Molecular basis for modulation of the p53 target selectivity by KLF4

The tumour suppressor p53 controls transcription of various genes involved in apoptosis, cell-cycle arrest, DNA repair and metabolism. However, its DNA-recognition specificity is not nearly sufficient to explain binding to specific locations in vivo. Here, we present evidence that KLF4 increases the DNA-binding affinity of p53 through the formation of a loosely arranged ternary complex on DNA. This effect depends on the distance between the response elements of KLF4 and p53. Using nuclear magnetic resonance and fluorescence techniques, we found that the amino-terminal domain of p53 interacts with the KLF4 zinc fingers and mapped the interaction site. The strength of this interaction was increased by phosphorylation of the p53 N-terminus, particularly on residues associated with regulation of cell-cycle arrest genes. Taken together, the cooperative binding of KLF4 and p53 to DNA exemplifies a regulatory mechanism that contributes to p53 target selectivity

Insights into congenital stationary night blindness based on the structure of G90D rhodopsin

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/1/embr201344.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/2/embr201344.reviewer_comments.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102109/3/embr201344-sup-0001.pd

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

Dissecting the Relation between a Nuclear Receptor and GATA: Binding Affinity Studies of Thyroid Hormone Receptor and GATA2 on TSHβ Promoter

Background: Much is known about how genes regulated by nuclear receptors (NRs) are switched on in the presence of a ligand. However, the molecular mechanism for gene down-regulation by liganded NRs remains a conundrum. The interaction between two zinc-finger transcription factors, Nuclear Receptor and GATA, was described almost a decade ago as a strategy adopted by the cell to up-or down-regulate gene expression. More recently, cell-based assays have shown that the Zn-finger region of GATA2 (GATA2-Zf) has an important role in down-regulation of the thyrotropin gene (TSH beta) by liganded thyroid hormone receptor (TR). Methodology/Principal Findings: In an effort to better understand the mechanism that drives TSH beta down-regulation by a liganded TR and GATA2, we have carried out equilibrium binding assays using fluorescence anisotropy to study the interaction of recombinant TR and GATA2-Zf with regulatory elements present in the TSH beta promoter. Surprisingly, we observed that ligand (T3) weakens TR binding to a negative regulatory element (NRE) present in the TSH beta promoter. We also show that TR may interact with GATA2-Zf in the absence of ligand, but T3 is crucial for increasing the affinity of this complex for different GATA response elements (GATA-REs). Importantly, these results indicate that TR complex formation enhances DNA binding of the TR-GATA2 in a ligand-dependent manner. Conclusions: Our findings extend previous results obtained in vivo, further improving our understanding of how liganded nuclear receptors down-regulate gene transcription, with the cooperative binding of transcription factors to DNA forming the core of this process.Medical Research Council (MRC), UKConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazi

Monitoring of breast cancer progression via aptamer-based detection of circulating tumor cells in clinical blood samples

Introduction: Breast cancer (BC) diagnostics lack noninvasive methods and procedures for screening and monitoring disease dynamics. Admitted CellSearch® is used for fluid biopsy and capture of circulating tumor cells of only epithelial origin. Here we describe an RNA aptamer (MDA231) for detecting BC cells in clinical samples, including blood. The MDA231 aptamer was originally selected against triple-negative breast cancer cell line MDA-MB-231 using cell-SELEX.Methods: The aptamer structure in solution was predicted using mFold program and molecular dynamic simulations. The affinity and specificity of the evolved aptamers were evaluated by flow cytometry and laser scanning microscopy on clinical tissues from breast cancer patients. CTCs were isolated form the patients’ blood using the developed method of aptamer-based magnetic separation. Breast cancer origin of CTCs was confirmed by cytological, RT-qPCR and Immunocytochemical analyses.Results: MDA231 can specifically recognize breast cancer cells in surgically resected tissues from patients with different molecular subtypes: triple-negative, Luminal A, and Luminal B, but not in benign tumors, lung cancer, glial tumor and healthy epithelial from lungs and breast. This RNA aptamer can identify cancer cells in complex cellular environments, including tumor biopsies (e.g., tumor tissues vs. margins) and clinical blood samples (e.g., circulating tumor cells). Breast cancer origin of the aptamer-based magnetically separated CTCs has been proved by immunocytochemistry and mammaglobin mRNA expression.Discussion: We suggest a simple, minimally-invasive breast cancer diagnostic method based on non-epithelial MDA231 aptamer-specific magnetic isolation of circulating tumor cells. Isolated cells are intact and can be utilized for molecular diagnostics purposes

Proteomics-Based Regression Model for Assessing the Development of Chronic Lymphocytic Leukemia

The clinical course of chronic lymphocytic leukemia (CLL) is very ambiguous, showing either an indolent nature of the disease or having latent dangerous progression, which, if diagnosed, will require an urgent therapy. The prognosis of the course of the disease and the estimation of the time of therapy initiation are crucial for the selection of a successful treatment strategy. A reliable estimating index is needed to assign newly diagnosed CLL patients to the prognostic groups. In this work, we evaluated the comparative expressions of proteins in CLL blood cells using a label-free quantification by mass spectrometry and calculated the integrated proteomic indexes for a group of patients who received therapy after the blood sampling over different periods of time. Using a two-factor linear regression analysis based on these data, we propose a new pipeline for evaluating model development for estimation of the moment of therapy initiation for newly diagnosed CLL patients

Binding of labelled N-terminal p53 peptides to KLF4 367–479.

*<p>Experiments were carried out at low ionic strength (110 mM) in order to reliably measure the binding constant. NMR experiments confirmed binding at physiological ionic strength.</p

Identification of the binding interface in KLF4 367–479.

<p>A: 2D ¹H,¹⁵N-HSQC spectrum for KLF4 (367–479) (red), labelled with resonance assignments, overlaid with a spectrum observed in the presence of 300 µM p53 (1–57) pT55 as a ligand (cyan). B: Chemical shift perturbations of KLF4 (367–479) residues upon addition of p53 (1–57) pT55. No signal was observed for Ser440 (blue) in the presence of p53.</p

KLF4 enhances the DNA-binding affinity of p53.

<p>A: DNA constructs generated and principle of cooperative fluorescence anisotropy titrations. Fluorescein (*, star), a p53 RE (<i>P</i>, dark grey), a spacer (<i>n</i>, light grey), and a KLF4 RE (<i>K</i>, black) compose the labelled DNA. The affinity of p53 towards DNA is measured in the presence (+ KLF4) and absence (- KLF4) of KLF4. B: Example p53 titration data using *P30K as DNA in the presence (circles) and absence (squares) of KLF4 in FA285 buffer. C: Normalised p53 DNA-binding affinity in the presence of 400 nM KLF4 as a function of the distance between the p53 RE and the KLF4 RE.</p

KLF4 and p53 directly interact.

<p>A: Domain structure of p53 and KLF4. Folded domains are shown in grey. p53 comprises an N-terminal domain (NTD) consisting of the transactivation domains 1 and 2 (TAD1, TAD2) and the proline-rich domain (PRD), a DNA-binding domain (DBD), a tetramerisation domain (TD), and a C-terminal domain (CTD). KLF4 domain boundaries for the transcriptional activation domain (AD) and inhibitory domain (ID) are approximate. Three zinc fingers (ZF) are encoded at the C-terminus. B: Normalised sedimentation coefficient distributions measured by FDSV-AUC. 225 nM FlAsH-labelled KLF4 in the absence (black) and in presence of 0.5 µM (grey), 5 µM (dotted line) and 50 µM (dashed line) unlabelled p53. Engineered, neutrally stabilised quadruple mutant M133L/V203A/N239Y/N268D p53 was used throughout this study. C: Direct interaction between p53 and KLF4. Fluorescence anisotropy titration at 110 mM total ionic strength with FlAsH-labelled KLF4 as probe and p53 as titrant. D: Fluorescence anisotropy titrations with a labelled p53 RE (*P). No binding is observed if KLF4 is titrated into a DNA-only solution (circles). If a *P-p53 complex is used as a probe (note higher anisotropy value, squares, FA285 buffer), a binding event can be observed.</p