Lessons from LIMK1 enzymology and their impact on inhibitor design

LIM domain kinase 1 (LIMK1) is a key regulator of actin dynamics. It is thereby a potential therapeutic target for the prevention of fragile X syndrome and amyotrophic lateral sclerosis. Herein, we use X-ray crystallography and activity assays to describe how LIMK1 accomplishes substrate specificity, to suggest a unique ‘rock-and-poke’ mechanism of catalysis and to explore the regulation of the kinase by activation loop phosphorylation. Based on these findings, a differential scanning fluorimetry assay and a RapidFire mass spectrometry activity assay were established, leading to the discovery and confirmation of a set of small-molecule LIMK1 inhibitors. Interestingly, several of the inhibitors were inactive towards the closely related isoform LIMK2. Finally, crystal structures of the LIMK1 kinase domain in complex with inhibitors (PF-477736 and staurosporine, respectively) are presented, providing insights into LIMK1 plasticity upon inhibitor binding

A Photometric Metallicity Estimate of the Virgo Stellar Overdensity

We determine photometric metal abundance estimates for individual main-sequence stars in the Virgo Overdensity (VOD), which covers almost 1000 deg^2 on the sky, based on a calibration of the metallicity sensitivity of stellar isochrones in the gri filter passbands using field stars with well-determined spectroscopic metal abundances. Despite the low precision of the method for individual stars, we derive [Fe/H] = -2.0 +/-0.1 (internal) +/-0.5 (systematic) for the metal abundance of the VOD from photometric measurements of 0.7 million stars in the Northern Galactic hemisphere with heliocentric distances from ~10 kpc to ~20 kpc. The metallicity of the VOD is indistinguishable, within Delta [Fe/H] < 0.2, from that of field halo stars covering the same distance range. This initial application suggests that the SDSS gri passbands can be used to probe the properties of main-sequence stars beyond ~10 kpc, complementing studies of nearby stars from more metallicity-sensitive color indices that involve the u passband.Comment: 5 pages, 3 figures, Accepted for publication in ApJ Letter

Signatures of minor mergers in the Milky Way disc I: The SEGUE stellar sample

It is now known that minor mergers are capable of creating structure in the phase-space distribution of their host galaxy's disc. In order to search for such imprints in the Milky Way, we analyse the SEGUE F/G-dwarf and the Schuster et al. (2006) stellar samples. We find similar features in these two completely independent stellar samples, consistent with the predictions of a Milky Way minor-merger event. We next apply the same analyses to high-resolution, idealised N-body simulations of the interaction between the Sagittarius dwarf galaxy and the Milky Way. The energy distributions of stellar particle samples in small spatial regions in the host disc reveal strong variations of structure with position. We find good matches to the observations for models with a mass of Sagittarius' dark matter halo progenitor $\lessapprox 10^{11}$ M$_{\odot}$. Thus, we show that this kind of analysis could be used to provide unprecedentedly tight constraints on Sagittarius' orbital parameters, as well as place a lower limit on its mass.Comment: 14 pages, 9 figures, 2 tables. Revised to reflect accepted versio

Human With No Lysine Kinase 3 (WNK3): A Target Enabling Package (TEP)

The Target Enabling Package (TEP) programme's foundation is built upon the recognition that genetic data is proving to be a powerful tool for target validation. As such, TEPs provide a critical mass of reagents and knowledge on a protein target to allow rapid biochemical and chemical exploration and characterisation of proteins with genetic linkage to key disease areas. TEPs provide an answer to the missing link between genomics and chemical biology, provide a starting point for chemical probe generation and therefore catalyse new biology and disease understanding with the ultimate aim of enabling translation collaborations and target/ drug discovery. We are committed to generating and making available 24 high-quality TEPs by June 2020.SUMMARY OF PROJECT Kinases WNK1-4 regulate cation-chloride cotransporters via phosphorylation of SPAK and OSR1 and thereby control salt homeostasis, cell volume and blood pressure. Gain of function mutations in WNK kinases are found in Gordon’s hypertension syndrome suggesting the WNK pathway as a therapeutic target. WNK3 inhibition in particular has also been shown to reduce cerebral injury after Ischemic stroke. Here we present assays and crystal structures that define (i) the molecular basis for disease mutations; (ii) the multiple functional domains of WNK kinases and their protein interactions; (iii) the binding of small molecule kinase inhibitors and a potential allosteric pocket.The work performed at the SGC has been funded by a grant from the Wellcome [106169/ZZ14/Z]

Light Curve Templates and Galactic Distribution of RR Lyrae Stars from Sloan Digital Sky Survey Stripe 82

We present an improved analysis of halo substructure traced by RR Lyrae stars in the SDSS stripe 82 region. With the addition of SDSS-II data, a revised selection method based on new ugriz light curve templates results in a sample of 483 RR Lyrae stars that is essentially free of contamination. The main result from our first study persists: the spatial distribution of halo stars at galactocentric distances 5--100 kpc is highly inhomogeneous. At least 20% of halo stars within 30 kpc from the Galactic center can be statistically associated with substructure. We present strong direct evidence, based on both RR Lyrae stars and main sequence stars, that the halo stellar number density profile significantly steepens beyond a Galactocentric distance of ~30 kpc, and a larger fraction of the stars are associated with substructure. By using a novel method that simultaneously combines data for RR Lyrae and main sequence stars, and using photometric metallicity estimates for main sequence stars derived from deep co-added u-band data, we measure the metallicity of the Sagittarius dSph tidal stream (trailing arm) towards R.A.2h-3h and Dec~0 deg to be 0.3 dex higher ([Fe/H]=-1.2) than that of surrounding halo field stars. Together with a similar result for another major halo substructure, the Monoceros stream, these results support theoretical predictions that an early forming, smooth inner halo, is metal poor compared to high surface brightness material that have been accreted onto a later-forming outer halo. The mean metallicity of stars in the outer halo that are not associated with detectable clumps may still be more metal-poor than the bulk of inner-halo stars, as has been argued from other data sets.Comment: Submitted to ApJ, 68 pages, 26 figures, supplemental material (light curves, templates, animation) can be downloaded from http://www.astro.washington.edu/bsesar/S82_RRLyr.htm

The mass and anisotropy profiles of galaxy clusters from the projected phase space density: testing the method on simulated data

We present a new method of constraining the mass and velocity anisotropy profiles of galaxy clusters from kinematic data. The method is based on a model of the phase space density which allows the anisotropy to vary with radius between two asymptotic values. The characteristic scale of transition between these asymptotes is fixed and tuned to a typical anisotropy profile resulting from cosmological simulations. The model is parametrized by two values of anisotropy, at the centre of the cluster and at infinity, and two parameters of the NFW density profile, the scale radius and the scale mass. In order to test the performance of the method in reconstructing the true cluster parameters we analyze mock kinematic data for 20 relaxed galaxy clusters generated from a cosmological simulation of the standard LCDM model. We use Bayesian methods of inference and the analysis is carried out following the Markov Chain Monte Carlo approach. The parameters of the mass profile are reproduced quite well, but we note that the mass is typically underestimated by 15 percent, probably due to the presence of small velocity substructures. The constraints on the anisotropy profile for a single cluster are in general barely conclusive. Although the central asymptotic value is determined accurately, the outer one is subject to significant systematic errors caused by substructures at large clustercentric distance. The anisotropy profile is much better constrained if one performs joint analysis of at least a few clusters. In this case it is possible to reproduce the radial variation of the anisotropy over two decades in radius inside the virial sphere.Comment: 11 pages, 10 figures, accepted for publication in MNRA

Crystal Structure of the PIM2 Kinase in Complex with an Organoruthenium Inhibitor

BACKGROUND: The serine/threonine kinase PIM2 is highly expressed in human leukemia and lymphomas and has been shown to positively regulate survival and proliferation of tumor cells. Its diverse ATP site makes PIM2 a promising target for the development of anticancer agents. To date our knowledge of catalytic domain structures of the PIM kinase family is limited to PIM1 which has been extensively studied and which shares about 50% sequence identity with PIM2. PRINCIPAL FINDINGS: Here we determined the crystal structure of PIM2 in complex with an organoruthenium complex (inhibition in sub-nanomolar level). Due to its extraordinary shape complementarity this stable organometallic compound is a highly potent inhibitor of PIM kinases. SIGNIFICANCE: The structure of PIM2 revealed several differences to PIM1 which may be explored further to generate isoform selective inhibitors. It has also demonstrated how an organometallic inhibitor can be adapted to the binding site of protein kinases to generate highly potent inhibitors. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

A Complete Spectroscopic Survey of the Milky Way Satellite Segue 1: The Darkest Galaxy

We present the results of a comprehensive Keck/DEIMOS spectroscopic survey of the ultra-faint Milky Way satellite galaxy Segue 1. We have obtained velocity measurements for 98.2% of the stars within 67 pc (10 arcmin, or 2.3 half-light radii) of the center of Segue 1 that have colors and magnitudes consistent with membership, down to a magnitude limit of r=21.7. Based on photometric, kinematic, and metallicity information, we identify 71 stars as probable Segue 1 members, including some as far out as 87 pc. After correcting for the influence of binary stars using repeated velocity measurements, we determine a velocity dispersion of 3.7^{+1.4}_{-1.1} km/s, with a corresponding mass within the half-light radius of 5.8^{+8.2}_{-3.1} x 10^5 Msun. The stellar kinematics of Segue 1 require very high mass-to-light ratios unless the system is far from dynamical equilibrium, even if the period distribution of unresolved binary stars is skewed toward implausibly short periods. With a total luminosity less than that of a single bright red giant and a V-band mass-to-light ratio of 3400 Msun/Lsun, Segue 1 is the darkest galaxy currently known. We critically re-examine recent claims that Segue 1 is a tidally disrupting star cluster and that kinematic samples are contaminated by the Sagittarius stream. The extremely low metallicities ([Fe/H] < -3) of two Segue 1 stars and the large metallicity spread among the members demonstrate conclusively that Segue 1 is a dwarf galaxy, and we find no evidence in favor of tidal effects. We also show that contamination by the Sagittarius stream has been overestimated. Segue 1 has the highest measured dark matter density of any known galaxy and will therefore be a prime testing ground for dark matter physics and galaxy formation on small scales.Comment: 24 pages, 4 tables, 11 figures (10 in color). Submitted for publication in ApJ. V3 revised according to comments from the refere

p53 Interacts with RNA Polymerase II through Its Core Domain and Impairs Pol II Processivity In Vivo

The tumor suppressor p53 principally functions as a gene-specific transcription factor. p53 triggers a variety of anti-proliferative programs by activating or repressing the transcription of effector genes in response to genotoxic stress. To date, much effort has been placed on understanding p53's ability to affect transcription in the context of its DNA-binding activity. How p53 regulates transcriptional output independent of DNA binding is less well understood. Here we provide evidence that human p53 can physically interact with the large subunit of RNA polymerase II (Pol II) both in in vitro interaction assays and in whole cell extracts, and that this interaction is mediated (at least in part) through p53's core DNA-binding domain and the Ser5-phosphorylated CTD of Pol II. Ectopic expression of p53, combined with mutations in transcription elongation factors or exposure to drugs that inhibit Pol II elongation, elicit sickness or lethality in yeast cells. These phenotypes are suppressed by oncogenic point mutations within p53's core domain. The growth phenotypes raise the possibility that p53 impairs Pol II elongation. Consistent with this, a p53-dependent increase in Pol II density is seen at constitutively expressed genes without a concomitant increase in transcript accumulation. Additionally, p53-expressing yeast strains exhibit reduced transcriptional processivity at an episomal reporter gene; this inhibitory activity is abolished by a core domain point mutation. Our results suggest a novel mechanism by which p53 can regulate gene transcription, and a new biological function for its core domain that is susceptible to inactivation by oncogenic point mutations

Tidal Signatures in the Faintest Milky Way Satellites: The Detailed Properties of Leo V, Pisces II and Canes Venatici II

We present deep wide-field photometry of three recently discovered faint Milky Way satellites: Leo V, Pisces II, and Canes Venatici II. Our main goals are to study the structure and star formation history of these dwarfs; we also search for signs of tidal disturbance. The three satellites have similar half-light radii ($\sim 60-90$ pc) but a wide range of ellipticities. Both Leo V and CVn II show hints of stream-like overdensities at large radii. An analysis of the satellite color-magnitude diagrams shows that all three objects are old ($>$ 10 Gyr) and metal-poor ([Fe/H] $\sim -2$), though neither the models nor the data have sufficient precision to assess when the satellites formed with respect to cosmic reionization. The lack of an observed younger stellar population (\la 10 Gyr) possibly sets them apart from the other satellites at Galactocentric distances \ga 150 kpc. We present a new compilation of structural data for all Milky Way satellite galaxies and use it to compare the properties of classical dwarfs to the ultra-faints. The ellipticity distribution of the two groups is consistent at the $\sim$2-$\sigma$ level. However, the faintest satellites tend to be more aligned toward the Galactic center, and those satellites with the highest ellipticity (\ga 0.4) have orientations ($\Delta \theta_{GC}$) in the range $20^{\circ} \lesssim \Delta \theta_{GC} \lesssim 40^{\circ}$. This latter observation is in rough agreement with predictions from simulations of dwarf galaxies that have lost a significant fraction of their dark matter halos and are being tidally stripped.Comment: 25 pages, 7 figures, ApJ accepted; version updated to match ApJ accepte