Tumor suppressor p53: analysis of wild-type and mutant p53 complexes.

It has been suggested that the dominant effect of mutant p53 on tumor progression may reflect the mutant protein binding to wild-type p53, with inactivation of suppressor function. To date, evidence for wild-type/mutant p53 complexes involves p53 from different species. To investigate wild-type/mutant p53 complexes in relation to natural tumor progression, we sought to identify intraspecific complexes, using murine p53. The mutant phenotype p53-246(0) was used because this phenotype is immunologically distinct from wild-type p53-246+ and thus permits immunological analysis for wild-type/mutant p53 complexes. The p53 proteins were derived from genetically defined p53 cDNAs expressed in vitro and also from phenotypic variants of p53 expressed in vivo. We found that the mutant p53 phenotype was able to form a complex with the wild type when the two p53 variants were cotranslated. When mixed in their native states (after translation), the wild-type and mutant p53 proteins did not exhibit any binding affinity for each other in vitro. Under identical conditions, complexes of wild-type human and murine p53 proteins were formed. For murine p53, both the wild-type and mutant p53 proteins formed high-molecular-weight complexes when translated in vitro. This oligomerization appeared to involve the carboxyl terminus, since truncated p53 (amino acids 1 to 343) did not form complexes. We suggest that the ability of the mutant p53 phenotype to complex with wild type during cotranslation may contribute to the transforming function of activated mutants of p53 in vivo.     

Mutant p53 exerts oncogenic effects through microRNAs and their target gene networks

MicroRNAs are potent regulators of gene expression and modulate multiple cellular processes including proliferation, differentiation and apoptosis. A number of microRNAs have been shown to be regulated by p53, the most frequently mutated gene in human cancer. It is has been demonstrated that some mutant p53 proteins not only lose tumor suppressor activity, but also acquire novel oncogenic functions that are independent of wild-type p53. In this review, we highlight recent evidences suggesting that some mutant p53 proteins regulate the expression of specific microRNAs to gain oncogenic functions and identify a gene network regulated by the microRNAs downstream of mutant p53.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

KSHV latent protein LANA2 inhibits sumo2 modification of p53

Tumor suppressor p53 plays a crucial antiviral role and targeting of p53 by viral proteins is a common mechanism involved in virus oncogenesis. The activity of p53 is tightly regulated at the post-translational levels through a myriad of modifications. Among them, modification of p53 by SUMO has been associated with the onset of cellular senescence. Kaposi´s sarcoma-associated herpesvirus (KSHV) expresses several proteins targeting p53, including the latent protein LANA2 that regulates polyubiquitylation and phosphorylation of p53. Here we show that LANA2 also inhibits the modification of p53 by SUMO2. Furthermore, we show that the reduction of p53-SUMO2 conjugation by LANA2, as well as the p53-LANA2 interaction, both require the SUMOylation of the viral protein and its interaction with SUMO or SUMOylated proteins in a non-covalent manner. Finally, we show that the control of p53-SUMO2 conjugation by LANA2 correlates with its ability to inhibit SUMO2- and type I interferon-induced senescence. These results highlight the importance of p53 SUMOylation in the control of virus infection and suggest that viral oncoproteins could contribute to viral infection and cell transformation by abrogating p53 SUMOylation.     

Surface-enhanced Raman scattering detection of wild-type and mutant p53 proteins at very low concentration in human serum

The development of ultrasensitive and rapid approaches to detect tumor markers at very low concentrations even in a physiological environment represents a challenge in nano-medicine. The p53 protein is at the center of the cellular network that protects organisms against the insurgence of tumors, most of which are related to alteration of p53 expression. Therefore p53 is regarded as a valuable prognostic marker whose detection at high sensitivity may considerably contribute to early diagnosis of cancers. In this work we have applied an analytical method based on surface enhanced Raman spectroscopy with high sensitivity and rapidity to improve traditional bioaffinity techniques. The Raman reporter bifunctional linker 4-aminothiophenol (4-ATP) first assembled onto 50 nm gold nanoparticles (Nps) has then been azotated to bind low concentration wild-type and two mutated forms of p53 proteins. The Raman signal enhancement of the resulting p53-(4-ATP-Np) systems has been used to identify the p53 molecules captured on a recognition substrate constituted by the azurin (Az) protein monolayer. Az has shown a strong association for both wild-type and mutated p53 proteins, allowing us to selectively detect these proteins at concentrations as low as 500 fM, in a human serum environment.     

Oligomerization of p53 Precedes its Association with Dynein and Nuclear Accumulation

Previous studies have identified several proteins that associate with microtubules and the dynein motor complex including p53, the glucocorticoid and the vitamin D receptors, and the APC (adenomatous polyposis coli) protein; but neither the residues important for this interaction nor the physical state of the proteins involved have been clarified. We observed in SN12C cells harboring a mutant p53 truncated at amino acid 336, impaired nuclear localization and impaired association with dynein. This finding was confirmed and extended by examining a series of truncated p53 proteins that identified residues 336 to 348 as crucial for association with dynein and nuclear transport. Point mutations identified the importance of residues involved in p53 oligomerization in this process, establishing a p53 oligomer as the cargo for dynein transport. The association of cytosolic p53 oligomers with dynein occurs independent of microtubules indicating that following this association, the p53/dynein complex then associates with microtubules and is transported to the peri-nuclear region. These studies suggest that mutations or modifications that affect p53 oligomerization not only interfere with DNA binding but also with its intracellular distribution. They also highlight the importance of an intact microtubule network in the trafficking of crucial cellular proteins.     

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.