Helical Packing Regulates Structural Transitions In Bax

Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a molecular switch to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions – in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a molecular switch to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors

Molecular mechanism of drug induced apoptosis and chemoresistance in estrogen receptor alpha +/- breast cancer cell lines: mcf-7 and mda-mb-231

It was recently shown that inhibition or downregulation of Bcl-2 represents a new therapeutic approach to by-pass chemoresistance mechanism in cancer cells. Therefore, we explored the potential of this approach in breast cancer cells; MCF-7 (drug-sensitive; p53 wild type) and MDA-MB-231 (drug-insensitive; p53 mutant). Cisplatin and paclitaxel induced apoptosis in a dose-dependent manner in both cell lines. Furthermore, silencing of Bcl-2 remarkably increased cisplatin and paclitaxel induced apoptosis. Dose dependent induction of apoptosis by cisplatin and paclitaxel was enhanced by the pre-treatment of these cells with HA14-1, a Bcl-2 inhibitor. Although the effect of cisplatin on cell death was significant in MCF-7 and MDA-MB- 231, paclitaxel was less potent only in MDA-MB-231 cells. To further understand the distinct role of drugs in breast cancer cells which were pre-treated with HA14-1, changes in mitochondrial membrane potential, caspase activation, and Bcl-2 family protein levels, generation of reactive oxygen species and lipid peroxidation were studied. The apoptotic effect of cisplatin with or without HA14- 1 pre-treatment was shown to be caspase-dependent in both cell lines. While proapoptotic Bcl-2 proteins (Bax, Puma, Bad) were found to be up-regulated, Bcl-2 and Bcl-xL were down-regulated when cells were pre-treated with HA14-1 followed by cisplatin or paclitaxel. MCF-7 and MDA-MB-231 cells overexpressing Bcl-2 displayed different responses upon drug-treatment. Although cisplatin could still induce apoptosis in Bcl-2 overexpressing MCF-7 cells by promoting pro-apoptotic Bcl-2 family members, Bcl-2 overexpression abrogated paclitaxel induced apoptosis in MCF-7 and MDA-MB-231 breast cancer cells, respectively. In conclusion, our findings suggest two important implications for understanding cisplatin and paclitaxel induced apoptosis mechanism and the potential role of Bcl-2 in this apoptotic pathway. First, the potentiating effect of Bcl-2 inhibitor (HA14-1) is drug and cell type specific and may not only depend on the inhibition of Bcl-2. Importantly, alteration of other pro-apoptotic or anti-apoptotic Bcl-2 family members may dictate the apoptotic response when HA14-1 is combined with chemotherapeutic drugs. Second, cisplatin activated a p53- regulated pro-apoptotic pathway to overcome Bcl-2 mediated resistance. These insights may be useful for the development of novel treatments for cancer cells overexpressing anti-apoptotic Bcl-2 proteins

Unravelling the molecular mechanism of Sorcin (SOluble Resistance-related Calcium binding proteIN)-dependent resistance to chemotherapeutic drugs in cancer and its network of interaction

Sorcin is calcium-binding oncoprotein overexpressed in several human tumors, is a marker of Multi-Drug Resistance (MDR), is highly expressed in chemoresistant cell lines, and confers MDR when overexpressed. Sorcin gene is in the same amplicon of glycoprotein-P (mdr1) and its silencing increases cancer cell sensitivity to chemotherapeutic drugs. Recently we solved the crystal structure of Sorcin in the apo and calcium-bound forms. Upon calcium binding, a large conformational change occurs, with the exposure of hydrophobic surfaces, that allows Sorcin interaction with molecular targets, some of them unravelled by a cutting-edge technique called Proteomic Peptide Phage Display (ProPPD). To elucidate Sorcin-dependent chemoresistance mechanisms, we: - used H1299 lung cancer cells, which express high amount of Sorcin; - silenced Sorcin expression through siRNA; - treated cells with doxorubicin to elucidate changes in the uptake process and in the biological response; - tested Sorcin affinity for doxorubicin through Surface Plasmon Resonance and fluorimetry experiments; -solved the crystal structure of doxorubicin-bound Sorcin. Our results show that, by direct and specific binding of doxorubicin, Sorcin can act as a buffer for the drug in the cytoplasm, enhance its accumulation outside the nucleus and then its extrusion through MDR1 pump. Indeed Sorcin silencing, in our cancer cells, increases the sensitivity towards the chemotherapeutic drug and subsequently cell death upon treatment

Enhancing Activity of the Anaphase Promoting Complex to Reverse Multiple Drug Resistant Behaviour in Breast Cancer

The development of multiple drug resistant (MDR) behaviour in cancer poses a significant challenge to effective treatment, yet this is not a rare event and occurs in up to 20% of breast, lung, colon, and hematological malignancies. MDR is defined as the presence of clinical resistance to cancer therapy including resistance to medical therapies not previously used. MDR can be innate, or acquired over the course of treatment. The underlying causes of MDR are clearly multifactorial, and there are several common themes that promote the behaviour: genomic instability, impaired stress response pathways, and dysregulated mitosis all promote MDR behavior. These cellular mechanisms exist as an interrelated network of pathways, as disruption of one often impacts another, such as the loss of mitotic regulation promoting genomic instability. This accumulation of mutations may then impair the activity of various stress responses pathways, thereby reducing the apoptotic response to chemotherapy and promoting MDR behaviour. The Anaphase Promoting Complex (APC) is an evolutionarily conserved, multi-subunit E3 ubiquitin ligase enzyme that targets selected proteins for proteasomal degradation during mitosis and G1. Its functions include activation of stress response pathways and regulated progression of mitosis, which protects the cell from genomic instability. Previously, a correlation had been established between dysfunction of the APC and cancer progression, primarily indicated by the accumulation of many APC substrates noted in multiple cancers. Recently, this relationship was directly implicated in the progression of cancer into an MDR state in canine lymphomas where relapse correlated with impaired APC function and remission restored APC activity. Therefore, we hypothesize that enhancing APC activity will subsequently restore chemosensitivity in MDR cancer. In yeast, novel peptides were identified that bound and activated the Saccharomyces cerevisiae (S. cerevisiae) APC. When stably expressed in the innately MDR breast cancer cell line MDA-MB-231, two of these peptides significantly enhanced APC activity. Significantly, peptide expression restored cytotoxic sensitivity to doxorubicin in this MDR cell population in accordance with relative APC activity, with greater APC activation producing greater chemosensitivity. An investigation of FOXO3A-dependent stress response pathways (suggested by previous studies) demonstrating a functional interaction between APC activity and yeast FOXO orthologs, to induce stress responses. We revealed FOXO3A signaling activity was increased upon APC-activating peptide expression, an elevation in FOXO3A activity, and therefore predicted a concurrent increase in stress-related apoptosis. As anticipated, peptide expression increased apoptosis with or without chemotherapy exposure. Peptide-dependent APC activity unexpectedly augmented mitotic dysregulation observed as an increased number of mitotic catastrophes, including chromosomal mis-segregation and micronuclei formation. The consequences of mitotic catastrophes are elevated genomic instability and DNA damage, which were found to be elevated. While this behaviour is typically considered to be tumorigenic, this phenomenon may enhance the cytotoxic effect of chemotherapy by inducing cell death via priming the cells with elevated genomic damage. Our results indicate that activation of the APC serves to enhance chemosensitivity in MDR cancer; however, the complicated mechanisms related to APC activity warrants further investigation into its relationship with MDR behaviour

Structural and functional interrogation of Anterior Gradient-2

Anterior Gradient-2 protein (AGR2) has recently been linked to the onset of several pathologies including asthma and inflammatory bowel disease. Most interestingly, it has been discovered to influence the transformation of cells and metastatic growth essential to cancer development, and has subsequently been linked to the development of resistance to anti-cancer therapeutics. AGR2 protein is overexpressed in a diverse range of human cancer types, and has been detected secreted into the extracellular milieu. Thus, AGR2 protein represents a compelling pro-oncogenic signalling intermediate in tumour emergence and endurance. This thesis presents an interdisciplinary approach including structural biology, cell biology and synthetic biology, and clinical studies to shed more light on the role of AGR2 in cancer development. Synthetic cell based reagents were developed to define the dominant pathways that are reprogrammed in a cell as a result of AGR2 synthesis. A cell panel was engineered incorporating the AGR2 (and mutants thereof) allele into the AGR2-null A375 cell line. These tools were then coupled to quantitative proteomics (SILAC) to unravel the mechanism whereby introduction of AGR2 alters cell phenotype, allowing identification of dominant pathways affected by AGR2 signalling. Using pathway analysis tools, the dominant pathway suppressed by wt-AGR2 expression highlighted the p53-signalling axis. DNA damage induced p53 stabilisation and p21 induction by cisplatin treatment confirmed the influence of AGR2 gene expression. Further data analysis identified the outlying protein expression changes identified by SILAC was the anti-viral cell cycle regulator TSG101 (tumour susceptibility gene 101), and confirmed by immunoblotting. Transfection and silencing studies of TSG101 confirmed that TSG101 attenuates p53 function. These data provide a mechanism to explain the most dominant pathways reprogrammed by AGR2 expression, incorporating ER stress response, proliferation markers and p53 pathway attenuation. Further advances were made in analysis of the function, regulation, and drugability of AGR2 protein. Assays were devised to define the subunit structure of AGR2 as a dimer unit; subsequent functional studies defined intrinsically disordered motifs that regulate stability of the dimer. A two-site sandwich microtiter assay (2SMTA) was designed to screen for self-peptides and mutations that regulate oligomer stability. These assays were used to identify the first biochemical property of AGR2 being that the dimer unit is required for maximal binding to the AAA+ protein, and well characterised AGR2 interactor, Reptin. In addition, based on this dimeric structure, a novel solution based dimerisation assay was developed to identify natural products that are able to disrupt the dimer suggesting that AGR2 itself can be targeted in principle with small molecules for therapeutic purposes

A study of the effects of taxol on the proliferation, differentiation and survival of the murine myeloid leukemia WEHI-3B JCS cells.

by Po Chu, Leung.Thesis submitted in: December 1999.Thesis (M.Phil.)--Chinese University of Hong Kong, 2000.Includes bibliographical references (leaves 141-169).Abstracts in English and Chinese.Acknowledgments --- p.iAbbreviation --- p.iiiAbstract --- p.viiChinese Abstract --- p.xTable of Contents --- p.xiiChapter Chapter 1: --- General IntroductionChapter 1.1 --- Hematopoiesis --- p.1Chapter 1.1.1 --- The Development of Hematopoietic Progenitor Cells --- p.1Chapter 1.1.2 --- Hematopoietic Growth Factors --- p.3Chapter 1.1.3 --- Transcriptionl Factors Involved in Lineage Commitment of Hematopoietic Progenitor Cells --- p.5Chapter 1.2 --- Leukemia --- p.7Chapter 1.2.1 --- Occurrence and Classification of Leukemia --- p.7Chapter 1.2.2 --- The Pathological Features and Etiology of Leukemia --- p.10Chapter 1.2.3 --- The Molecular Basis of Leukemia --- p.13Chapter 1.2.4 --- Current Therapeutic Strategies --- p.14Chapter 1.2.4.1 --- Conventional Therapies for Leukemia --- p.14Chapter 1.2.4.2 --- Induction of Cell Differentiation and Apoptosis for Treatment of Leukemia --- p.16Chapter 1.2.5 --- The Use of Murine Myelomonocytic Leukemia WEHI-3B JCS Cells As a Model for the Study of Leukemia Cell Proliferation， Differentiation and Survival --- p.22Chapter 1.3 --- Taxol: A Novel Anti-cancer Agent --- p.23Chapter 1.3.1 --- Discovery and Action Mechanism --- p.23Chapter 1.3.2 --- Metabolism and Toxicity of Taxol --- p.27Chapter 1.3.3 --- The Biological Activities of Taxol --- p.28Chapter 1.3.4 --- The Anti-tumor Effects of Taxol --- p.30Chapter 1.3.5 --- The Effects of Taxol on Leukemia --- p.31Chapter 1.4 --- Aims and Scopes of This Investigation --- p.32Chapter Chapter 2: --- Materials and MethodsChapter 2.1 --- Materials --- p.35Chapter 2.1.1 --- Mice --- p.35Chapter 2.1.3 --- "Culture Media,Buffer and Other Solutions" --- p.37Chapter 2.1.4 --- Radioisotope and Scintillation Fluid --- p.39Chapter 2.1.5 --- Taxol --- p.40Chapter 2.1.6 --- Recombinant Cytokines --- p.40Chapter 2.1.7 --- Vitamin Analogs --- p.42Chapter 2.1.8 --- Various Signal Transduction Pathway Activators and Inhibitors --- p.42Chapter 2.1.9 --- Monoclonal Antibodies and Buffers for Flow Cytometry --- p.43Chapter 2.1.10 --- Reagents and Chemicals for Gene Expression Study --- p.45Chapter 2.1.11 --- Chemical Solutions and Buffers for Western Blot --- p.50Chapter 2.1.12 --- Reagents for Colony Assay --- p.54Chapter 2.2 --- Methods --- p.55Chapter 2.2.1 --- Culture of Leukemia Cell Lines --- p.55Chapter 2.2.2 --- Treatment of Leukemia Cells with Various Drugs and Cytokines --- p.55Chapter 2.2.3 --- Cell Morphological Study --- p.55Chapter 2.2.4 --- Determination of Leukemia Cell Survival and Proliferation --- p.56Chapter 2.2.5 --- Colony Assay --- p.56Chapter 2.2.6 --- Flow Cytometry Analysis --- p.57Chapter 2.2.6.1 --- Surface Antigen Immunophenotyping --- p.57Chapter 2.2.6.2 --- Assay of Endocytic Activity --- p.58Chapter 2.2.6.3 --- Cell Cycle /DNA Content Evaluation --- p.58Chapter 2.2.7 --- Gene Expression Study --- p.59Chapter 2.2.7.1 --- Preparation of Total Cellular RNA --- p.59Chapter 2.2.7.2 --- Reverse Transcription --- p.60Chapter 2.2.7.3 --- Polymerase Chain Reaction (PCR) --- p.60Chapter 2.2.7.4 --- Agarose Gel Electrophoresis --- p.61Chapter 2.2.8 --- DNA Fragmentation Analysis --- p.61Chapter 2.2.9 --- Protein Expression Study --- p.62Chapter 2.2.9.1 --- Protein Extraction --- p.62Chapter 2.2.9.2 --- Quantification of the Protein --- p.62Chapter 2.2.9.3 --- Western Blot Analysis --- p.63Chapter 2.2.10 --- Statistical Analysis --- p.64Chapter Chapter 3: --- ResultsChapter 3.1 --- Effects of Taxol on the Proliferation and Apoptosis of the Murine Myeloid Leukemia Cells --- p.65Chapter 3.1.1 --- Growth-Inhibitory Effects of Taxol on Murine Myeloid Leukemia WEHI-3B JCS cells --- p.65Chapter 3.1.2 --- Cytotoxic Effects of Taxol on Murine Bone Marrow Cells and Myeloid Leukemia WEHI-3B JCS Cells --- p.69Chapter 3.1.3 --- Anti-proliferative Effect of Taxol on Different Leukemia Cell Lines --- p.72Chapter 3.1.4 --- Effects of Taxol on the Cell Cycle Kinetics of WEHI-3B JCS Cells --- p.81Chapter 3.1.5 --- Induction of DNA Fragmentation of WEHI-3B JCS cells by Taxol --- p.83Chapter 3.1.6 --- Effect of Taxol on the Clonogenicity of WEHI-3B JCS Cells In Vitro and Tumorigenicity In Vivo --- p.86Chapter 3.2 --- Effects of Taxol on the Induction of Monocytic Cell Differentiation in Murine Myeloid Leukemia Cells --- p.88Chapter 3.2.1 --- Morphological Changes in Taxol-Treated Murine Myelomonocytic Leukemia WEHI-3B JCS Cells --- p.88Chapter 3.2.2 --- Surface Antigen Immunophenotyping of Taxol-treated WE HI-3B cells --- p.91Chapter 3.2.3 --- Endocytic Activity of Taxol-treated WEHI-3B JCS cells --- p.95Chapter 3.3 --- Modulatory Effect of Taxol and Cytokines on the Proliferation of WEHI- 3B JCS Cells --- p.96Chapter 3.4 --- Modulatory Effect of Taxol and Physiological Differentiation Inducers on the Proliferation of WEHI-3B JCS cells --- p.103Chapter 3.5 --- The Possible Involvement of Protein Kinase C in the Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.106Chapter 3.6 --- Modulation of Apoptotic Gene Expression in Taxol-treated WEHI-3B JCS cells --- p.113Chapter 3.7 --- Modulatory Effects of Taxol on the Protein Expression of WEHI-3B JCS Cells --- p.119Chapter Chapter 4: --- Discussion and ConclusionsChapter 4.1 --- "Effects of Taxol on the Proliferation,Differentiation and Apoptosis of the Murine Myeloid Leukemia Cells" --- p.126Chapter 4.2 --- "Modulatory Effects of Taxol, Cytokines and Physiological Differentiation Inducers on the Proliferation of the Myelomonocytic Leukemia WEHI-3B JCS Cells" --- p.132Chapter 4.3 --- The Possible Involvement of Protein Kinase C in Anti-proliferative Activity of Taxol on WEHI-3B JCS Cells --- p.136Chapter 4.4 --- The Modulation of Apoptosis Gene Expression in Taxol-treated WEHI-3B JCS Cells --- p.137Chapter 4.5 --- The Modulation of Protein Expression in Taxol-treated WEHI-3B JCS Cells --- p.138Chapter 4.6 --- Conclusions and Future Perspectives --- p.139References --- p.14

Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4’s hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer

Analysis and inhibition of oncogenic RET-dependent signalling

The aim of this PhD project is to analyse the RET-dependent signalling pathway and to block it with small molecule inhibitors, thus promoting innovative approaches to cure papillary thyroid carcinoma (PTC). The first goal was to determine the three-dimensional structure of RET catalytic domain to obtain detailed knowledge of the target molecule. The Baculovirus expression system and purification procedure was optimised to produce high amount of recombinant protein (rRET) to perform biochemical characterization and crystallization attempts. rRET was active and properly folded, thus resulting a suitable reagent for inhibitors screening and crystallization experiments. The crystallization screening was performed using rRET (complete kinase domain) and three other constructs, in which different deletions were introduced. No crystals were obtained after many attempts in two independent laboratories. Concurrently, the members of 2-indolinone family, a promising class of compounds studied with molecular modelling, were screened in an ELISA-based assay to identify the best inhibitor of RET. SU5416 demonstrated an efficient inhibition of RET and was further analysed both in vitro and cellular systems, showing good specificity and selectivity. In animal model SU5416 blocked RET phosphorylation, indicating feasible pharmacological properties also in in vivo system. Since B-RAF hyperactivation leads to transformation of thyroid cells, we also used siRNA technology to investigate whether B-RAF represented a target for selective treatment and tested a new inhibitor that showed activity in B-RAF positive melanoma cell lines, in B-RAF and RET/PTC thyroid models. The current study demonstrated that B-RAF inhibition by siRNA and small molecule inhibitor determined re-expression of thyroid specific proteins, thus indicating a restoring of the differentiation process, and block of the proliferation. This work has proposed the rational identification of RET and B-RAF inhibitors by analysing their signalling pathway. These results raise the possibility that PTC could be treated effectively with small molecule inhibitors

The RNA-binding protein LARP1 as potential biomarker and therapeutic target in ovarian cancer

Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3’ untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer.Open Acces

Proteomic investigation of the MDM2 interactome and linear motif interactions

The oncoprotein MDM2 has an integral role in cancer development via multiple signalling pathways. Two proteomic mass spectrometry screens, label-free with spectral counting quantitation and 8-plex iTRAQ were used to identify proteins up or downregulated over time by the MDM2 targeting drug Nutlin. A subset of previously identified MDM2 binding partners were identified as altered after Nutlin treatment, along with proteins which have not as yet been linked to MDM2 or p53. Proteins altered two hours after Nutlin treatment were screened for sequence similarity to an MDM2 binding consensus motif based on the BOX-I region of p53. Peptides corresponding to this motif were validated for MDM2 binding, and the mode of binding investigated using competition ELISA and thermal denaturation assays. Known MDM2 ligands such as Nutlin were shown to have a range of effects on the binding of these newly identified MDM2 peptides, which may be attributed to allosteric regulation of MDM2. The effects of Nutlin on two full length proteins identified by the MS screens, CypB and NPM, were confirmed in vivo. In vitro binding of MDM2 to CypB and PK, which contain BOX-I like motifs, was also demonstrated validating proteomic mass spectrometry screens as a method to identify new protein-protein interactions. To further investigate the potential of linear motifs to modulate protein-protein interactions, a peptide aptamer targeting the protein AGR2 was tested for effect on AGR2 and p53 in a cancer cell line