rate constants are measured. Interestingly enough, the threeproteins are likely engaged in a ternary interaction, whose kinetics points out that binding of Azurin to p53 causes asignificant decrease of the Mdm2–p53 association rate constant and binding affinity, without hindering theaccessibility of Mdm2 to the binding pocket of p53. The Azurin-induced p53 conformational change, as demonstratedby circular dichroism, suggests that the protein may affect Mdm2–p53 association through an allostericmechanism, which could give an useful insight into designing new anticancer drugs.
Azurin modulates the association of Mdm2 with p53: SPR evidence from interaction of the full-length proteins
D'Orazi, Gabriella;
2011-01-01
Abstract
The tumour suppressor p53 plays a crucial role in cell stress response and its anticancer activity is mainlydown-regulated by the oncoprotein Mdm2 that, upon binding to p53, blocks its transcriptional activity andpromotes its ubiquitin-dependent degradation. Targeting Mdm2–p53 interaction is believed to be the most directof all p53-activating strategies to treat tumours in which p53 has retained its wild-type function. The bacterialprotein Azurin has been shown to bind p53, inhibiting cancer cell proliferation likely through a post-translationalincreasing of the p53 level. This apparent antagonist action with respect to the Mdm2–p53 functional interactionsuggests that binding of Azurin to p53 might interfere with the Mdm2–p53 association and, thus, preventing p53from degradation. Toward this end, a detailed kinetic characterization of the binding interaction of these threeproteins has been performed by surface plasmon resonance. The occurrence of specific binary interactions of bothAzurin andMdm2 with p53, as investigated more appropriately in their full-length conformation, is ascertained andthe corresponding association and dissociation rate constants are measured. Interestingly enough, the threeproteins are likely engaged in a ternary interaction, whose kinetics points out that binding of Azurin to p53 causes asignificant decrease of the Mdm2–p53 association rate constant and binding affinity, without hindering theaccessibility of Mdm2 to the binding pocket of p53. The Azurin-induced p53 conformational change, as demonstratedby circular dichroism, suggests that the protein may affect Mdm2–p53 association through an allostericmechanism, which could give an useful insight into designing new anticancer drugs.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
