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Defining dynamic protein–protein interactions in the ubiquitin conjugationreaction is a challenging research area. Generating peptide aptamers thattarget components such as ubiquitin itself, E1, E2, or E3 could provide toolsto dissect novel features of the enzymatic cascade. Next-generation deepsequencing platforms were used to identify peptide sequences isolated fromphage-peptide libraries screened against Ubiquitin and its ortholog NEDD8.In over three rounds of selection under differing wash criteria, over 13,000peptides were acquired targeting ubiquitin, while over 10,000 peptides wereselected against NEDD8. The overlap in peptides against these two proteinswas less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8toward linear peptide motifs. Two of these ubiquitin-binding peptides wereidentified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysishighlighted distinct modes of binding of the two different peptide aptamers.These data highlight the utility of using next-generation sequencing ofcombinatorial phage-peptide libraries to isolate peptide aptamers toward aprotein target that can be used as a chemical tool in a complex multi-enzymereaction

Background: The identification of mutated proteins in human cancer cellstermedproteogenomics requires several technologically independent research methodologies including genomics, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape.Methods: An isogenic melanoma cell with a p53-null genotype was createdby CRISPR/CAS9 system to determine how p53 gene inactivation affectsmutant proteome expression. A mutant peptide reference database wasdeveloped by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database.Results: Approximately 190 mutated peptides were enriched in wt-p53cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wtp53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes.Conclusion: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupledto the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells.General significance: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.

Additional file 4: Supplementary Table 1 Summary of the MDM2 protein-protein interactions known to be regulated by Nutlin-3. Abbreviations: SPR (surface plasmon resonance), NPM (MDM2-nucleophosmin interaction), CypB (cyclophilin B), GRK2 (G-protein-coupled receptor kinase 2), GPR17 (G-protein-coupled receptor), DLD (dihydrolipoamide dehydrogenase), YFP (yellow fluorescent protein), SWATH (sequential window acquisition of all theoretical mass spectra).

Survival analysis based on Tomm34 expression in epithelial ovarian cancers with p53 mutation. (A) Overall survival, (B) progression free survival and (C) post progression survival. (PDF 204 kb)

HDM2 and HDMX are two homologs essential for controlling p53 tumor suppressor activity under normal conditions. Both proteins bind different sites on the p53 N-terminus, and while HDM2 has E3 ubiquitin ligase activity towards p53, HDMX does not. Nevertheless, HDMX is required for p53 polyubiquitination and degradation, but the underlying molecular mechanism remains unclear. Alone, HDMX and HDM2 interact via their respective C-terminal RING domains but here we show that the presence of p53 induces an N-terminal interface under normal cellular conditions. This results in an increase in HDM2-mediated p53 polyubiquitination and degradation. The HDM2 inhibitor Nutlin-3 binds the N-terminal p53 binding pocket and is sufficient to induce the HDM2-HDMX interaction, suggesting that the mechanism depends on allosteric changes that control the multiprotein complex formation. These results demonstrate an allosteric interchange between three different proteins (HDMX-HDM2-p53) and help to explain the molecular mechanisms of HDM2-inhibitory drugs.

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. Published version

p53 is a tetrameric protein with a thermodynamically unstable deoxyribonucleic acid (DNA)‐binding domain flanked by intrinsically disordered regulatory domains that control its activity. The unstable and disordered segments of p53 allow high flexibility as it interacts with binding partners and permits a rapid on/off switch to control its function. The p53 tetramer can exist in multiple conformational states, any of which can be stabilized by a particular modification. Here, we apply the allostery model to p53 to ask whether evidence can be found that the “activating” C‐terminal phosphorylation of p53 stabilizes a specific conformation of the protein in the absence of DNA. We take advantage of monoclonal antibodies for p53 that measure indirectly the following conformations: unfolded, folded, and tetrameric. A double antibody capture enzyme linked‐immunosorbent assay was used to observe evidence of conformational changes of human p53 upon phosphorylation by casein kinase 2 in vitro. It was demonstrated that oligomerization and stabilization of p53 wild‐type conformation results in differential exposure of conformational epitopes PAb1620, PAb240, and DO12 that indicates a reduction in the “unfolded” conformation and increases in the folded conformation coincide with increases in its oligomerization state. These data highlight that the oligomeric conformation of p53 can be stabilized by an activating enzyme and further highlight the utility of the allostery model when applied to understanding the regulation of unstable and intrinsically disordered proteins.