Zinc Binding Catalytic Domain of Human Tankyrase 1

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substratebinding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular

Landscape of somatic allelic imbalances and copy number alterations in HER2-amplified breast cancer

Introduction: Human epidermal growth factor receptor 2 (HER2)-amplified breast cancer represents a clinically well-defined subgroup due to availability of targeted treatment. However, HER2-amplified tumors have been shown to be heterogeneous at the genomic level by genome-wide microarray analyses, pointing towards a need of further investigations for identification of recurrent copy number alterations and delineation of patterns of allelic imbalance. Methods: High-density whole genome array-based comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) array data from 260 HER2-amplified breast tumors or cell lines, and 346 HER2-negative breast cancers with molecular subtype information were assembled from different repositories. Copy number alteration (CNA), loss-of-heterozygosity (LOH), copy number neutral allelic imbalance (CNN-AI), subclonal CNA and patterns of tumor DNA ploidy were analyzed using bioinformatical methods such as genomic identification of significant targets in cancer (GISTIC) and genome alteration print (GAP). The patterns of tumor ploidy were confirmed in 338 unrelated breast cancers analyzed by DNA flow cytometry with concurrent BAC aCGH and gene expression data. Results: A core set of 36 genomic regions commonly affected by copy number gain or loss was identified by integrating results with a previous study, together comprising > 400 HER2-amplified tumors. While CNN-AI frequency appeared evenly distributed over chromosomes in HER2-amplified tumors, not targeting specific regions and often < 20% in frequency, the occurrence of LOH was strongly associated with regions of copy number loss. HER2-amplified and HER2-negative tumors stratified by molecular subtypes displayed different patterns of LOH and CNN-AI, with basal-like tumors showing highest frequencies followed by HER2-amplified and luminal B cases. Tumor aneuploidy was strongly associated with increasing levels of LOH, CNN-AI, CNAs and occurrence of subclonal copy number events, irrespective of subtype. Finally, SNP data from individual tumors indicated that genomic amplification in general appears as monoallelic, that is, it preferentially targets one parental chromosome in HER2-amplified tumors. Conclusions: We have delineated the genomic landscape of CNAs, amplifications, LOH, and CNN-AI in HER2-amplified breast cancer, but also demonstrated a strong association between different types of genomic aberrations and tumor aneuploidy irrespective of molecular subtype

Planet Formation Imager (PFI): science vision and key requirements

The Planet Formation Imager (PFI) project aims to provide a strong scientific vision for ground-based optical astronomy beyond the upcoming generation of Extremely Large Telescopes. We make the case that a breakthrough in angular resolution imaging capabilities is required in order to unravel the processes involved in planet formation. PFI will be optimised to provide a complete census of the protoplanet population at all stellocentric radii and over the age range from 0.1 to ~100 Myr. Within this age period, planetary systems undergo dramatic changes and the final architecture of planetary systems is determined. Our goal is to study the planetary birth on the natural spatial scale where the material is assembled, which is the "Hill Sphere" of the forming planet, and to characterise the protoplanetary cores by measuring their masses and physical properties. Our science working group has investigated the observational characteristics of these young protoplanets as well as the migration mechanisms that might alter the system architecture. We simulated the imprints that the planets leave in the disk and study how PFI could revolutionise areas ranging from exoplanet to extragalactic science. In this contribution we outline the key science drivers of PFI and discuss the requirements that will guide the technology choices, the site selection, and potential science/technology tradeoffs.S.K. acknowledges support from an STFC Rutherford Fellowship (ST/J004030/1) and Philip Leverhulme Prize (PLP-2013-110). Part of this work was carried out at the Jet Propulsion Laboratory, California Institute of Technology, under a contract with the National Aeronautics and Space Administration

Development of new NMR techniques and the structure of the N-terminal domain of Escherichia coli DnaB helicas

New techniques applicable in biomolecular nuclear magnetic resonance (NMR) spectroscopy were developed. The three-dimensional structure of the N-terminal domain of Escherichia coli DnaB helicase was determined at atomic resolution using heteronuclear multidimensional NMR techniques. Homonuclear semi-selective decoupling during acquisition improves the resolution of an NMR experiment and presents a convenient way of suppressing zero-quantum cross peaks in NOESY and ROESY experiments. The decoupling pulses can, however, result in decoupling sidebands due to the modulation of the effective magnetic field by the decoupling pulses. Two methods for the suppression of the decoupling sidebands were devised. Spin-state selective filters for the separation of doublet components into different subspectra were developed. The filters are versatile building blocks for NMR pulse sequences and can be used in a straightforward manner for the measurement of one-bond coupling constants and relaxation interference effects as well as for the design of TROSY type experiments. A single scan, sensitivity enhanced TROSY experiment with pulsed field gradients for coherence order selection was developed. Since the modified TROSY pulse sequence requires only a single scan (two FIDs per complex data point) it represents a sequence element suitable for inclusion in more complex NMR experiments. DnaB helicase is the primary replicatory helicase in Escherichia coli. Native DnaB is a hexamer of identical 52.3 kDa subunits. Each subunit is comprised of two domains. The structurally well-defined core of the N-terminal domain of DnaB was determined by NMR to comprise residues 24 to 136 by resonance assignments of the flexible residues in two DnaB fragments of different length, DnaB(1-142) and DnaB(1-161). A third construct, DnaB(24-136), was subsequently overexpressed and isotopically enriched for structure determination. The three-dimensional structure was calculated using the program DYANA and energy minimised using the program OPAL. The final 20 conformers had a root mean square deviation relative to the mean structure of 0.51 A for the backbone heavy atoms of residues 29-134. An estimate of the rotational correlation time of DnaB(24-136) and ultracentrifugation data indicated partial self-aggregation of DnaB(24-136) into dimers. Chemical shift changes of amide (15N-1H) groups were measured as a function of protein concentration and identified the dimerisation interface. The dissociation constant was estimated to be in the mM range. A model of the dimer was built based on the identification of the interaction surface and intermolecular nuclear Overhauser effects (NOEs) and is in agreement with residual dipolar couplings. An association-dissociation equilibrium for the N-terminal domain of DnaB has been proposed earlier from electron microscopy data and is thought to be of importance in the functional DnaB hexamer. Interestingly, DnaB(24-136) is structurally related to the primary dimerisation domain of Escherichia coli gyrase A, which may indicate functional similarities

NMR structure of the N-terminal domain of E. coli DnaB helicase: implications for structure rearrangements in the helicase hexamer

Background: DnaB is the primary replicative helicase in Escherichia coli. Native DnaB is a hexamer of identical subunits, each consisting of a larger C-terminal domain and a smaller N-terminal domain. Electron-microscopy data show hexamers with C6 or C3 symmetry, indicating large domain movements and reversible pairwise association. Results: The three-dimensional structure of the N-terminal domain of E. coli DnaB was determined by nuclear magnetic resonance (NMR) spectroscopy. Structural similarity was found with the primary dimerisation domain of a topoisomerase, the gyrase A subunit from E. coli. A monomer-dimer equilibrium was observed for the isolated N-terminal domain of DnaB. A dimer model with C2 symmetry was derived from intermolecular nuclear Overhauser effects, which is consistent with all available NMR data. Conclusions: The monomer-dimer equilibrium observed for the N-terminal domain of DnaB is likely to be of functional significance for helicase activity, by participating in the switch between C6 and C3 symmetry of the helicase hexamer

PARP inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/PARP3

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1