Coevolution of actin and associated proteins: an α-actinin-like protein in a cyanobacterium (Spirulina platensis)

Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells. Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria. Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e. Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins. We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins. We also report for the first time that α-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein. Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an α-actinin-like protein using specific antibodies.     

Molecular Modeling of the NPY Binding Site on the Y1 Receptor

A three-dimensional model of the neuropeptide Y (NPY) - rat Y₁ (rY₁) receptor complex and of the NPY _13-36 - rY₁ receptor complex was constructed by molecular modeling based on the electron density projection map of rhodopsin and on site-directed mutagenesis studies of neuropeptide receptors. In order to further guide the modeling, the nucleotide sequences encoding Trp287, Cys295 and His297 in the third extracellular loop of the rY₁ receptor, were altered by site-directed mutagenesis experiments. Single-point mutated receptors were expressed in COS-7 cells, and tested for their ability to bind radio labelled NPY (³H-NPY). Mutations of Trp287 and His297 completely abolished binding of ³H-NPY. The Cys295Ser mutation only slightly decreased the binding of ³H-NPY, suggesting that the involvement of Cys295 in a disulphide bond is not essential for maintaining the correct three-dimensional structure of the binding site for NPY. Molecular dynamics simulations of NPY-rY₁ receptor interactions suggested that Asp199, Asp103 and Asp286 in the receptor interact, respectively, with Lys4, Arg33 and Arg35 of NPY. The simulations also suggested that His297 acts as a hydrogen acceptor from Arg35 in NPY, and that Tyr1 of NPY interacts with a binding pocket on the receptor formed by Asn115, Asp286, Trp287 and His297. Tyr36 in NPY interacted both with Thr41 and Tyr99 via hydrogen bonds, and also with Asn296, His297 and Phe301. The present study suggests that amino acid residues at the extracellular end of the transmembrane helices and in the extracellular loops are strongly involved in binding to NPY and NPY_13-36.Electronic Supplementary Material available.     

N-Carbamoyl-α-Amino Acids Rather than Free α-Amino Acids Formation in the Primitive Hydrosphere: A Novel Proposal for the Emergence of Prebiotic Peptides

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/ NH₃ in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L^–1, T = 25 °C, pH = 8, [NH₃] very low), this system leads to 99.9% of -hydroxyacetonitrile and 0.1% of -aminoacetonitrile (precursor of the -amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of -aminonitriles which shift the initial equilibrium in favor of the -aminonitrile pathway. The first reaction catalyzed by formaldehyde generates -aminoamides, precursors of -aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl--aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the -aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl--amino acid seems then to be the fatal issue of the HCHO/HCN/NH₃ system that nature used to perform its evolution. These N-protected -amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.     

Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction

Anisomycin, a translational inhibitor secreted by Streptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH₂-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun, junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-α), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-α, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.     

Interaction of COP1 and UVR8 regulates UV‐B‐induced photomorphogenesis and stress acclimation in Arabidopsis

The ultraviolet‐B (UV‐B) portion of the solar radiation functions as an environmental signal for which plants have evolved specific and sensitive UV‐B perception systems. The UV‐B‐specific UV RESPONSE LOCUS 8 (UVR8) and the multifunctional E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) are key regulators of the UV‐B response. We show here that uvr8‐null mutants are deficient in UV‐B‐induced photomorphogenesis and hypersensitive to UV‐B stress, whereas overexpression of UVR8 results in enhanced UV‐B photomorphogenesis, acclimation and tolerance to UV‐B stress. By using sun simulators, we provide evidence at the physiological level that UV‐B acclimation mediated by the UV‐B‐specific photoregulatory pathway is indeed required for survival in sunlight. At the molecular level, we demonstrate that the wild type but not the mutant UVR8 and COP1 proteins directly interact in a UV‐B‐dependent, rapid manner in planta. These data collectively suggest that UV‐B‐specific interaction of COP1 and UVR8 in the nucleus is a very early step in signalling and responsible for the plant's coordinated response to UV‐B ensuring UV‐B acclimation and protection in the natural environment.