The tumor suppressor protein, p53, is either mutated or absent in >50% of cancers and is negatively regulated by the mouse double minute (MDM2) protein. Understanding and inhibition of the MDM2-p53 interaction are, therefore, critical for developing novel chemotherapeutics, which are currently limited because of a lack of appropriate study tools. We present a nanosensing approach to investigate full-length MDM2 interactions with p53, thus providing an allosteric assay for identifying binding ligands. Surface-enhanced Raman scattering (SERS)-active nanoparticles, functionalized with a p53 peptide mimic (peptide 12.1), display biologically specific aggregation following addition of MDM2. Nanoparticle assembly is competitively inhibited by the N-terminal MDM2-binding ligands peptide 12.1 and Nutlin-3. This study reports nanoparticle assembly through specific protein-peptide interactions that can be followed by SERS. We demonstrate solution-based MDM2 allosteric interaction studies that use the full-length protein.assay system | biosensing | nano-assembly | protein interaction studies | Raman spectroscopyT he p53 tumor suppressor protein is often referred to as the "guardian of the genome" owing to its key role in cell-cycle regulation and its activity in a number of different cancer pathways (1-3). The antiproliferative action of p53 arises from the induction of cell-cycle arrest and apoptosis in cells subjected to DNA damage in response to stress. As such, p53 is central to protecting cells from uncontrolled growth and malignant transformation with inactivation or mutation of p53 found in over 50% of human cancers (1-3). Under nonstressed conditions, mouse double minute (MDM)2 negatively regulates p53, primarily through the ubiquitin degradation pathway but also via transrepression (4, 5). Studies have shown up-regulation of MDM2 in malignancies where p53 is fully functional, making the MDM2-p53 feedback loop of great interest in chemotherapeutic studies (6, 7).MDM2 is a multidomain protein that exerts E3-ligase activity on a number of proteins, including p53 (5, 8, 9). The ubiquitination activity of MDM2 is a multisubunit process whereby the N-terminal hydrophobic pocket and central acidic domain of MDM2 are used in p53 binding, to the N-terminal and central domains of p53, respectively (10). The complexity of this multisubunit interaction makes it relatively difficult to investigate the allosteric nature of MDM2. This is made more difficult by the problems in acquiring a structure of full-length MDM2 because of intrinsically disordered regions on the protein that reduce likelihood of crystallization. Focus solely on the N-terminal domain of MDM2 has identified Nutlin-3-type molecules that activate p53 in cells; however, these also act as allosteric agonists that stimulate rather than inhibit p53 ubiquitination (10-15). The putative dimerization of MDM2 through the C-terminal really interesting new gene (RING) domain is reported to be critical for E3-ligase activity; however, molecular reasoning for this is not f...

Better understanding of GBM signalling networks in-vivo would help develop more physiologically relevant ex vivo models to support therapeutic discovery. A “functional proteomics” screen was undertaken to measure the specific activity of a set of protein kinases in a two-step cell-free biochemical assay to define dominant kinase activities to identify potentially novel drug targets that may have been overlooked in studies interrogating GBM-derived cell lines. A dominant kinase activity derived from the tumour tissue, but not patient-derived GBM stem-like cell lines, was Bruton tyrosine kinase (BTK). We demonstrate that BTK is expressed in more than one cell type within GBM tissue; SOX2-positive cells, CD163-positive cells, CD68-positive cells, and an unidentified cell population which is SOX2-negative CD163-negative and/or CD68-negative. The data provide a strategy to better mimic GBM tissue ex vivo by reconstituting more physiologically heterogeneous cell co-culture models including BTK-positive/negative cancer and immune cells. These data also have implications for the design and/or interpretation of emerging clinical trials using BTK inhibitors because BTK expression within GBM tissue was linked to longer patient survival.

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