Recognition and Activation of the Plant AKT1 Potassium Channel by the Kinase CIPK23.

Plant growth largely depends on the maintenance of adequate intracellular levels of potassium (K+). The families of 10 Calcineurin B-Like (CBL) calcium sensors and 26 CBL-Interacting Protein Kinases (CIPKs) of Arabidopsis (Arabidopsis thaliana) decode the calcium signals elicited by environmental inputs to regulate different ion channels and transporters involved in the control of K+ fluxes by phosphorylation-dependent and -independent events. However, the detailed molecular mechanisms governing target specificity require investigation. Here, we show that the physical interaction between CIPK23 and the noncanonical ankyrin domain in the cytosolic side of the inward-rectifier K+ channel AKT1 regulates kinase docking and channel activation. Point mutations on this domain specifically alter binding to CIPK23, enhancing or impairing the ability of CIPK23 to regulate channel activity. Our data demonstrate the relevance of this protein-protein interaction that contributes to the formation of a complex between CIPK23/CBL1 and AKT1 in the membrane for the proper regulation of K+ transport.

Interference of the complex between NCS-1 and Ric8a with phenothiazines regulates synaptic function and is an approach for fragile X syndrome.

The protein complex formed by the Ca2+ sensor neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor protein Ric8a coregulates synapse number and probability of neurotransmitter release, emerging as a potential therapeutic target for diseases affecting synapses, such as fragile X syndrome (FXS), the most common heritable autism disorder. Using crystallographic data and the virtual screening of a chemical library, we identified a set of heterocyclic small molecules as potential inhibitors of the NCS-1/Ric8a interaction. The aminophenothiazine FD44 interferes with NCS-1/Ric8a binding, and it restores normal synapse number and associative learning in a Drosophila FXS model. The synaptic effects elicited by FD44 feeding are consistent with the genetic manipulation of NCS-1. The crystal structure of NCS-1 bound to FD44 and the structure-function studies performed with structurally close analogs explain the FD44 specificity and the mechanism of inhibition, in which the small molecule stabilizes a mobile C-terminal helix inside a hydrophobic crevice of NCS-1 to impede Ric8a interaction. Our study shows the drugability of the NCS-1/Ric8a interface and uncovers a suitable region in NCS-1 for development of additional drugs of potential use on FXS and related synaptic disorders.

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling.

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

Structural basis of the regulatory mechanism of the plant CIPK family of protein kinases controlling ion homeostasis and abiotic stress.

Plant cells have developed specific protective molecular machinery against environmental stresses. The family of CBL-interacting protein kinases (CIPK) and their interacting activators, the calcium sensors calcineurin B-like (CBLs), work together to decode calcium signals elicited by stress situations. The molecular basis of biological activation of CIPKs relies on the calcium-dependent interaction of a self-inhibitory NAF motif with a particular CBL, the phosphorylation of the activation loop by upstream kinases, and the subsequent phosphorylation of the CBL by the CIPK. We present the crystal structures of the NAF-truncated and pseudophosphorylated kinase domains of CIPK23 and CIPK24/SOS2. In addition, we provide biochemical data showing that although CIPK23 is intrinsically inactive and requires an external stimulation, CIPK24/SOS2 displays basal activity. This data correlates well with the observed conformation of the respective activation loops: Although the loop of CIPK23 is folded into a well-ordered structure that blocks the active site access to substrates, the loop of CIPK24/SOS2 protrudes out of the active site and allows catalysis. These structures together with biochemical and biophysical data show that CIPK kinase activity necessarily requires the coordinated releases of the activation loop from the active site and of the NAF motif from the nucleotide-binding site. Taken all together, we postulate the basis for a conserved calcium-dependent NAF-mediated regulation of CIPKs and a variable regulation by upstream kinases.

The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

Fulminant septic shock due to community-acquired pneumonia caused by Legionella pneumophila SG1 Olda OLDA ST1. Case report.

Legionellers' desease accounts for 1-8 % of cases of severe community-acquired pneumonia (CAP). Legionella spp. Is the causative organism that can result in respiratory failure, multi-organ dysfunction, sepsis, and death. Therefore, rapid diagnosis and efficient treatment are crucial. We report the clinical and microbiology study of a patient with community-acquired pneumonia caused by Legionella pneumophila, with fatal outcome. After death, the strain causing the infection was identified as Legionella pneumophila serogroup 1, Olda OLDA phenotype and sequence-type 1. This is the first reported case of septic shock and death associated with an isolate of these characteristics.

A simple method for direct mercury analysis in dried blood spots (DBS) samples for human biomonitoring studies.

Human exposure to mercury can have serious health effects, especially in vulnerable groups such as children and fetuses. The use of dried blood spot (DBS) samples to collect capillary blood greatly facilitates sample collection and fieldwork, being a less invasive alternative to blood collection by venipuncture, needing a small volume of sample, and does not require specialized medical staff. Moreover, DBS sampling reduces logistical and financial barriers related to transport and storage of blood samples. We propose here a novel method to analyze total mercury in DBS samples in a Direct Mercury Analyzer (DMA) that allow the control of the volume of the DBS samples. This method has shown good results in terms of precision (<6% error), accuracy (<10% coefficient of variation) and recovery (75-106%). The applicability of the method in human biomonitoring (HBM) was demonstrated in a pilot study involving 41 adults aged 18-65. Mercury concentrations of DBS samples from capillary blood collected by finger prick (real DBS samples) were determined in the DMA and compared with those determined in whole blood (venous blood) by ICP-MS, the method usually used in HBM. The sampling procedure was also validated by comparison of real DBS samples and DBS generated artificially in the laboratory by depositing venous samples in cellulose cards (laboratory DBS). There were no statistically significant differences in the results obtained using both methodologies (DMA: Geometric Mean (confidence interval 95%) = 3.87 (3.12-4.79) µg/L; ICP-MS: Geometric Mean (confidence interval 95%) = 3.46 (2.80-4.27) µg/L). The proposed method is an excellent alternative to be applied in clinical settings as screening methodology for assessing mercury exposure in vulnerable groups, such us pregnant woman, babies and children.

Deciphering the Inhibition of the Neuronal Calcium Sensor 1 and the Guanine Exchange Factor Ric8a with a Small Phenothiazine Molecule for the Rational Generation of Therapeutic Synapse Function Regulators.

Protein-protein interactions (PPIs) are known to play an essential role between the neuronal calcium sensor 1 (NCS-1) and the guanine exchange factor Ric8a to regulate synapse function, emerging as a druggable interface for synaptopathies such as the fragile X syndrome (FXS). Recently, the phenothiazine FD44 has been identified as an inhibitor of this PPI, decreasing the abnormally high synapse number and enhancing associative learning in a FXS animal model. Here, we have integrated advanced experimental and computational studies to obtain important structural insights into Drosophila NCS-1/FD44 recognition to understand the basis of its affinity and specificity and generate improved PPI regulators. This has allowed the identification of a new small drug-like molecule, IGS-1.76, which efficiently inhibits the human NCS-1/Ric8a complex with improved binding potency. The crystal structure of the Drosophila NCS-1/IGS-1.76 complex demonstrates that the new inhibitor, although chemically different from FD44, shares the same mechanism of action and constitutes a new hit candidate for FXS.

Blockade of nicotinic receptors of bovine adrenal chromaffin cells by nanomolar concentrations of atropine.

Nanomolar concentrations of atropine have been considered up to now to be selective for blockade of muscarinic receptors for acetylcholine. A collateral finding indicated to us that these low concentrations of atropine could also target the neuronal nicotinic receptors. We report here a detailed study on this novel property of atropine. Catecholamine release, measured on-line with amperometry in chromaffin cells stimulated with acetylcholine pulses was blocked by atropine in a competitive manner. To corroborate a direct action of atropine on nicotinic receptors, we have employed N,N-dimethyl-N'-phenyl-piperazinium (DMPP), a pure nicotinic receptor agonist; atropine blocked its secretory responses with an IC50 of 2.04 nM. Nicotinic currents, recorded with the whole cell configuration of the patch-clamp technique were blocked by atropine in a concentration-dependent manner (IC50 of 11 nM), also showing a competitive nature. Nicotinic receptor currents in oocytes expressing bovine alpha7 and alpha3beta4 nicotinic receptors were blocked by atropine with an IC50 of 11.2 and 46.8 nM, respectively. Atropine (30 nM) also decreased the increment of the cytosolic calcium concentrations after stimulation with 30 microM DMPP in bovine chromaffin cells. However, action potentials evoked by DMPP were not modified by atropine. Our results demonstrate that nicotinic currents and their downstream consequences (i.e. cytosolic calcium elevations and catecholamine release) were blocked by nanomolar concentrations of atropine; although the blockade was partial, it must be considered when using atropine to study cholinergic neurotransmission, particularly at synapses where both nicotinic and muscarinic receptors are present i.e., the adrenal medulla and autonomic ganglia.

Preliminary crystallographic analysis of the ankyrin-repeat domain of Arabidopsis thaliana AKT1: identification of the domain boundaries for protein crystallization.

The Arabidopsis thaliana K(+) transporter 1 (AKT1) participates in the maintenance of an adequate cell potassium (K(+)) concentration. The CBL-interacting protein kinase 23 (CIPK23) activates AKT1 for K(+) uptake under low-K(+) conditions. This process is mediated by the interaction between the cytosolic ankyrin-repeat (AR) domain of AKT1 and the kinase domain of CIPK23. However, the precise boundaries of the AR domain and the residues responsible for the interaction are still unknown. Here, the optimization procedure to obtain an AR domain construct suitable for crystallization and the preliminary crystallographic analysis of the obtained crystals are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 34.83, b = 65.89, c = 85.44 Å, and diffracted to 1.98 Šresolution.