Small molecule induced reactivation of mutant p53 in cancer cells

The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Here, we have identified a compound, PK7088, which is active in vitro: PK7088 bound to the mutant with a dissociation constant of 140 μM and raised its melting temperature, and we have determined the binding mode of a close structural analogue by X-ray crystallography. We showed that PK7088 is biologically active in cancer cells carrying the Y220C mutant by a battery of tests. PK7088 increased the amount of folded mutant protein with wild-type conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. We suggest a set of criteria for assigning activation of p53

p63 expression in normal skin and usual cutaneous carcinomas

Background: p63 is a p53 homologue that is mapped to chromosome 3q27. This gene encodes six different isoforms, which have either transactivating or dominant negative effects on p53-reporter genes. It has been described that in contrast to p53, p63 seems not to be associated with tumor predisposition, as neither p63 knockout mouse models nor germline p63 mutations are related to an increased risk of tumorigenesis. It has been demonstrated that p63 is a reliable keratinocyte stem cell marker and that it is involved in the maintenance of the stem cell population. Scant data on p63 expression in normal skin, basal cell carcinomas (BCCs), keratoacanthomas and squamous cell carcinomas (SCCs) have been reported. We herein evaluated p63 expression in 16 BCCs, one keratoacanthoma and 13 SCCs. Methods: Immunohistochemistry according to the streptavidinbiotin-peroxidase technique, using the antibody 4A4 raised against all p63 isoforms, was performed. p63 expression was evaluated in epidermal cells and skin appendages. Semi-quantitative evaluation (–, π, ππ, πππ) of p63 expression in BCCs, keratoacanthoma and SCCs was carried out. Only nuclear expression was considered as specific. Results: p63 was expressed in the nuclei of epidermal basal and suprabasal cells, in the cells of the germinative hair matrix and the external root sheath of hair follicles, in the basal cells of the sebaceous gland and in the myoepithelial/basal cells of the sweat glands. All terminally differentiated cells were negative for p63. All BCCs showed ππto πππ immunoreactivity. At variance, keratoacanthomas and grade I and II SCCs showed variable p63 reactivity in a basal layerlike distribution, whereas undifferentiated cells of grade III SCCs showed ππto πππ positivity. A grade IV spindle SCC showed π immunoreactivity. The SCCs in situ showed remarkable expression of p63 in all cell layers. Terminally differentiated squamous cells were either negative or showed only focal immunoreactivity in the carcinomas. Conclusions: p63 is consistently expressed in the basal cells of epidermis and cutaneous appendages, including the basal/ myoepithelial cells of sweat glands. Based on our findings, the balance of probabilities favors that p63 might play a role in the pattern of differentiation and in the oncogenesis of usual carcinomas of the skin.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/5386/2001. Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI)

Moonlighting Proteins Hal3 and Vhs3 Form a Heteromeric PPCDC with Ykl088w in Yeast CoA Biosynthesis

Premi a l'excel·lència investigadora. 2010Unlike most other organisms, the essential five-step Coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the gene(s) encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remains unidentified. Sequence homology analyses suggest three candidates, namely Ykl088w, Hal3 and Vhs3, as putative PPCDC enzymes in Saccharomyces cerevisiae. Interestingly, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3/Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer which consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3/Vhs3 as moonlighting proteins, involved in both CoA biosynthesis and protein phosphatase regulation