The MYC oncoprotein directly interacts with its chromatin cofactor PNUTS to recruit PP1 phosphatase.

Despite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remain an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation, chromatin occupancy, and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. By NMR we identified a distinct peptide motif within MB0 that interacts with PNUTS residues 1-148, a functional unit, here termed PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its MYC-binding patch. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo, leading to elevated MYC phosphorylation. These data demonstrate that the MB0 region of MYC directly interacts with the PAD of PNUTS, which provides new insight into the control mechanisms of MYC as a regulator of gene transcription and a pervasive cancer driver.

Small-angle x-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model.

In this work, we used small-angle x-ray and neutron scattering to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa transcriptional regulator MexR, a member of the multiple antibiotics resistance regulator (MarR) family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterization of both free and DNA-bound MarR proteins. Here, we show how combined neutron and x-ray small-angle scattering of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.

Production and characterisation of modularly deuterated UBE2D1-Ub conjugate by small angle neutron and X-ray scattering.

This structural study exploits the possibility to use modular protein deuteration to facilitate the study of ubiquitin signalling, transfer, and modification. A protein conjugation reaction is used to combine protonated E2 enzyme with deuterated ubiquitin for small angle X-ray and neutron scattering with neutron contrast variation. The combined biomolecules stay as a monodisperse system during data collection in both protonated and deuterated buffers indicating long stability of the E2-Ub conjugate. With multiphase ab initio shape restoration and rigid body modelling, we reconstructed the shape of a E2-Ub-conjugated complex of UBE2D1 linked to ubiquitin via an isopeptide bond. Solution X-ray and neutron scattering data for this E2-Ub conjugate in the absence of E3 jointly indicate an ensemble of open and backbent states, with a preference for the latter in solution. The approach of combining protonated and labelled proteins can be used for solution studies to assess localization and movement of ubiquitin and could be widely applied to modular Ub systems in general.

Adipokines and Vascular Endothelial Growth Factor in Normal Human Breast Tissue in Vivo - Correlations and Attenuation by Dietary Flaxseed.

Exposure to sex steroids increases the risk of breast cancer but the exact mechanisms are yet to be elucidated. Events in the microenvironment are important for carcinogenesis. Diet containing phytoestrogens can affect the breast microenvironment and alter the risk of breast cancer. It has previously been shown that estrogen regulates extracellular levels of leptin, adiponectin, and VEGF in normal breast tissue in vivo. Whether these proteins correlate in breast tissue in vivo or if diet addition of flaxseed, a major source of phytoestrogens in Western diets, alters adipokine levels in breast tissue are unknown. We used microdialysis to sample proteins of normal human breast tissue and abdominal subcutaneous fat in situ in 34 pre-and postmenopausal women. In vitro, co-culture of breast cancer cells and primary human adipocytes was used. In vivo, in normal breast tissue, a significant positive correlation between VEGF and leptin was detected. No correlations were found in fat tissue. Co-culture of adipocytes and breast cancer cells per se increased the secretion of VEGF and leptin and enhanced the effects of estradiol compared to culture of either cell type alone. In vitro, inhibition of VEGF diminished the release of leptin while inhibition of leptin had no influence on VEGF secretion. The levels of leptin decreased and adiponectin increased after a dietary addition of 25 g of flaxseed/day for one menstrual cycle. We conclude that VEGF and leptin correlate significantly in normal human breast tissue in vivo and that dietary addition of flaxseed affect adipokine levels in the breast.

Multiple direct interactions of TBP with the MYC oncoprotein.

Transcription factor c-MYC is a potent oncoprotein; however, the mechanism of transcriptional regulation via MYC-protein interactions remains poorly understood. The TATA-binding protein (TBP) is an essential component of the transcription initiation complex TFIID and is required for gene expression. We identify two discrete regions mediating MYC-TBP interactions using structural, biochemical and cellular approaches. A 2.4 -Å resolution crystal structure reveals that human MYC amino acids 98-111 interact with TBP in the presence of the amino-terminal domain 1 of TBP-associated factor 1 (TAF1TAND1). Using biochemical approaches, we have shown that MYC amino acids 115-124 also interact with TBP independently of TAF1TAND1. Modeling reveals that this region of MYC resembles a TBP anchor motif found in factors that regulate TBP promoter loading. Site-specific MYC mutants that abrogate MYC-TBP interaction compromise MYC activity. We propose that MYC-TBP interactions propagate transcription by modulating the energetic landscape of transcription initiation complex assembly.

Author Correction: Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cytotoxic unsaturated electrophilic compounds commonly target the ubiquitin proteasome system.

A large number of natural products have been advocated as anticancer agents. Many of these compounds contain functional groups characterized by chemical reactivity. It is not clear whether distinct mechanisms of action can be attributed to such compounds. We used a chemical library screening approach to demonstrate that a substantial fraction (~20%) of cytotoxic synthetic compounds containing Michael acceptor groups inhibit proteasome substrate processing and induce a cellular response characteristic of proteasome inhibition. Biochemical and structural analyses showed binding to and inhibition of proteasome-associated cysteine deubiquitinases, in particular ubiquitin specific peptidase 14 (USP14). The results suggested that compounds bind to a crevice close to the USP14 active site with modest affinity, followed by covalent binding. A subset of compounds was identified where cell death induction was closely associated with proteasome inhibition and that showed significant antineoplastic activity in a zebrafish embryo model. These findings suggest that proteasome inhibition is a relatively common mode of action by cytotoxic compounds containing Michael acceptor groups and help to explain previous reports on the antineoplastic effects of natural products containing such functional groups.

Estradiol affects extracellular leptin:adiponectin ratio in human breast tissue in vivo.

CONTEXT: Exposure to sex steroids is associated with increased breast cancer risk, and adipokines, leptin and adiponectin have been implicated in cancer progression. However, it is not known whether sex steroids affect adipokine secretion in breast tissue. OBJECTIVE: To elucidate the role of estrogen and tamoxifen on adipokine release in normal human breast tissue and breast cancer. SETTING AND DESIGN: Microdialysis sampling was used to collect extracellular in vivo leptin and adiponectin from normal human breast tissue in premenopausal healthy volunteers during the menstrual cycle and in postmenopausal women before tamoxifen treatment and after 6 weeks of treatment. In women with breast cancer, microdialysis was performed intratumorally before surgery. In addition, whole normal breast tissue biopsies were cultured ex vivo, and murine breast cancer models were evaluated. RESULTS: In normal breast tissue, plasma estradiol negatively correlated with local extracellular adiponectin levels (r = -0.34; P < .05) and positively correlated with leptin (r = 0.37; P < .05) and leptin:adiponectin ratio (r = 0.38; P < .05). In postmenopausal women, tamoxifen treatment increased adiponectin (P < 0.05) and decreased leptin (P < .01) and the leptin:adiponectin ratio (P < .01). These in vivo results were confirmed in breast tissue biopsies cultured ex vivo. In patients with breast cancer, extracellular leptin was higher (P < .01) and adiponectin lower (P < .05) in tumors than in normal adjacent breast tissue. In a murine model of breast cancer, estrogen exposure increased leptin secretion (P < .05). CONCLUSIONS: Estrogen exposure may have a critical role in the regulation of adipokines in human breast tissue and may serve as therapeutic targets for treatment and prevention.

Estradiol, tamoxifen, and flaxseed alter IL-1ß and IL-1Ra levels in normal human breast tissue in vivo.

INTRODUCTION: Sex steroid exposure increases the risk of breast cancer by unclear mechanisms. Diet modifications may be one breast cancer prevention strategy. The proinflammatory cytokine family of IL-1 is implicated in cancer progression. IL-1Ra is an endogenous inhibitor of the proinflammatory IL-1α and IL-1ß. OBJECTIVE: The objective of this study was to elucidate whether estrogen, tamoxifen, and/or diet modification altered IL-1 levels in normal human breast tissue. DESIGN AND METHODS: Microdialysis was performed in healthy women under various hormone exposures, tamoxifen therapy, and diet modifications and in breast cancers of women before surgery. Breast tissue biopsies from reduction mammoplasties were cultured. RESULTS: We show a significant positive correlation between estradiol and in vivo levels of IL-1ß in breast tissue and abdominal sc fat, whereas IL-1Ra exhibited a significant negative correlation with estradiol in breast tissue. Tamoxifen or a dietary addition of 25 g flaxseed per day resulted in significantly increased levels of IL-1Ra in the breast. These results were confirmed in ex vivo culture of breast biopsies. Immunohistochemistry of the biopsies did not reveal any changes in cellular content of the IL-1s, suggesting that mainly the secreted levels were affected. In breast cancer patients, intratumoral levels of IL-1ß were significantly higher compared with normal adjacent breast tissue. CONCLUSION: IL-1 may be under the control of estrogen in vivo and may be attenuated by antiestrogen therapy and diet modifications. The increased IL-1ß in breast cancers of women strongly suggests IL-1 as a potential therapeutic target in breast cancer treatment and prevention.