UNRAVELLING THE MOLECULAR MECHANISMS OF IMPAIRED LIMB DEVELOPMENT: ROLE OF DNP63A IN SPLIT-HAND/FOOT MALFORMATION TYPE 4 AND IN THALIDOMIDE TERATOGENICITY
Creators: name: MOLINARI, ELISA
Publisher: name: Universit� degli Studi di Milano
Year: 2015

description: The p63 transcription factor, homolog to the p53 tumour suppressor, plays a key role in limb, epithelial and cranio-facial development. p63 activity and stability are tightly modulated to guarantee correct development of such structures and an impairment of this regulative mechanism can result in severe malformations. The control of p63 function is achieved not only through the regulation of its gene expression but also through a complex network of post-translational modifications. Acetylation, phosphorylation and ubiquitination affect p63 half-life, the specificity and efficiency of protein-protein interactions and overall modulate the transcriptional activity of the protein. Here we describe two pathways that, by post-translationally modifying ?Np63? protein, modulate its stability and function. One such pathway involves FGF8, c-Abl and p300 which cooperate in controlling the stability and function of ?Np63? protein by leading to its acetylation on lysine 193 (K193). Interestingly, K193 is mutated into glutamic acid (K193E) in patients affected by Split Hand/Foot Malformation (SHFM) type 4. c-Abl kinase activity is required to transduce the signal induced by FGF8 leading to ?Np63? stabilization and transcriptional activation, through its acetylation mediated by p300. The ?Np63?-K193E mutant, which cannot be acetylated by this pathway, displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ?Np63? target genes. Our results, elucidating an important regulatory pathway activated by FGF8 and essential for ?Np63? activation and stabilization, shed new light on the molecular mechanism that could be at the basis of the SHFM4 pathogenesis. The other pathway that we here present is a degradative pathway promoted by the teratogenic drug thalidomide that leads to proteasome-mediated degradation of ?Np63?, resulting in a lack of activation of ?Np63? development-related target genes. In cell lines, thalidomide treatment induces a downregulation of ?Np63? protein via the action of GSK3 kinase and FBWX7 ubiquitin ligase. Upon thalidomide treatment, GSK3 kinase is required to phosphorylate ?Np63? on the residues serine 383 and threonine 397. This phosphorylation is recognized as a signal by FBWX7 which in turn ubiquitinates ?Np63?, leading to its proteasome-mediated degradation. Thalidomide treatment induces a downregulation of ?Np63? protein levels also in vivo in zebrafish embryos, where it results in a phenotypical and molecular impairment of fin development. Importantly, the microinjection of zp63-mRNA into zebrafish embryos treated with thalidomide rescues both the phenotypical and molecular defects induced by the drug, indicating that the downregulation of ?Np63? is, at least in part, at the bases of thalidomide-induced malformations. Our results, by demonstrating that ?Np63? is a molecular target of thalidomide teratogenicity, provide a fundamental missing piece in the description of the drug molecular mechanism of action. Thalidomide is now used for the treatment of multiple myeloma and leprosy: a better understanding of its mechanism of action might pave the way for the design of related compounds with equal therapeutic properties but devoid of teratogenic activity.