Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes

The effect of simulated ischemia [hypoxia, no glucose, extracellular pH (pH(o)) 6.4] on cGMP synthesis induced by stimulation of soluble (sGC) or particulate guanylyl cyclase (pGC) was investigated in adult rat cardiomyocytes. Intracellular cGMP content was measured after stimulation of sGC by S-nitroso-N-penicillamine (SNAP) or stimulation of pGC by natriuretic peptides [urodilatin (Uro), atrial natriuretic peptide (ANP), or C-type natriuretic peptide (CNP)] for 1 min in the presence of phosphodiesterase inhibitors. After 2 h of simulated ischemia, a decrease of >50% was observed in pGC-dependent cGMP synthesis, but no significant change was observed in sGC-dependent cGMP synthesis. The reduction in cGMP synthesis caused by simulated ischemia was mimicked by extracellular acidosis (pH(o) 6.4), which decreased pGC-mediated cGMP synthesis without altering sGC-mediated cGMP synthesis. An extreme sensitivity of pGC activity to low pH was also observed in membrane cell fractions. Hypoxia without acidosis (pH(o) 7.4) profoundly depressed cellular ATP content but did not change the response to SNAP, Uro, or ANP (selective agonists of pGC type A receptor). Only cGMP synthesis in response to CNP (a selective agonist of pGC type B receptor) was significantly reduced by ATP depletion. These data support the relevance of intracellular pH as a modulator of cGMP and suggest that, in ischemic cardiomyocytes, synthesis of cGMP would be mainly nitric oxide dependent.     

Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation

Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC). Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation. Rho activity was measured in tubular epithelial cells by affinity pulldown assay. Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity. Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho. Changes in [Na+] and [K+] at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response. Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors. Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B. ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells. ML-7 had a smaller inhibitory effect. In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response. Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation. However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation.     

Sodium Channel Toxins and Neurotransmitter Release

Voltage-dependent sodium channels (VDSC) are an important class of ion channels in excitable cells, where they are responsible for the generation and conduction of action potential. In addition, the release of neurotransmitters from nerve terminals is influenced by sodium channel activity. The function of VDSC is subject to modulation by various neurotoxins, such as scorpion toxins, which have long been used as tools in the investigation of neurotransmitter release. This opens an interesting perspective concerning modulation of neurotransmission via pharmacological manipulation of sodium channel properties, which can lead to a better understanding of their physiological and pathological roles. Here we briefly review the studies of neurotoxins acting on sodium channels, focusing primarily on the view of the mechanisms of neurotransmitter release.     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

Edaphic races and phylogenetic taxa in the Lasthenia californica complex (Asteraceae: Heliantheae): an hypothesis of parallel evolution

Lasthenia californica sensu Ornduff consists of two races that differ in their flavonoid pigments and edaphic tolerances. Recent phylogenetic studies of Lasthenia have revealed that members of L. californica sensu Ornduff belong to two phylogenetic species. The relationship of the edaphic races to these new species and to each other is the focus of this study. Characterization of flavonoid profiles and phylogenetic placement of 33 populations demonstrates that races and phylogenetic taxa are not concordant, suggesting that one or both edaphic races evolved in parallel in the two clades. We hypothesize an edaphically linked ecological role for flavonoid differences that first revealed the existence of two races.     

The structural basis for kainoid selectivity at AMPA receptors revealed by low-mode docking calculations

The kainoids are a class of excitatory and excitotoxic pyrrolidine dicarboxylates that act at ionotropic glutamate receptors. The kainoids bind kainate receptors with high affinity and, while binding affinity is lower at AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, they are active in functional assays at this receptor subtype as well. However, kainoids are only partial agonists at AMPA receptors. Currents evoked by kainoids have been described as either slowly desensitizing, partially desensitizing, or non-desensitizing. Recently acquired X-ray crystal structures of the ligand binding domain of the iGluR2, AMPA sensitive receptor suggest that differences in ligand-receptor interactions may influence functional properties of an agonist. In an effort to identify important ligand-receptor interactions of various kainoids, we have conducted a series of low-mode docking searches of AMPA agonists in the iGluR2 binding domain. Kainic acid exhibited alternate low-lying geometries, with loss of hydrogen bonds to domain 2, which may represent a dissociation route not available to other kainoids. The most potent of the kainoids are capable of forming hydrogen bonding interactions that span the two domains of the receptor. In particular, a hydrogen bond between the domoic acid C6' carboxylic acid and Ser652 may prevent a peptide bond rotation that is associated with the desensitized state of the receptor.     

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.     

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.