Background: Transthyretin (TTR) aggregation is associated with systemic amyloidosis. Results: Res... more Background: Transthyretin (TTR) aggregation is associated with systemic amyloidosis. Results: Residue replacements on the F and H strands hinder TTR aggregation. Conclusion: The F and H strands are aggregation-driving segments of TTR. The binding of designed peptides inhibits protein aggregation. Significance: We point the way to new therapeutic approaches against TTR aggregation by using peptides to block amyloid segments.
Torulene, a C(40) carotene, is the precursor of the end product of the Neurospora carotenoid path... more Torulene, a C(40) carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C(35) xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C(35) apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce beta-apo-4'-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of gamma-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.
Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (... more Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue-long (IF7) and the 149-residue-long (IF17). In this work, we studied both the IF7 and IF17 by Nuclear Magnetic Resonance (NMR), and we described their binding to GS, by using NMR and biolayer interferometry (BLI). We assigned the backbone nuclei of all residues of IF7. Analysis of chemical shifts and the (15)N-{(1)H}-NOEs at two field strengths suggest that IF7 region Thr3-Arg13, and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The 2D (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting firstly with IF7 were Gly34, Lys39 and Phe41 (and/or Trp42), close to those regions that appeared ordered. We also determined the kons and koffs for the binding reactions to GS of both IF7 and IF17, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding reaction of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model (GS +IF7 ↔ GS : IF7 (see manuscript file) ); (ii) there is a strong electrostatic component in the apparent kon; and, (iii) the binding is not diffusion-limited.
In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glut... more In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K(D) ~1 μM) than that measured for IF7 (~0.3 μM). The K(D)s determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.
La glutamina sintetasa de tipo I (GS) de Synechocystis sp. PCC 6803 se regula a nivel transcripci... more La glutamina sintetasa de tipo I (GS) de Synechocystis sp. PCC 6803 se regula a nivel transcripcional y postraduccional en función del balance carbono/nitrógeno celular. La regulación postraduccional se lleva a cabo mediante la interacción reversible proteína-proteína con dos factores inactivantes, IF7 e IF17 (1). En el caso de Anabaena sp. PCC 7120, existe un único factor inactivante denominado IF7A, homólogo a IF7 de Synechocystis. La GS de Synechocystis es inactivada por IF7, IF17 e IF7A, mientras que la GS de Anabaena sólo es inactivada por su propio factor, IF7A. Basándonos en esta observación, hemos construído cuatro proteínas quiméricas entre la GS de Synechocystis y la de Anabaena. El estudio comparado de estas proteínas nos ha permitido localizar una región carboxilo terminal de 56 residuos de aminoácidos responsable de la interacción GS/IFs. Por otra parte conocemos la naturaleza electrostática de dicha interacción y se han identificado tres residuos de arginina de los fac...
Background: Transthyretin (TTR) aggregation is associated with systemic amyloidosis. Results: Res... more Background: Transthyretin (TTR) aggregation is associated with systemic amyloidosis. Results: Residue replacements on the F and H strands hinder TTR aggregation. Conclusion: The F and H strands are aggregation-driving segments of TTR. The binding of designed peptides inhibits protein aggregation. Significance: We point the way to new therapeutic approaches against TTR aggregation by using peptides to block amyloid segments.
Torulene, a C(40) carotene, is the precursor of the end product of the Neurospora carotenoid path... more Torulene, a C(40) carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C(35) xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C(35) apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce beta-apo-4'-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of gamma-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.
Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (... more Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue-long (IF7) and the 149-residue-long (IF17). In this work, we studied both the IF7 and IF17 by Nuclear Magnetic Resonance (NMR), and we described their binding to GS, by using NMR and biolayer interferometry (BLI). We assigned the backbone nuclei of all residues of IF7. Analysis of chemical shifts and the (15)N-{(1)H}-NOEs at two field strengths suggest that IF7 region Thr3-Arg13, and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The 2D (1)H-(15)N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting firstly with IF7 were Gly34, Lys39 and Phe41 (and/or Trp42), close to those regions that appeared ordered. We also determined the kons and koffs for the binding reactions to GS of both IF7 and IF17, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding reaction of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model (GS +IF7 ↔ GS : IF7 (see manuscript file) ); (ii) there is a strong electrostatic component in the apparent kon; and, (iii) the binding is not diffusion-limited.
In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glut... more In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K(D) ~1 μM) than that measured for IF7 (~0.3 μM). The K(D)s determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.
La glutamina sintetasa de tipo I (GS) de Synechocystis sp. PCC 6803 se regula a nivel transcripci... more La glutamina sintetasa de tipo I (GS) de Synechocystis sp. PCC 6803 se regula a nivel transcripcional y postraduccional en función del balance carbono/nitrógeno celular. La regulación postraduccional se lleva a cabo mediante la interacción reversible proteína-proteína con dos factores inactivantes, IF7 e IF17 (1). En el caso de Anabaena sp. PCC 7120, existe un único factor inactivante denominado IF7A, homólogo a IF7 de Synechocystis. La GS de Synechocystis es inactivada por IF7, IF17 e IF7A, mientras que la GS de Anabaena sólo es inactivada por su propio factor, IF7A. Basándonos en esta observación, hemos construído cuatro proteínas quiméricas entre la GS de Synechocystis y la de Anabaena. El estudio comparado de estas proteínas nos ha permitido localizar una región carboxilo terminal de 56 residuos de aminoácidos responsable de la interacción GS/IFs. Por otra parte conocemos la naturaleza electrostática de dicha interacción y se han identificado tres residuos de arginina de los fac...
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