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Background and Objectives: Esophageal cancer has one of the worst prognoses of all cancers, mainly due to its aggressive growth pattern and late presentation. Using radiotherapy, chemotherapy, or even surgery it is only possible to cure a small minority and so alternative strategies need to be evaluated. In this study we aim to investigate the feasibility of using liposomes to transfect genes into human esophageal biopsies at high enough levels to achieve a biological effect.Methods: Human esophageal biopsies were transfected with constructs encoding Green Fluorescent Protein or human wild type p53. Expression of Green Fluorescent Protein within these samples was determined by FACScan analysis and confocal microscopy, whereas p53 expression was assessed by Western blotting and Reverse Transcriptase Polymerase Chain Reaction, probing for p53 and a downstream effector WAF1/p21.Results: Confocal microscopy verified the expression of Green Fluorescent Protein in human esophageal biopsies in cells 7 to 10 layers deep. Green Fluorescent Protein expression has also been demonstrated by FACScan analysis. The successful introduction and expression of wild type p53 has been shown in normal esophageal biopsies by Western blotting and RT-PCR and in Barrett's esophageal biopsies by Western blotting alone. In addition, we have demonstrated an increase in the expression of WAF1/p21 in 50% of biopsies transfected with p53.Conclusion: Liposome-mediated transfer of genes into human esophageal cells is feasible and can be performed at a sufficient dose to achieve expression of downstream genes. [ABSTRACT FROM AUTHOR]

Background: PD-L1 expression on cancer cells is an important mechanism of tumor immune escape, and immunotherapy targeting the PD-L1/PD1 interaction is a common treatment option for patients with melanoma. However, many patients do not respond to treatment and novel predictors of response are emerging. One suggested modifier of PD-L1 is the p53 pathway, although the relationship of p53 pathway function and activation is poorly understood., Methods: The study was performed on human melanoma cell lines with various p53 status. We investigated PD-L1 and proteins involved in IFNγ signaling by immunoblotting and mRNA expression, as well as membrane expression of PD-L1 by flow cytometry. We evaluated differences in the ability of NK cells to recognize and kill target tumor cells on the basis of p53 status. We also investigated the influence of proteasomal degradation and protein half-life, IFNγ signaling and p53 activation on biological outcomes, and performed bioinformatic analysis using available data for melanoma cell lines and melanoma patients., Results: We demonstrate that p53 status changes the level of membrane and total PD-L1 protein through IRF1 regulation and show that p53 loss influences the recently discovered SOX10/IRF1 regulatory axis. Bioinformatic analysis identified a dependency of SOX10 on p53 status in melanoma, and a co-regulation of immune signaling by both transcription factors. However, IRF1/PD-L1 regulation by p53 activation revealed complicated regulatory mechanisms that alter IRF1 mRNA but not protein levels. IFNγ activation revealed no dramatic differences based on TP53 status, although dual p53 activation and IFNγ treatment confirmed a complex regulatory loop between p53 and the IRF1/PD-L1 axis., Conclusions: We show that p53 loss influences the level of PD-L1 through IRF1 and SOX10 in an isogenic melanoma cell model, and that p53 loss affects NK-cell cytotoxicity toward tumor cells. Moreover, activation of p53 by MDM2 inhibition has a complex effect on IRF1/PD-L1 activation. These findings indicate that evaluation of p53 status in patients with melanoma will be important for predicting the response to PD-L1 monotherapy and/or dual treatments where p53 pathways participate in the overall response., (© 2024. The Author(s).)

Robotically assisted proteomics provides insights into the regulation of multiple proteins achieving excellent spatial resolution. However, developing an effective method for spatially resolved quantitative proteomics of formalin fixed paraffin embedded tissue (FFPE) in an accessible and economical manner remains challenging. We introduce non-robotic In-insert FFPE proteomics approach, combining glass insert FFPE tissue processing with spatial quantitative data-independent mass spectrometry (DIA). In-insert approach identifies 450 proteins from a 5 µm thick breast FFPE tissue voxel with 50 µm lateral dimensions covering several tens of cells. Furthermore, In-insert approach associated a keratin series and moesin (MOES) with prolactin-induced protein (PIP) indicating their prolactin and/or estrogen regulation. Our data suggest that PIP is a spatial biomarker for hormonally triggered cytoskeletal remodeling, potentially useful for screening hormonally affected hotspots in breast tissue. In-insert proteomics represents an alternative FFPE processing method, requiring minimal laboratory equipment and skills to generate spatial proteotype repositories from FFPE tissue., (© 2024. The Author(s).)

Interferon induced transmembrane proteins (IFITMs) play a dual role in the restriction of RNA viruses and in cancer progression, yet the mechanism of their action remains unknown. Currently, there is no data about the basic biochemical features or biophysical properties of the IFITM1 protein. In this work, we report on description and biochemical characterization of three conformational variants/oligomeric species of recombinant IFITM1 protein derived from an Escherichia coli expression system. The protein was extracted from the membrane fraction, affinity purified, and separated by size exclusion chromatography where two distinct oligomeric species were observed in addition to the expected monomer. These species remained stable upon re-chromatography and were designated as "dimer" and "oligomer" according to their estimated molecular weight. The dimer was found to be less stable compared to the oligomer using circular dichroism thermal denaturation and incubation with a reducing agent. A two-site ELISA and HDX mass spectrometry suggested the existence of structural motif within the N-terminal part of IFITM1 which might be significant in oligomer formation. Together, these data show the unusual propensity of recombinant IFITM1 to naturally assemble into very stable oligomeric species whose study might shed light on IFITM1 anti-viral and pro-oncogenic functions in cells., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Tumor antigens can emerge through multiple mechanisms, including translation of noncoding genomic regions. This noncanonical category of tumor antigens has recently gained attention; however, our understanding of how they recur within and between cancer types is still in its infancy. Therefore, we developed a proteogenomic pipeline based on deep learning de novo mass spectrometry (MS) to enable the discovery of noncanonical MHC class I-associated peptides (ncMAP) from noncoding regions. Considering that the emergence of tumor antigens can also involve posttranslational modifications (PTM), we included an open search component in our pipeline. Leveraging the wealth of MS-based immunopeptidomics, we analyzed data from 26 MHC class I immunopeptidomic studies across 11 different cancer types. We validated the de novo identified ncMAPs, along with the most abundant PTMs, using spectral matching and controlled their FDR to 1%. The noncanonical presentation appeared to be 5 times enriched for the A03 HLA supertype, with a projected population coverage of 55%. The data reveal an atlas of 8,601 ncMAPs with varying levels of cancer selectivity and suggest 17 cancer-selective ncMAPs as attractive therapeutic targets according to a stringent cutoff. In summary, the combination of the open-source pipeline and the atlas of ncMAPs reported herein could facilitate the identification and screening of ncMAPs as targets for T-cell therapies or vaccine development., (©2023 The Authors; Published by the American Association for Cancer Research.)

Interferons (IFNs) are important cytokines that regulate immune responses through the activation of hundreds of genes, including interferon-induced transmembrane proteins (IFITMs). This evolutionarily conserved protein family includes five functionally active homologs in humans. Despite the high sequence homology, IFITMs vary in expression, subcellular localization and function. The initially described adhesive and antiproliferative or pro-oncogenic functions of IFITM proteins were diluted by the discovery of their antiviral properties. The large set of viruses that is inhibited by these proteins is constantly expanding, as are the possible mechanisms of action. In addition to their beneficial antiviral effects, IFITM proteins are often upregulated in a broad spectrum of cancers. IFITM proteins have been linked to most hallmarks of cancer, including tumor cell proliferation, therapeutic resistance, angiogenesis, invasion, and metastasis. Recent studies have described the involvement of IFITM proteins in antitumor immunity. This review summarizes various levels of IFITM protein regulation and the physiological and pathological functions of these proteins, with an emphasis on tumorigenesis and antitumor immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Friedlová, Zavadil Kokáš, Hupp, Vojtěšek and Nekulová.)

The IFITM restriction factors play a role in cancer cell progression through undefined mechanisms. We investigate new protein-protein interactions for IFITM1/3 in the context of cancer that would shed some light on how IFITM1/3 attenuate the expression of targeted proteins such as HLA-B. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the SRSF1 splicing factor and transporter of mRNA to the ribosome. Using in situ proximity ligation assays, we confirmed a predominant cytosolic protein-protein association for SRSF1 and IFITM1/3. Accordingly, IFITM1/3 interacted with HLA-B mRNA in response to IFNγ stimulation using RNA-protein proximity ligation assays. In addition, RT-qPCR assays in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that HLA-B gene expression at the mRNA level does not account for lowered HLA-B protein synthesis in response to IFNγ. Complementary, shotgun RNA sequencing did not show major transcript differences between IFITM1/IFITM3 null cells and wt-SiHa cells. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to wild type. It was partially reverted by IFITM1/3 complementation. Our data link IFITM1/3 proteins to HLA-B mRNA and SRSF1 and, all together, our results begin to elucidate how IFITM1/3 catalyze the synthesis of target proteins. IFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. Our study has identified new proteins associated with IFITMs which support their role in mediating protein expression; a pivotal function that is highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 affect the expression of targeted proteins; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs in mediating protein abundance is relevant, as it has the potential for regulating the expression of viral and oncogenic proteins.

Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there are a few reports on DIA mass spectrometry (MS) approaches for proteome analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, to facilitate detection and quantification of immune- and glioblastoma (GBM)-relevant proteins from FFPE patient materials, we established a simple and precise DIA-MS workflow. We first evaluated different lysis buffers for their efficiency in protein extractions from FFPE GBM tissues. Our results showed that more than 1700 proteins were detected and over 1400 proteins were quantified from GBM FFPE tissue microdissections. GBM-relevant proteins (e.g., GFAP, FN1, VIM, and MBP) were quantified with high precision (median coefficient of variation <12%). In addition, immune-related proteins (e.g., ILF2, MIF, and CD38) were consistently detected and quantified. The strategy holds great potential for routinizing protein quantification in FFPE tissue samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5 V569E and MAP4K5 V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.

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., (© 2021 Al Shboul et al.)

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

CHIP is an E3-ubiquitin ligase that contributes to healthy aging and has been characterized as neuroprotective. To elucidate dominant CHIP-dependent changes in protein steady-state levels in a patient-derived human neuronal model, CHIP function was ablated using gene-editing and an unbiased proteomic analysis conducted to compare knock-out and wild-type isogenic induced pluripotent stem cell (iPSC)-derived cortical neurons. Rather than a broad effect on protein homeostasis, loss of CHIP function impacted on a focused cohort of proteins from actin cytoskeleton signaling and membrane integrity networks. In support of the proteomics, CHIP knockout cells had enhanced sensitivity to induced membrane damage. We conclude that the major readout of CHIP function in cortical neurons derived from iPSC of a patient with elevate α-synuclein, Parkinson's disease and dementia, is the modulation of substrates involved in maintaining cellular "health". Thus, regulation of the actin cytoskeletal and membrane integrity likely contributes to the neuroprotective function(s) of CHIP., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors.)

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

The fundamentals of how protein-protein/RNA/DNA interactions influence the structures and functions of the workhorses from the cells have been well documented in the 20th century. A diverse set of methods exist to determine such interactions between different components, particularly, the mass spectrometry (MS) methods, with its advanced instrumentation, has become a significant approach to analyze a diverse range of biomolecules, as well as bring insights to their biomolecular processes. This review highlights the principal role of chemistry in MS-based structural proteomics approaches, with a particular focus on the chemical cross-linking of protein-protein/DNA/RNA complexes. In addition, we discuss different methods to prepare the cross-linked samples for MS analysis and tools to identify cross-linked peptides. Cross-linking mass spectrometry (CLMS) holds promise to identify interaction sites in larger and more complex biological systems. The typical CLMS workflow allows for the measurement of the proximity in three-dimensional space of amino acids, identifying proteins in direct contact with DNA or RNA, and it provides information on the folds of proteins as well as their topology in the complexes. Principal CLMS applications, its notable successes, as well as common pipelines that bridge proteomics, molecular biology, structural systems biology, and interactomics are outlined.

SARS-CoV-2, or COVID-19, has a devastating effect on our society, both in terms of quality of life and death rates; hence, there is an urgent need for developing safe and effective therapeutics against SARS-CoV-2. The most promising strategy to fight against this deadly virus is to develop an effective vaccine. Internalization of SARS-CoV-2 into the human host cell mainly occurs through the binding of the coronavirus spike protein (a trimeric surface glycoprotein) to the human angiotensin-converting enzyme 2 (ACE2) receptor. The spike-ACE2 protein-protein interaction is mediated through the receptor-binding domain (RBD) of the spike protein. Mutations in the spike RBD can significantly alter interactions with the ACE2 host receptor. Due to its important role in virus transmission, the spike RBD is considered to be one of the key molecular targets for vaccine development. In this study, a spike RBD-based subunit vaccine was designed by utilizing a ferritin protein nanocage as a scaffold. Several fusion protein constructs were designed in silico by connecting the spike RBD via a synthetic linker (different sizes) to different ferritin subunits (H-ferritin and L-ferritin). The stability and the dynamics of the engineered nanocage constructs were tested by extensive molecular dynamics simulation (MDS). Based on our MDS analysis, a five amino acid-based short linker (S-Linker) was the most effective for displaying the spike RBD over the surface of ferritin. The behavior of the spike RBD binding regions from the designed chimeric nanocages with the ACE2 receptor was highlighted. These data propose an effective multivalent synthetic nanocage, which might form the basis for new vaccine therapeutics designed against viruses such as SARS-CoV-2.

A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3, and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Using light chain-7 as a reference sequence, hydrogen deuterium exchange mass spectrometry was used to identify the dominant CDR region implicated in CD20 antigen binding. Early in the deuteration reaction, the CD20 antigen suppressed deuteration at CDR3 (VH). In later time points, deuterium suppression occurred at CDR2 (VH) and CDR2 (VL), with the maintenance of the CDR3 (VH) interaction. These data suggest that CDR3 (VH) functions as the dominant antigen docking motif and that antibody aggregation is induced at later time points after antigen binding. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry. These data support the further development of an engineered, synthetic canine-murine monoclonal antibody, focused on CDR3 (VH), for use as a canine lymphoma therapeutic that mimics the human-murine chimeric anti-CD20 antibody Rituximab., (© 2021 The Author(s).)

An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an "up" and "down" state. In addition, the trimer cavity may function as a "bouncing spring" that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools., Competing Interests: The authors declare no competing interests.

In recent years, a lot of scientific interest has focused on cancer immunotherapy. Although chronic inflammation has been described as one of the hallmarks of cancer, acute inflammation can actually trigger the immune system to fight diseases, including cancer. Toll-like receptor (TLR) ligands have long been used as adjuvants for traditional vaccines and it seems they may also play a role enhancing efficiency of tumor immunotherapy. The aim of this perspective is to discuss the effects of TLR stimulation in cancer, expression of various TLRs in different types of tumors, and finally the role of TLRs in anti-cancer immunity and tumor rejection., (Copyright © 2019 Urban-Wojciuk, Khan, Oyler, Fåhraeus, Marek-Trzonkowska, Nita-Lazar, Hupp and Goodlett.)

Interferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape., (Copyright © 2019. Published by Elsevier Inc.)

Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit "rim" of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric Reptin Y340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. SIGNIFICANCE: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a 'hot-spot' protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape., (Copyright © 2019. Published by Elsevier B.V.)

Death Associated Protein Kinase 1 (DAPK1) is an important signaling kinase mediating the biological effect of multiple natural biomolecules such as IFN-γ, TNF-α, curcumin, etc. DAPK1 is degraded through both ubiquitin-proteasomal and lysosomal degradation pathways. To investigate the crosstalk between these two DAPK1 degradation pathways, we carried out a screen using a set of ubiquitin E2 siRNAs at the presence of Tuberous Sclerous 2 (TSC2) and identified that the small ubiquitin-like molecule (SUMO) pathway is able to regulate the protein levels of DAPK1. Inhibition of the SUMO pathway enhanced DAPK1 protein levels and the minimum domain of DAPK1 protein required for this regulation is the kinase domain, suggesting that the SUMO pathway regulates DAPK1 protein levels independent of TSC2. Suppression of the SUMO pathway did not enhance DAPK1 protein stability. In addition, mutation of the potential SUMO conjugation sites on DAPK1 kinase domain did not alter its protein stability or response to SUMO pathway inhibition. These data suggested that the SUMO pathway does not regulate DAPK1 protein degradation. The exact molecular mechanism underlying this regulation is yet to be discovered., Competing Interests: The authors have no conflicts of interest to declare.

AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Ubiquitin is a key component of the regulatory network that maintains gene expression in eukaryotes, yet the molecular mechanism(s) by which non-degradative ubiquitination modulates transcriptional activator (TA) function is unknown. Here endogenous p53, a stress-activated transcription factor required to maintain health, is stably monoubiquitinated, following pathway activation by IR or Nutlin-3 and localized to the nucleus where it becomes tightly associated with chromatin. Comparative structure-function analysis and in silico modelling demonstrate a direct role for DNA-binding domain (DBD) monoubiquitination in TA activation. When attached to the DBD of either p53, or a second TA IRF-1, ubiquitin is orientated towards, and makes contact with, the DNA. The contact is made between a predominantly cationic surface on ubiquitin and the anionic DNA. Our data demonstrate an unexpected role for ubiquitin in the mechanism of TA-activity enhancement and provides insight into a new level of transcriptional regulation.

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Monoclonal antibodies are leading agents for therapeutic treatment of human diseases, but are limited in use by the paucity of clinically relevant models for validation. Sporadic canine tumours mimic the features of some human equivalents. Developing canine immunotherapeutics can be an approach for modeling human disease responses. Rituximab is a pioneering agent used to treat human hematological malignancies. Biologic mimics that target canine CD20 are just being developed by the biotechnology industry. Towards a comparative canine-human model system, we have developed a novel anti-CD20 monoclonal antibody (NCD1.2) that binds both human and canine CD20. NCD1.2 has a sub-nanomolar Kd as defined by an octet red binding assay. Using FACS, NCD1.2 binds to clinically derived canine cells including B-cells in peripheral blood and in different histotypes of B-cell lymphoma. Immunohistochemical staining of canine tissues indicates that the NCD1.2 binds to membrane localized cells in Diffuse Large B-cell lymphoma, Marginal Zone Lymphoma, and other canine B-cell lymphomas. We cloned the heavy and light chains of NCD1.2 from hybridomas to determine whether active scaffolds can be acquired as future biologics tools. The VH and VL genes from the hybridomas were cloned using degenerate primers and packaged as single chains (scFv) into a phage-display library. Surprisingly, we identified two scFv (scFv-3 and scFv-7) isolated from the hybridoma with bioactivity towards CD20. The two scFv had identical VH genes but different VL genes and identical CDR3s, indicating that at least two light chain mRNAs are encoded by NCD1.2 hybridoma cells. Both scFv-3 and scFv-7 were cloned into mammalian vectors for secretion in CHO cells and the antibodies were bioactive towards recombinant CD20 protein or peptide. The scFv-3 and scFv-7 were cloned into an ADEPT-CPG2 bioconjugate vector where bioactivity was retained when expressed in bacterial systems. These data identify a recombinant anti-CD20 scFv that might form a useful tool for evaluation in bioconjugate-directed anti-CD20 immunotherapies in comparative medicine.

Motivation: Proteins often recognize their interaction partners on the basis of short linear motifs located in disordered regions on proteins' surface. Experimental techniques that study such motifs use short peptides to mimic the structural properties of interacting proteins. Continued development of these methods allows for large-scale screening, resulting in vast amounts of peptide sequences, potentially containing information on multiple protein-protein interactions. Processing of such datasets is a complex but essential task for large-scale studies investigating protein-protein interactions., Results: The software tool presented in this article is able to rapidly identify multiple clusters of sequences carrying shared specificity motifs in massive datasets from various sources and generate multiple sequence alignments of identified clusters. The method was applied on a previously published smaller dataset containing distinct classes of ligands for SH3 domains, as well as on a new, an order of magnitude larger dataset containing epitopes for several monoclonal antibodies. The software successfully identified clusters of sequences mimicking epitopes of antibody targets, as well as secondary clusters revealing that the antibodies accept some deviations from original epitope sequences. Another test indicates that processing of even much larger datasets is computationally feasible., Availability and Implementation: Hammock is published under GNU GPL v. 3 license and is freely available as a standalone program (from http://www.recamo.cz/en/software/hammock-cluster-peptides/) or as a tool for the Galaxy toolbox (from https://toolshed.g2.bx.psu.edu/view/hammock/hammock). The source code can be downloaded from https://github.com/hammock-dev/hammock/releases., Contact: muller@mou.cz, Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2015. Published by Oxford University Press.)

Proteins targeted to secretory pathway enter the endoplasmic reticulum where they undergo post-translational modification and subsequent quality control executed by exquisite catalysts of protein folding, protein disulphide isomerases (PDIs). These enzymes can often provide strict conformational protein folding solutions to highly cysteine-rich cargo as they facilitate disulphide rearrangement in the endoplasmic reticulum. Under conditions when PDI substrates are not isomerised properly, secreted proteins can accumulate in the endoplasmic reticulum leading to endoplasmic reticulum stress initiation with implications for human disease development. Anterior Gradient-2 (AGR2) is an endoplasmic reticulum-resident PDI superfamily member that has emerged as a dominant effector of basic biological properties in vertebrates including blastoderm formation and limb regeneration. AGR2 perturbation in mammals influences disease processes including cancer progression and drug resistance, asthma, and inflammatory bowel disease. This review will focus on the molecular characteristics, function, and regulation of AGR2, views on its emerging biological functions and misappropriation in disease, and prospects for therapeutic intervention into endoplasmic reticulum-resident protein folding pathways for improving the treatment of human disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Developing drug-like molecules to inhibit the interactions formed by disordered proteins is desirable due to the high correlation of disorder with protein implicated in disease, but is challenging due in part to the lack of atomistically resolved and resolvable structures from conformationally dynamic systems. Ion mobility mass spectrometry (IM-MS) is well-positioned to assess protein ligand interactions along with the effect of a given inhibitor on conformation. Here we demonstrate the use of IM-MS to characterize the effect of two inhibitors RITA and Nutlin-3 on their respective binding partners: p53 and MDM2. RITA binds N-terminal transactivation domain of p53 (Np53) weakly, preventing direct observation of the complex in the gas phase. Nonetheless, upon incubation with RITA, we observe an alteration in the charge state distribution and in the conformational distributions adopted by Np53 in the gas phase. This finding supports the hypothesis that RITAs mode of action proceeds via a conformational change in p53. Circular dichroism corroborates our gas phase findings, showing a slight increase in secondary structure content on ligand incubation, and HDX-MS experiments also highlight the dynamic properties of this protein. Using the same approach we present data to show the effect of Nutlin-3 binding to the N-terminal domain of MDM2 (N-MDM2), N-MDM2 presents as at least two conformational families in the absence of Nutlin-3. Upon Nutlin-3 binding, the protein undergoes a compaction event similar to that exhibited by RITA on Np53. This multi-technique approach highlights the inherent disorder in these systems; and in particular exemplifies the power of IM-MS as a technique to study transient interactions between small molecule inhibitors and intrinsically disordered proteins.

Melanoma antigen A (MAGE-A) proteins comprise a structurally and biochemically similar sub-family of Cancer/Testis antigens that are expressed in many cancer types and are thought to contribute actively to malignancy. MAGE-A proteins are established regulators of certain cancer-associated transcription factors, including p53, and are activators of several RING finger-dependent ubiquitin E3 ligases. Here, we show that MAGE-A2 associates with MDM2, a ubiquitin E3 ligase that mediates ubiquitylation of more than 20 substrates including mainly p53, MDM2 itself, and MDM4, a potent p53 inhibitor and MDM2 partner that is structurally related to MDM2. We find that MAGE-A2 interacts with MDM2 via the N-terminal p53-binding pocket and the RING finger domain of MDM2 that is required for homo/hetero-dimerization and for E2 ligase interaction. Consistent with these data, we show that MAGE-A2 is a potent inhibitor of the E3 ubiquitin ligase activity of MDM2, yet it does not have any significant effect on p53 turnover mediated by MDM2. Strikingly, however, increased MAGE-A2 expression leads to reduced ubiquitylation and increased levels of MDM4. Similarly, silencing of endogenous MAGE-A expression diminishes MDM4 levels in a manner that can be rescued by the proteasomal inhibitor, bortezomid, and permits increased MDM2/MDM4 association. These data suggest that MAGE-A proteins can: (i) uncouple the ubiquitin ligase and degradation functions of MDM2; (ii) act as potent inhibitors of E3 ligase function; and (iii) regulate the turnover of MDM4. We also find an association between the presence of MAGE-A and increased MDM4 levels in primary breast cancer, suggesting that MAGE-A-dependent control of MDM4 levels has relevance to cancer clinically.

Developing approaches to discover protein-protein interactions (PPIs) remains a fundamental challenge. A chemical biology platform is applied here to identify novel PPIs for the AAA+ superfamily oncoprotein reptin. An in silico screen coupled with chemical optimization provided Liddean, a nucleotide-mimetic which modulates reptin's oligomerization status, protein-binding activity and global conformation. Combinatorial peptide phage library screening of Liddean-bound reptin with next generation sequencing identified interaction motifs including a novel reptin docking site on the p53 tumor suppressor protein. Proximity ligation assays demonstrated that endogenous reptin forms a predominantly cytoplasmic complex with its paralog pontin in cancer cells and Liddean promotes a shift of this complex to the nucleus. An emerging view of PPIs in higher eukaryotes is that they occur through a striking diversity of linear peptide motifs. The discovery of a compound that alters reptin's protein interaction landscape potentially leads to novel avenues for therapeutic development.

Mouse double minute 2 (MDM2) has a phosphorylation site within a lid motif at Ser17 whose phosphomimetic mutation to Asp17 stimulates MDM2-mediated polyubiquitination of p53. MDM2 lid deletion, but not Asp17 mutation, induced a blue shift in the λ(max) of intrinsic fluorescence derived from residues in the central domain including Trp235, Trp303, Trp323, and Trp329. This indicates that the Asp17 mutation does not alter the conformation of MDM2 surrounding the tryptophan residues. In addition, Phe235 mutation enhanced MDM2 binding to p53 but did not stimulate its ubiquitination function, thus uncoupling increases in p53 binding from its E3 ubiquitin ligase function. However, the Asp17 mutation in MDM2 stimulated its discharge of the UBCH5a-ubiquitin thioester adduct (UBCH5a is a ubiquitin-conjugating enzyme E2D 1 UBC4/5 homolog yeast). This stimulation of ubiquitin discharge from E2 was independent of the p53 substrate. There are now four known effects of the Asp17 mutation on MDM2: (i) it alters the conformation of the isolated N-terminus as defined by NMR; (ii) it induces increased thermostability of the isolated N-terminal domain; (iii) it stimulates the allosteric interaction of MDM2 with the DNA-binding domain of p53; and (iv) it stimulates a novel protein-protein interaction with the E2-ubiquitin complex in the absence of substrate p53 that, in turn, increases hydrolysis of the E2-ubiquitin thioester bond. These data also suggest a new strategy to disrupt MDM2 function by targeting the E2-ubiquitin discharge reaction., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Linear motifs mediate protein-protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors., (Copyright © 2014 Elsevier Inc. All rights reserved.)

AGR2 forms an ER-resident signalling axis in cell development, limb regeneration, and in human diseases like asthma and cancer, yet molecular mechanisms underlying its effects remain largely undefined. A single integrated Flippase recombination target (FRT) site was engineered within the AGR2-non expressing A375 cell line to allow integration of a constitutively expressed AGR2 alleles. This allows an analysis of how AGR2 protein expression reprogrammes intracellular signalling. The engineered expression of AGR2 had marginal impact on global transcription signalling, compared to its paralogue AGR3. However, expression of AGR2 had a significant impact on remodelling the cellular proteome using a triple-labelled SILAC protocol. 29 045 peptides were detected for the identification and relative quantitation of 3003 proteins across the experimental conditions. Ingenuity Pathway annotation highlighted the dominant pathway suppressed by wt-AGR2 was the p53-signalling axis. DNA damage induced p53 stabilization and p21 induction by cisplatin treatment confirmed that wt-AGR2 expression suppressed the p53 pathway. The furthest outlying SILAC protein expression change induced by AGR2 was the anti-viral and cell cycle regulator tumour susceptibility gene 101 (TSG101), confirmed by immunoblotting. Transfection of TSG101 into MCF7 (AGR2+, oestrogen dependent), A549 (AGR2+, oestrogen independent) or A375 (AGR2-) cells confirmed that TSG101 attenuates p53 signalling. These systems wide screens suggest that the most dominant landscape reprogrammed by low levels of AGR2 protein is the cellular proteome, rather than the transcriptome, and provide focus for evaluating its role in proteostasis.

Sarcomas are rare forms of cancer with a high unmet clinical need that develop in connective tissue, such as muscle, bone, nerves, cartilage, and fat. The outcome for patients is poor, with surgery and postoperative radiotherapy the standard treatment for patients. A better understanding of the molecular pathology of sarcoma may allow for the development of novel therapeutics. There are dozens of sarcoma subtypes where there is a need for targetted therapeutics, with the most commonly studied including Ewing's sarcoma and osteosarcoma. Here we initiate a proteomics-based target-discovery program to define "dominant" pro-oncogenic signaling targets in the most common sarcoma in adults: high-grade pleiomorphic soft tissue sarcoma. We have carried out a proteome screen using tandem mass tag isobaric labeling on three high-grade undifferentiated pleomorphic sarcoma biopsies from different tissue sites. We identified the commonly dysregulated proteins within the three sarcomas and further validated the most penetrant receptor as CLIC1, using immunohistochemistry arising from two different population cohorts representing over 300 patients. The dominant expression of CLIC1 in a broad range of human sarcomas suggests that studying this relatively unexplored signaling pathway might provide new insights into disease mechanism and facilitate the development of new CLIC1 targeted therapeutics.

Death associated protein kinase 1 (DAPK) is an important serine/theoreine kinase involved in various cellular processes such as apoptosis, autophagy and inflammation. DAPK expression and activity are misregulated in multiple diseases including cancer, neuronal death, stoke, et al. Methylation of the DAPK gene is common in many types of cancer and can lead to loss of DAPK expression. In this review, we summarize the pathological status and functional roles of DAPK in disease and compare the published reagents that can manipulate the expression or activity of DAPK. The pleiotropic functions of DAPK make it an intriguing target and the barriers and opportunities for targeting DAPK for future clinical application are discussed.

Osteosarcoma is the most common primary bone tumour of both children and dogs. It is an aggressive tumour in both species with a rapid clinical course leading ultimately to metastasis. In dogs and children distant metastasis occurs in >80% of individuals treated by surgery alone. Both canine and human osteosarcoma has been shown to contain a sub-population of cancer stem cells (CSCs), which may drive tumour growth, recurrence and metastasis, suggesting that naturally occurring canine osteosarcoma could act as a preclinical model for the human disease. Here we report the successful isolation of CSCs from primary canine osteosarcoma, as well as established cell lines. We show that these cells can form tumourspheres, and demonstrate relative resistance to chemotherapy. We demonstrate similar results for the human osteosarcma cell lines, U2OS and SAOS2. Utilizing the Affymetrix canine microarray, we are able to definitively show that there are significant differences in global gene expression profiles of isolated osteosarcoma stem cells and the daughter adherent cells. We identified 13,221 significant differences (p = 0.05), and significantly, COX-2 was expressed 141-fold more in CSC spheres than daughter adherent cells. To study the role of COX-2 expression in CSCs we utilized the COX-2 inhibitors meloxicam and mavacoxib. We found that COX-2 inhibition had no effect on CSC growth, or resistance to chemotherapy. However inhibition of COX-2 in daughter cells prevented sphere formation, indicating a potential significant role for COX-2 in tumour initiation.

Reactivation of the wild-type p53 pathway is one key goal aimed at developing targeted therapeutics in the cancer research field. Although most p53 protein kinases form 'p53-activating' signals, there are few kinases whose action can contribute to the inhibition of p53, as Casein kinase 1 (CK1) and Checkpoint kinase 1 (CHK1). Here we report on a pyrazolo-pyridine analogue showing activity against both CK1 and CHK1 kinases that lead to p53 pathway stabilisation, thus having pharmacological similarities to the p53-activator Nutlin-3. These data demonstrate the emerging potential utility of multivalent kinase inhibitors., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2. A two site-sandwich microtiter assay ((2S) MTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the (2S) MTA assay. A DSS-crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (more stable dimer), and monomeric AGR2(E60A) revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2(E60A) mutant. Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its' biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its' oncogenic potential., (© 2013 The Protein Society.)

MDM2 is a multidomain protein that functions as an E3 ubiquitin ligase, transcription repressor, mRNA-binding protein, translation factor, and molecular chaperone. The small molecule Nutlin-3 has been engineered to bind to the N-terminal hydrophobic pocket domain of MDM2. This binding of Nutlin-3 has two consequences: (i) antagonistic effects through competitive disruption of the MDM2-p53 complex and (ii) agonist effects that allosterically stabilize MDM2 protein-protein interactions that increase p53 ubiquitination as well as nucleophosmin deoligomerization. We present a methodology using a hydrogen/deuterium (H/D) exchange platform that measures Nutlin-3 binding to the N-terminal domain of MDM2 (MDM2(1-126)) in order to begin to develop dynamic assays that evaluate MDM2 allostery. In order to localize the regions in MDM2 being suppressed by Nutlin-3, MDM2 was incubated with the ligand and H/D amide exchange was measured after pepsin digestion. One dynamic segment containing amino acids 55-60 exhibited slower deuterium exchange after Nutlin-3 binding, reflecting ligand binding within the hydrophobic pocket. However, another dominant suppression of H/D exchange was observed in a motif from amino acids 103-107 that reflects surface hydrophobic residues surrounding the hydrophobic pocket of MDM2. In order to explore the consequences of this latter Nutlin-3 interaction site on MDM2, the Y104G and L107G mutant series was constructed. The MDM2(Y104G) and MDM2(L107G) mutants were fully active in p53 binding. However, the authentic p53-derived peptide:MDM2(Y104G) complex exhibited partial resistance to Nutlin-3 inhibition, while the p53-mimetic 12.1 peptide:MDM2(Y104G) complex retained normal Nutlin-3 responsiveness. These data reveal the existence of a second functional Nutlin-3-binding site in a surface hydrophobic patch of MDM2, flanking the hydrophobic pocket. This reveals two modes of peptide binding by MDM2 and highlights the utility of H/D exchange as an assay for measuring allosteric effects in MDM2., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The pro-metastatic protein anterior gradient-2 (AGR2) was previously demonstrated as a predictive factor of poor response to tamoxifen treatment. In this study we aimed to delineate the key signalling pathway that may contribute to regulation of AGR2 protein induction in order to identify novel targets to overcome tamoxifen resistance in tumour cells. Together, our data identify PDPK1-AKT as a pro-oncogenic signalling pathway that triggers AGR2 protein induction in response to tamoxifen and suggest that AKT inhibitors could be used as part of a therapeutic strategy to treat tamoxifen resistant, AGR2 over-expressing cancers., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cancer stem cells were identified in a feline mammary carcinoma cell line by demonstrating expression of CD133 and utilising the tumour sphere assay. A population of cells was identified that had an invasive, mesenchymal phenotype, expressed markers of pluripotency and enhanced tumour formation in the NOD-SCID mouse and chick embryo models. This population of feline mammary carcinoma stem cells was resistant to chemotherapy and radiation, possibly due to aberrant activation of the ATM/p53 DNA damage pathway. Epithelial-mesenchymal transition was a feature of the invasive phenotype. These data demonstrate that cancer stem cells are a feature of mammary cancer in cats., (Copyright © 2012 Elsevier Ltd. All rights reserved.)