Interaction of an anticancer peptide fragment of azurin with p53 and its isolated domains studied by atomic force spectroscopy

Abstract

p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.

MDM2–MDM4 molecular interaction investigated by atomic force spectroscopy and surface plasmon resonance

“…The specificity of the MDM2-MDM4 interaction is clearly revealed both by the F* values and the unbinding frequency, obtained by force measurements, and by the x β and the k off values, obtained by fitting with the Bell-Evans model, which are in the range usually reported for specific biological interactions. 24,33,35,36,43 Moreover, the lifetime τ (τ =1/k off ) of the MDM2-MDM4 interaction is .70 times longer than τ of the MDM2-p53 association; such a value suggests that the heterodimer is available for several cycles of association and dissociation with p53 before the displacement of the MDM2-MDM4 complex occurs. 34 The relatively long lifetime of the complex with respect to that of MDM2-p53 might be consistent with the efficacy of the heterodimer in the p53 downregulation.…”

“…36 To complete the kinetic profile of the interaction, we also estimated the association rate constant (k on ) for the MDM2-MDM4 complex according to the expression k on = N A × V eff /t 0.5 , where N A is the Avogadro's number, V eff is the effective volume of a halfsphere with a radius r eff around the tip, and t 0.5 is the time for the half-maximal binding probability, given by t 0.5 =2 r eff /ν, where ν is the approach speed of the cantilever. 42,43 Accordingly, a k on of approximately 10 4 M -1 s -1 was obtained. The assessment of both the dissociation and association rate constants allowed us to determine an equilibrium dissociation constant K D , calculated as K D = k off /k on , of approximately 10 -6 M for the MDM2-MDM4 complex.…”

A nanotechnological, molecular-modeling, and immunological approach to study the interaction of the anti-tumorigenic peptide p28 with the p53 family of proteins

“…4,5 Once bound to either wild type or mutated p53, 6,7 p28 blocks the binding of the E3 ubiquitin ligase Cop1 and induces its autodegradation. This increases the level and activity of p53 and the expression of its downstream cell cycle targets p21 and p27 significantly, inhibiting the cancer cell cycle at G 2 /M.…”

“…This increases the level and activity of p53 and the expression of its downstream cell cycle targets p21 and p27 significantly, inhibiting the cancer cell cycle at G 2 /M. 4,5,8 The structural similarity between p53 DBD and that of its homologues p63 and p73 suggests that p28 could also bind to either p63 or p73 and potentially enhance the reported tumor suppressive activity of individual isomers 4,7,9 through a similar mechanism of post-translational stabilization that alters their ubiquitination. 23 In addition to autoregulatory feedback loops, polyubiquitination regulates the stability of the p53 family of proteins through a series of E3 ubiquitin ligases divided into three subfamilies: the Homologous to E6-AP Carboxyl Terminus (HECT) domain-containing E3s, the Really Interesting New Gene (RING) finger domain-containing E3s, and the U box E3s.…”

“…20,29,30 We used atomic force spectroscopy (AFS) to determine the binding characteristics between p28, p63, and p73 at the single molecule level with piconewton sensitivity, in near native conditions without labeling. This strategy was integrated with dynamic modeling and a molecular immunologic approach to demonstrate that p28 also binds stably and with high affinity to the DBD of p63 and p73 but, unlike p53, 6,7 does not uniformly increase the intracellular level of either by inhibiting the binding of HECT or RING E3 ubiquitin ligases.…”