Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

SummaryCardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic αF-αG loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase

Un modelo para el análisis de las mediaciones rituales y su relación con los procesos de cambio social

El artículo propone un modelo para el estudio de las mediaciones rituales que asume un doble desafío: a) articular las múltiples dimensiones de la acción ritual de un modo flexible, conservando su complejidad y variedad y b) dar unidad a su diversidad fenomenológica, preservando sus diferencias. El concepto de “mediación” se propone por su versatilidad y perspectiva, que le permiten actuar como común denominador de las diversas articulaciones que presentan los sistemas rituales con los elementos de su entorno sociocultural y natural. Éste se orienta a explicar el trabajo ritual a partir de sus relaciones con los elementos del contexto y las situaciones comunicativas donde ejecuta el rito y seguir su dinámica de transformaciones en el marco de procesos históricos.This article proposes a model for the study of the ritual mediations that assumes a double challenge: a) to articulate the multiple dimensions of the ritual action in a flexible way, preserving its complexity and variety and b) to unify its phenomenological diversity, preserving the differences. The concept of “mediation” is proposed because of its versatility and perspective. These characteristics allow the mediation to act as a common factor of the diverse articulations that the ritual systems present with its sociocultural and natural environment. This one is oriented to explain the ritual work from its relations with the elements of the context and the communicative situations where it executes the rite and follows its dynamic of transformations in the frame of historical proceses

Guanylyl cyclase sees the light

Cyclic guanosine monophosphate (cGMP) is a critical second messenger that regulates cardiovascular function and vision in humans. Two recent papers, including one in BMC Structural Biology, have revealed atomic structures of the enzymes that catalyze the synthesis of cGMP providing new clues about the molecular basis of substrate specificity and allosteric regulation in nucleotide cyclases

Generating Interpretable Fuzzy Controllers using Particle Swarm Optimization and Genetic Programming

Autonomously training interpretable control strategies, called policies, using pre-existing plant trajectory data is of great interest in industrial applications. Fuzzy controllers have been used in industry for decades as interpretable and efficient system controllers. In this study, we introduce a fuzzy genetic programming (GP) approach called fuzzy GP reinforcement learning (FGPRL) that can select the relevant state features, determine the size of the required fuzzy rule set, and automatically adjust all the controller parameters simultaneously. Each GP individual's fitness is computed using model-based batch reinforcement learning (RL), which first trains a model using available system samples and subsequently performs Monte Carlo rollouts to predict each policy candidate's performance. We compare FGPRL to an extended version of a related method called fuzzy particle swarm reinforcement learning (FPSRL), which uses swarm intelligence to tune the fuzzy policy parameters. Experiments using an industrial benchmark show that FGPRL is able to autonomously learn interpretable fuzzy policies with high control performance.Comment: Accepted at Genetic and Evolutionary Computation Conference 2018 (GECCO '18

Structural and Functional Characterization of Hyper-Phosphorylated GRK5 Protein Expressed From E. coli

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 interacts with GPCRs. Although past studies used mammalian and insect cells to produce GRK5, this study aims to use E. coli cells to discover more about GRK5’s structure and function. Previous studies revealed E. coli produce a hyper-phosphorylated version of the GRK5 protein. We attempted to crystalize this GRK5 produced from E. coli to reveal its conformation in a phosphorylated state that we hypothesize to be similar to its form when bound to GPCRs. We also tested the functionality of this GRK5 to reveal the effects of phosphorylation. We genetically edited the GRK5 gene in multiple E. coli samples to create GRK5 with less phosphorylation sites and tested activity levels by measuring the phosphorylation of GPCRs mediated by each GRK5 variant. Successfully creating an E. coli system for structural and functional analysis of GRK5 would help reduce time and costs for GRK5 research, and it could speed up the full understanding of the interactions between GRK5 and GPCRs

Room temperature ferromagnetic-like behavior in Mn-implanted and post-annealed InAs layers deposited by Molecular Beam Epitaxy

We report on the magnetic and structural properties of Ar and Mn implanted InAs epitaxial films grown on GaAs (100) by Molecular Beam Epitaxy (MBE) and the effect of Rapid Thermal Annealing (RTA) for 30 seconds at 750C. Channeling Particle Induced X- ray Emission (PIXE) experiments reveal that after Mn implantation almost all Mn atoms are subsbtitutional in the In-site of the InAs lattice, like in a diluted magnetic semiconductor (DMS). All of these samples show diamagnetic behavior. But, after RTA treatment the Mn-InAs films exhibit room-temperature magnetism. According to PIXE measurements the Mn atoms are no longer substitutional. When the same set of experiments were performed with As as implantation ion all of the layers present diamagnetism without exception. This indicates that the appearance of room-temperature ferromagnetic-like behavior in the Mn-InAs-RTA layer is not related to lattice disorder produce during implantation, but to a Mn reaction produced after a short thermal treatment. X-ray diffraction patterns (XRD) and Rutherford Back Scattering (RBS) measurements evidence the segregation of an oxygen deficient-MnO2 phase (nominally MnO1.94) in the Mn-InAs-RTA epitaxial layers which might be on the origin of room temperature ferromagnetic-like response observed.Comment: 16 pages, 5 figures. Acepted in J. Appl. Phy

Input from Torus Longitudinalis Drives Binocularity and Spatial Summation in Zebrafish Optic Tectum

Background: A continued effort in neuroscience aims to understand the way brain circuits consisting of diverse neuronal types generate complex behavior following sensory input. A common feature of vertebrate visual systems is that lower-order and higher-order visual areas are reciprocally connected. Feedforward projections confer visual responsiveness to higher-order visual neurons while feedback projections likely serve to modulate responses of lower-order visual neurons in a context-dependent manner. Optic tectum is the largest first-order visual brain area in zebrafish and is reciprocally connected with the torus longitudinalis (TL), a second-order visual brain area that does not receive retinal input. A functional role for feedback projections from TL to tectum has not been identified. Here we aim to understand how this feedback contributes to visual processing. Results: In this study, we demonstrate that TL feedback projections to tectum drive binocular integration and spatial summation in a defined tectal circuit. We performed genetically targeted, cell type-specific functional imaging in tectal pyramidal neurons (PyrNs) and their two input neuron populations: retinal ganglion cells (RGCs) and neurons in TL. We find that PyrNs encode gradual changes in scene luminance using a complement of three distinct response classes that encode different light intensity ranges. Functional imaging of RGC inputs to tectum suggest that these response classes originate in the retina and RGC input specifies PyrN functional classes. In contrast, TL input serves to endow PyrNs with large, compound receptive fields that span both retinal hemifields. Conclusions: These findings reveal a novel role for the zebrafish TL in driving binocular integration and spatial summation in tectal PyrNs. The neural circuit we describe generates a population of tectal neurons with large receptive fields tailored for detecting changes in the visual scene

Engineering Bioluminescent Sensors of Cyclic AMP to Study Opioid Signaling

Opioids are small signaling molecules which bind to opioid receptors on the surface of cells. The kappa opioid receptor (KOR) is one of three major types of opioid receptors found in human neurons. When an opioid binds to a KOR, a variety of biochemical signaling pathways are activated inside the cell. Each of these pathways are associated with different physiological effects of KOR activation. The production of a small signaling molecule, cyclic adenosine monophosphate (cAMP), is known to be inhibited during KOR activation of the analgesic (pain-killing) signaling pathway. The ability to interrogate the individual responses of KOR signaling pathways in a living mammal would greatly improve our understanding of how opioids work in the brain. To this end, we have developed a biosensor functioning via bioluminescent resonance energy transfer (BRET) as a tool for both fluorescent and luminescent ratiometric quantification of cAMP. We couple two fluorescent proteins, emitting at different wavelengths, to a luciferase which provides chemiluminescent excitation energy for the complex. The intensity of the two emitted wavelengths vary inversely to each other in response to the presence of cAMP. Calculating the ratio of the two emission intensities creates a metric for cAMP concentration that is normalized to the concentration of our sensor, allowing quantitative comparison across trials. The application of our sensor for dual-color live-cell microscopy was demonstrated in mammalian cells using fluorescence and bioluminescence microscopy. Further proof-of-principle studies in KOR-expressing mammalian cells demonstrates the viability of our sensor for live-cell KOR signaling

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity