Mimicry of p53 mono-ubiquitylation

Abstract

The tumor suppressor p53 is subjected to various posttranslational modifications (PTMs) that affect various properties of p53 including half-life, function and subcellular localization. The C-terminal 30 amino acids of p53 are assumed to play an important role in regulating p53's function(s). Consistent with this hypothesis, the C-terminal region is subjected to various PTMs including acetylation, phosphorylation, methylation, ubiquitylation, neddylation or sumoylation. However, the actual contribution of individual PTMs to p53 function and regulation remains mostly unclear. To gain deeper insights into p53 regulation by mono-ubiquitylation, site-specifically mono-ubiquitylated p53 needs to be generated. Moreover, non-hydrolysable mono-ubiquitylated p53 allows to identify proteins that interact with p53 dependent on its modification status in cell lysates since the modification cannot be hydrolysed by deubiquitylating enzymes. During the work of this thesis, various forms of p53-ubiquitin (Ub) conjugates were generated by utilizing different bioorthogonal strategies. Initially, we employed Cu(I)-catalyzed alkyne-azide cycloaddition (CuAAC) to link p53 and Ub, as it is already used regularly to generate Ub dimers, Ub chains and other Ub conjugates. Therefore, p53 and Ub were functionalized with an alkyne- and azide-reactive group, respectively, by amber codon suppression and selective pressure incorporation. The bioorthogonal reaction results in a triazol linkage, mimicking the native isopeptide bond. After the generation of p53-Ub site-specifically linked at position 372, we investigated the interaction of this conjugate with the E3 ligase complex E6/E6AP. No ubiquitylation of p53-372Ub or Cu-treated p53 was observered, indicating a conformational change of p53. Thus, we focused our efforts with CuAAC to a truncated form of p53 (CTD) encompassing amino acid 325-393. p53 CTD mutants, which harbor specific lysine to cysteine mutations, were functionalized with an alkyne by Michael addition of propargyl acrylate. CuAAC allowed to generate reasonable amounts of Ub-linked p53 CTD at positions 372 and 386. Due to possible modulation of the interactome of mono-ubiquitylated p53, a quantitative affinity enrichment experiment with cell lysate was performed. By using p53 CTD, we were able to identify 176 already known p53 interactors. In particular, mono-ubiquitylated p53 CTD enabled to link p53 via physical interactions to lysosomal degradation, endosomal trafficking and DNA repair. Additionally, we changed the biorthogonal strategy for full-length p53 to metal-free oxime ligation. Oxime ligation is based on the reaction of a keto or aldehyde moiety with an aminooxy moiety. Thus, we incorporated keto-lysine in p53 and a Boc-protected aminooxy-lysine derivative in Ub using amber codon suppression. This enabled the generation of a p53-Ub conjugate site-specifically linked at position 120. The oxime-linked p53-Ub was proven to be functional by studies with the E3 ligase complex E6/E6AP. Notably, preliminary DNA binding studies hint to an altered DNA binding behavior, at least with respect to the p21 response element. Taken together, in this thesis different chemical approaches to investigate the effect of site-specifc mono-ubiquitylation on p53 functions were established. The produced p53 conjugates were characterized in respect to DNA binding, identification of cellular binding partners and the effect of E3 ligase (E6/E6AP or Hdm2)-mediated ubiquitiylation.