Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent protein kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has an unique consensus substrate phosphorylation motif compared to conventional CDKs. To elucidate the structure and inhibitor binding properties of this atypical CDK we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multi-targeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts

Interrogating the CD27:CD70 axis in αCD40-dependent control of pancreatic adenocarcinoma

Immune checkpoint blockade immunotherapy has radically changed patient outcomes in multiple cancer types. Pancreatic cancer is one of the notable exceptions, being protected from immunotherapy by a variety of mechanisms, including the presence of a dense stroma and immunosuppressive myeloid cells. Previous studies have demonstrated that CD40 stimulation can remodel the tumor microenvironment in a manner that promotes effector immune cell responses and can cooperate with immune checkpoint inhibition for durable tumor control mediated by T cells. Here we confirm the capability of this combination therapy to dramatically, and durably, control pancreatic cancer growth in an orthotopic model and that the immune memory to this cancer is primarily a function of CD4+ T cells. We extend this understanding by demonstrating that recruitment of recently primed T cells from the draining lymph nodes is not necessary for the observed control, suggesting that the pre-existing intra-tumoral cells respond to the combination therapy. Further, we find that the efficacy of CD40 stimulation is not dependent upon CD70, which is commonly induced on dendritic cells in response to CD40 agonism. Finally, we find that directly targeting the receptor for CD70, CD27, in combination with the TLR3 agonist polyIC, provides some protection despite failing to increase the frequency of interferon gamma-secreting T cells

CDKL Family Kinases Have Evolved Distinct Structural Features and Ciliary Function

Various kinases, including a cyclin-dependent kinase (CDK) family member, regulate the growth and functions of primary cilia, which perform essential roles in signaling and development. Neurological disorders linked to CDK-Like (CDKL) proteins suggest that these underexplored kinases may have similar functions. Here, we present the crystal structures of human CDKL1, CDKL2, CDKL3, and CDKL5, revealing their evolutionary divergence from CDK and mitogen-activated protein kinases (MAPKs), including an unusual ?J helix important for CDKL2 and CDKL3 activity. C. elegans CDKL-1, most closely related to CDKL1-4 and localized to neuronal cilia transition zones, modulates cilium length; this depends on its kinase activity and ?J helix-containing C terminus. Human CDKL5, linked to Rett syndrome, also localizes to cilia, and it impairs ciliogenesis when overexpressed. CDKL5 patient mutations modeled in CDKL-1 cause localization and/or cilium length defects. Together, our studies establish a disease model system suggesting cilium length defects as a pathomechanism for neurological disorders, including epilepsy

The accretion origin of the Milky Way's stellar halo

We have used data from the Sloan Digital Sky Survey (SDSS) Data Release 5 to explore the overall structure and substructure of the stellar halo of the Milky Way using about 4 million color-selected main sequence turn-off stars. We fit oblate and triaxial broken power-law models to the data, and found a `best-fit' oblateness of the stellar halo 0.5<c/a<0.8, and halo stellar masses between Galactocentric radii of 1 and 40kpc of (3.7+/-1.2)x10^8 M_sun. The density profile of the stellar halo is approximately r^{-3}; it is possible that the power law slope is shallower inside 20kpc and steeper outside that radius. Yet, we found that all smooth and symmetric models were very poor fits to the distribution of stellar halo stars because the data exhibit a great deal of spatial substructure. We quantified deviations from a smooth oblate/triaxial model using the RMS of the data around the model profile on scales >~100pc, after accounting for the (known) contribution of Poisson uncertainties. The fractional RMS deviation of the actual stellar distribution from any smooth, parameterized halo model is >~40%: hence, the stellar halo is highly structured. We compared the observations with simulations of galactic stellar halos formed entirely from the accretion of satellites in a cosmological context by analysing the simulations in the same way as the data. While the masses, overall profiles, and degree of substructure in the simulated stellar halos show considerable scatter, the properties and degree of substructure in the Milky Way's halo match well the properties of a `typical' stellar halo built exclusively out of the debris from disrupted satellite galaxies. Our results therefore point towards a picture in which an important fraction of the Milky Way's stellar halo has been accreted from satellite galaxies.Comment: Submitted to the Astrophysical Journal. 14 pages; 11 figure

The DUF1669 domain of FAM83 family proteins anchor casein kinase 1 isoforms

Members of the casein kinase 1 (CK1) family of serine-threonine protein kinases are implicated in the regulation of many cellular processes, including the cell cycle, circadian rhythms, and Wnt and Hedgehog signaling. Because these kinases exhibit constitutive activity in biochemical assays, it is likely that their activity in cells is controlled by subcellular localization, interactions with inhibitory proteins, targeted degradation, or combinations of these mechanisms. We identified members of the FAM83 family of proteins as partners of CK1 in cells. All eight members of the FAM83 family (FAM83A to FAM83H) interacted with the α and α-like isoforms of CK1; FAM83A, FAM83B, FAM83E, and FAM83H also interacted with the δ and ε isoforms of CK1. We detected no interaction between any FAM83 member and the related CK1γ1, CK1γ2, and CK1γ3 isoforms. Each FAM83 protein exhibited a distinct pattern of subcellular distribution and colocalized with the CK1 isoform(s) to which it bound. The interaction of FAM83 proteins with CK1 isoforms was mediated by the conserved domain of unknown function 1669 (DUF1669) that characterizes the FAM83 family. Mutations in FAM83 proteins that prevented them from binding to CK1 interfered with the proper subcellular localization and cellular functions of both the FAM83 proteins and their CK1 binding partners. On the basis of its function, we propose that DUF1669 be renamed the polypeptide anchor of CK1 domain

Two Stellar Components in the Halo of the Milky Way

The halo of the Milky Way provides unique elemental abundance and kinematic information on the first objects to form in the Universe, which can be used to tightly constrain models of galaxy formation and evolution. Although the halo was once considered a single component, evidence for its dichotomy has slowly emerged in recent years from inspection of small samples of halo objects. Here we show that the halo is indeed clearly divisible into two broadly overlapping structural components -- an inner and an outer halo -- that exhibit different spatial density profiles, stellar orbits and stellar metallicities (abundances of elements heavier than helium). The inner halo has a modest net prograde rotation, whereas the outer halo exhibits a net retrograde rotation and a peak metallicity one-third that of the inner halo. These properties indicate that the individual halo components probably formed in fundamentally different ways, through successive dissipational (inner) and dissipationless (outer) mergers and tidal disruption of proto-Galactic clumps.Comment: Two stand-alone files in manuscript, concatenated together. The first is for the main paper, the second for supplementary information. The version is consistent with the version published in Natur

Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i-->i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, alpha-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable alpha-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ~9 kcal/mol, but this was compensated by increased conformational entropy (TDS ≤ 7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by alpha-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases

Measuring the Impacts of Community-based Grasslands Management in Mongolia's Gobi

We assessed a donor-funded grassland management project designed to create both conservation and livelihood benefits in the rangelands of Mongolia's Gobi desert. The project ran from 1995 to 2006, and we used remote sensing Normalized Differential Vegetation Index data from 1982 to 2009 to compare project grazing sites to matched control sites before and after the project's implementation. We found that the productivity of project grazing sites was on average within 1% of control sites for the 20 years before the project but generated 11% more biomass on average than the control areas from 2000 to 2009. To better understand the benefits of the improved grasslands to local people, we conducted 280 household interviews, 8 focus group discussions, and 31 key informant interviews across 6 districts. We found a 12% greater median annual income as well as a range of other socioeconomic benefits for project households compared to control households in the same areas. Overall, the project generated measurable benefits to both nature and people. The key factors underlying project achievements that may be replicable by other conservation projects include the community-driven approach of the project, knowledge exchanges within and between communities inside and outside the country, a project-supported local community organizer in each district, and strong community leadership

LSST Science Book, Version 2.0

A survey that can cover the sky in optical bands over wide fields to faint magnitudes with a fast cadence will enable many of the exciting science opportunities of the next decade. The Large Synoptic Survey Telescope (LSST) will have an effective aperture of 6.7 meters and an imaging camera with field of view of 9.6 deg^2, and will be devoted to a ten-year imaging survey over 20,000 deg^2 south of +15 deg. Each pointing will be imaged 2000 times with fifteen second exposures in six broad bands from 0.35 to 1.1 microns, to a total point-source depth of r~27.5. The LSST Science Book describes the basic parameters of the LSST hardware, software, and observing plans. The book discusses educational and outreach opportunities, then goes on to describe a broad range of science that LSST will revolutionize: mapping the inner and outer Solar System, stellar populations in the Milky Way and nearby galaxies, the structure of the Milky Way disk and halo and other objects in the Local Volume, transient and variable objects both at low and high redshift, and the properties of normal and active galaxies at low and high redshift. It then turns to far-field cosmological topics, exploring properties of supernovae to z~1, strong and weak lensing, the large-scale distribution of galaxies and baryon oscillations, and how these different probes may be combined to constrain cosmological models and the physics of dark energy.Comment: 596 pages. Also available at full resolution at http://www.lsst.org/lsst/sciboo

Target 2035-update on the quest for a probe for every protein

Twenty years after the publication of the first draft of the human genome, our knowledge of the human proteome is still fragmented. The challenge of translating the wealth of new knowledge from genomics into new medicines is that proteins, and not genes, are the primary executers of biological function. Therefore, much of how biology works in health and disease must be understood through the lens of protein function. Accordingly, a subset of human proteins has been at the heart of research interests of scientists over the centuries, and we have accumulated varying degrees of knowledge about approximately 65% of the human proteome. Nevertheless, a large proportion of proteins in the human proteome (∼35%) remains uncharacterized, and less than 5% of the human proteome has been successfully targeted for drug discovery. This highlights the profound disconnect between our abilities to obtain genetic information and subsequent development of effective medicines. Target 2035 is an international federation of biomedical scientists from the public and private sectors, which aims to address this gap by developing and applying new technologies to create by year 2035 chemogenomic libraries, chemical probes, and/or biological probes for the entire human proteome