RESUMO
The tumor suppressor p53 is regulated by various posttranslational modifications including different types of ubiquitylation, which exert distinct effects on p53. While modification by ubiquitin chains targets p53 for degradation, attachment of single ubiquitin moieties (mono-ubiquitylation) affects the intracellular location of p53 and/or its interaction with chromatin. However, how this is achieved at the molecular level remains largely unknown. Similarly, since p53 can be ubiquitylated at different lysine residues, it remains unclear if the eventual effect depends on the position of the lysine modified. Here, we combined genetic code expansion with oxime ligation to generate p53 site-specifically mono-ubiquitylated at position 120. We found that mono-ubiquitylation at this position neither interferes with p53 ubiquitylation by the E3 ligases HDM2 and E6AP in complex with the viral E6 oncoprotein nor affects p53 binding to a cognate DNA sequence. Thus, ubiquitylation per se does not affect physiologically relevant properties of p53.
Assuntos
Proteínas Oncogênicas Virais , Proteína Supressora de Tumor p53 , Lisina/metabolismo , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteína Supressora de Tumor p53/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Deregulation of the ubiquitin ligase E6AP is causally linked to the development of human disease, including cervical cancer. In complex with the E6 oncoprotein of human papillomaviruses, E6AP targets the tumor suppressor p53 for degradation, thereby contributing to carcinogenesis. Moreover, E6 acts as a potent activator of E6AP by a yet unknown mechanism. However, structural information explaining how the E6AP-E6-p53 enzyme-substrate complex is assembled, and how E6 stimulates E6AP, is largely missing. Here, we develop and apply different crosslinking mass spectrometry-based approaches to study the E6AP-E6-p53 interplay. We show that binding of E6 induces conformational rearrangements in E6AP, thereby positioning E6 and p53 in the immediate vicinity of the catalytic center of E6AP. Our data provide structural and functional insights into the dynamics of the full-length E6AP-E6-p53 enzyme-substrate complex, demonstrating how E6 can stimulate the ubiquitin ligase activity of E6AP while facilitating ubiquitin transfer from E6AP onto p53.
Assuntos
Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Sítios de Ligação , Humanos , Espectrometria de Massas , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Especificidade por Substrato , UbiquitinaçãoRESUMO
POU-V class proteins like Oct4 are crucial for keeping cells in an undifferentiated state. An Oct4 homologue in Xenopus laevis, Oct25, peaks in expression during early gastrulation, when many cells are still uncommitted. Nevertheless, extensive morphogenesis is taking place in all germ layers at that time. Phenotypical analysis of embryos with Oct25 overexpression revealed morphogenesis defects, beginning during early gastrulation and resulting in spina-bifida-like axial defects. Analysis of marker genes and different morphogenesis assays show inhibitory effects on convergence and extension and on mesoderm internalization. On a cellular level, cell-cell adhesion is reduced. On a molecular level, Oct25 overexpression activates expression of PAPC, a functional inhibitor of the cell adhesion molecule EP/C-cadherin. Intriguingly, Oct25 effects on cell-cell adhesion can be restored by overexpression of EP/C-cadherin or by inhibition of the PAPC function. Thus, Oct25 affects morphogenesis via activation of PAPC expression and subsequent functional inhibition of EP/C-cadherin.
