Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rhoassociated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention

Ligand binding mechanism in steroid receptors; from conserved plasticity to differential evolutionary constraints

Steroid receptor drugs have been available for more than half a century, but details 24 of the ligand binding mechanism has remained elusive. We solved X-ray structures of 25 the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at 26 helix 6-7 region that extend the ligand binding pocket towards the receptor surface. 27 Since none of the endogenous ligands exploit this region, we hypothesized that it 28 constitutes an integral part of the binding event. Extensive all atom unbiased ligand 29 exit and entrance simulations corroborate a ligand binding pathway that gives the 30 observed structural plasticity a key functional role. Kinetic measurements reveal that 31 the receptor residence time correlate with structural rearrangements observed in both 32 structures and simulations. Ultimately, our findings reveal why nature has conserved 33 the capacity to open up this region and highlight how differences in the details of the 34 ligand entry process result in differential evolutionary constraints across the steroid 35 receptors.This study was supported by The European Research Council (2009-Adg25027-535 PELE) to V.G and by the SEV-2011-00067 grant of the Severo Ochoa Program. We 536 would like to acknowledge our AstraZeneca colleagues J. Hartleib, R.Unwin and 537 R.Knöll for helpful discussions. We also thank N. Blomberg (ELIXIR) and R. Neutze 538 (University of Gothenburg) for careful reading of the manuscript.Peer ReviewedPostprint (author's final draft

Transport management - a Swedish case study of organizational processes and performance

Transport represents a critical link for lean supply chains from forests to mills. The management processes developed by individual organizations are often a result of their supply context and mill service demands. The goals of this study were to i) provide a general process map for transport management in cut-to-length supply systems and ii) quantify the effect of varying planning and control cycles on organization performance. The key performance indicator for organizational performance was weekly delivery fulfillment, and the study was limited to the non-winter months when there is greatest variation in road trafficability. The process map documents operational planning and control activities at monthly, weekly, and daily levels. Two main variants of planning activities were found. These had diverging effects on delivery fulfillment as transport distances increased but could also facilitate varying degrees of responsiveness in core supply areas. Regarding the control cycle, the number of follow-up activities used by managers increased with the number of mills/terminals served and their total weekly delivery volume

Energy Transduction and Sensory Signalling in Haloarchaea Structural Characterisation of the Photocycles of Bacteriorhodopsin and Sensory Rhodopsin II

Archaeal rhodopsins belong a subfamily of heptahelical transmembrane proteins. All contain a buried retinal chromophore covalently bound to a conserved lysine residue in helix G via a protonated Schiff base. Upon light-activation, retinal is isomerised from an all-trans to a 13-cis configuration, which initiates a sequence of specific structural rearrangements. For bacteriorhodopsin, this results in the vectorial transport of a proton across the cell membrane. For sensory rhodopsin II this initiates a photosignal, which is communicated to a tightly bound transducer molecule. The transducer, in turn, triggers a phosphorylation cascade inducing a negative phototaxis response in the host archaea. The kinetic crystallography studies presented in this thesis reveal the early structural rearrangements of bacteriorhodopsin\u27s photocycle, leading up to the primary proton transfer between the protonated Schiff base and the negatively charged Asp85. Upon photoactivation, retinal isomerisation induces a structural rearrangement in the attached lysine residue which disorders a key water molecule. In the resting state, this water molecule is hydrogen bonded to both the primary proton donor and acceptor. Its removal initiates a sequence of events, reversing the relative proton affinities of the primary proton transfer groups. Moreover, a local bend in helix C, partially driven by the mutual electrostatic attraction between the Schiff base and Asp85, facilitates the creation of a low barrier pathway for the proton transfer. From the early rearrangements observed here and the structural results from later intermediates, a model for vectorial proton pumping is proposed. The mechanism of proton pumping by bacteriorhodopsin is contrasted against the early structural rearrangements observed in the photocycle of sensory rhodopsin II. As indicated by their strong structural relationship, the mechanism for the primary proton transfer event appears to be conserved. Moreover, the putative binding site for the cognate transducer suggests a common structural theme connecting the latter half of the bacteriorhodopsin photocycle and the mechanism of signal relay in sensory rhodopsin II. However, slight modifications of the retinal binding pocket lead to subtle differences in the early relaxation of photoisomerised retinal. These results illustrate how the two systems have been optimised for the different functions of energy transduction and sensory signalling

Pedestrian Group Tracking Using the GM-PHD Filter

A GM-PHD filter is used for pedestrian tracking in a crowdsurveillance application. The purpose is to keep track of thedifferent groups over time as well as to represent the shape ofthe groups and the number of people within the groups. In-put data to the GM-PHD filter are detections using a state ofthe art algorithm applied to video frames from the PETS 2012benchmark data. In a first step, the detections in the framesare converted from image coordinates to world coordinates.This implies that groups can be defined in physical units interms of distance in meters and speed differences in metersper second. The GM-PHD filter is a Bayesian framework thatdoes not form tracks of individuals. Its output is well suitedfor clustering of individuals into groups. The results demon-strate that the GM-PHD filter has the capability of estimatingthe correct number of groups with an accurate representationof their sizes and shapes.CADICSCUA

Pedestrian Group Tracking Using the GM-PHD Filter

A GM-PHD filter is used for pedestrian tracking in a crowdsurveillance application. The purpose is to keep track of thedifferent groups over time as well as to represent the shape ofthe groups and the number of people within the groups. In-put data to the GM-PHD filter are detections using a state ofthe art algorithm applied to video frames from the PETS 2012benchmark data. In a first step, the detections in the framesare converted from image coordinates to world coordinates.This implies that groups can be defined in physical units interms of distance in meters and speed differences in metersper second. The GM-PHD filter is a Bayesian framework thatdoes not form tracks of individuals. Its output is well suitedfor clustering of individuals into groups. The results demon-strate that the GM-PHD filter has the capability of estimatingthe correct number of groups with an accurate representationof their sizes and shapes.CADICSCUA

Unraveling the Allosteric Cross-Talk Between Coactivator Peptide and Ligand Binding Site in Glucocorticoid Receptor

Glucocorticoid receptor (GR) is a nuclear receptor that controls critical biological processes by regulating thetranscription of specific genes. There is a known allosteric cross-talk between the ligand and coregulator bindingsites within the GR ligand binding domain that is crucial for the control of the functional response. However, themolecular mechanisms underlying such an allosteric control remain elusive. Here, molecular dynamics (MD)simulations, bioinformatic analysis and biophysical measurements are integrated to capture the structural anddynamic features of the allosteric cross-talk within GR. We identified a network of evolutionarily conservedresidues that enables the allosteric signal transduction, in agreement with experimental data. MD simulationsclarify how such network is dynamically interconnected and offer a mechanistic explanation of how the differentpeptides affect the intensity of the allosteric signal. This study provides useful insights to elucidate the GRallosteric regulation, ultimately, posing the foundation for designing novel drugs.</div

Backbone 1H, 13C, and 15N resonance assignments of the ligand binding domain of the human wildtype glucocorticoid receptor and the F602S mutant variant

The glucocorticoid receptor (GR) is a nuclear hormone receptor that regulates key genes controlling development, metabolism, and the immune response. GR agonists are efficacious for treatment of inflammatory, allergic, and immunological disorders. Steroid hormone binding to the ligand-binding domain (LBD) of GR is known to change the structural and dynamical properties of the receptor, which in turn control its interactions with DNA and various co-regulators and drive the pharmacological response. Previous biophysical studies of the GR LBD have required the use of mutant forms to overcome issues with limited protein stability and high aggregation propensity. However, these mutant variants are known to also influence the functional response of the receptor. Here we report a successful protocol for protein expression, purification, and NMR characterization of the wildtype human GR LBD. We achieved chemical shift assignments for 90% of the LBD backbone resonances, with 216 out of 240 non-proline residues assigned in the 1H–15N TROSY spectrum. These advancements form the basis for future investigations of allosteric effects in GR signaling