Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) are two energy-consuming enzymes of the Calvin–Benson cycle, whose regulation is crucial for the global balance of the ...photosynthetic process under different environmental conditions. In oxygen phototrophs, GAPDH and PRK regulation involves the redox-sensitive protein CP12. In the dark, oxidized chloroplast thioredoxins trigger the formation of a GAPDH/CP12/PRK complex in which both enzyme activities are down-regulated. In this report, we show that free GAPDH (A4-isoform) and PRK are also inhibited by oxidants like H2O2, GSSG and GSNO. Both in the land plant Arabidopsis thaliana and in the green microalga Chlamydomonas reinhardtii, both enzymes can be glutathionylated as shown by biotinylated-GSSG assay and MALDI-ToF mass spectrometry. CP12 is not glutathionylated but homodisulfides are formed upon oxidant treatments. In Arabidopsis but not in Chlamydomonas, the interaction between oxidized CP12 and GAPDH provides full protection from oxidative damage. In both organisms, preformed GAPDH/CP12/PRK complexes are protected from GSSG or GSNO oxidation, and in Arabidopsis also from H2O2 treatment. Overall, the results suggest that the role of CP12 in oxygen phototrophs needs to be extended beyond light/dark regulation, and include protection of enzymes belonging to Calvin–Benson cycle from oxidative stress.
•Plant enzymes GAPDH and PRK are inactivated by thiol oxidation.•The regulatory protein CP12 forms intramolecular disulfides upon oxidation.•In Arabidopsis, CP12 oxidation prevents GAPDH inactivation via complex formation.•Preformed GAPDH/CP12/PRK ternary complex protects enzymes from oxidative stress.•Complexed enzymes are more protected in Arabidopsis than in Chlamydomonas.
Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, ...organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member,
AMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that
AMY3 activity was sensitive to ROS, such as H
O
. Treatments with H
O
inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized
AMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated
AMY3 and performing oxidation experiments of Cys-to-Ser variants of
AMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the p
values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (p
= 5.70 ± 0.28; p
= 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of
AMY3 is proposed.
Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion ...to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Integrating carbon nanoparticles (CNPs) with proteins to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science. The ...comprehension of CNP–protein interactions requires the still-missing identification and characterization of the ‘binding pocket’ for the CNPs. Here, using Lysozyme and C60 as model systems and NMR chemical shift perturbation analysis, a protein–CNP binding pocket is identified unambiguously in solution and the effect of the binding, at the level of the single amino acid, is characterized by a variety of experimental and computational approaches. Lysozyme forms a stoichiometric 1:1 adduct with C60 that is dispersed monomolecularly in water. Lysozyme maintains its tridimensional structure upon interaction with C60 and only a few identified residues are perturbed. The C60 recognition is highly specific and localized in a well-defined pocket.
Scleractinian coral skeletons are composed mainly of aragonite in which a small percentage of organic matrix (OM) molecules is entrapped. It is well known that in corals the mineral deposition occurs ...in a biological confined nucleation site, but it is still unclear to what extent the calcification is controlled by OM molecules. Hence, the shape, size and organization of skeletal crystals from the fiber level through the colony architecture, were also attributed to factors as diverse as nucleation site mineral supersaturation and environmental factors in the habitat. In this work the OMs were extracted from the skeleton of three colonial corals, Acropora digitifera, Lophelia pertusa and Montipora caliculata. A. digitifera has a higher calcification rate than the other two species. OM molecules were characterized and their CaCO3 mineralization activity was evaluated by experiments of overgrowth on coral skeletons and of precipitation from solutions containing OM soluble and insoluble fractions and magnesium ions. The precipitates were characterized by spectroscopic and microscopic techniques. The results showed that the OM molecules of the three coral share similar features, but differ from those associated with mollusk shells. However, A. digitifera OM shows peculiarities from those from L. pertusa and M. caliculata. The CaCO3 overgrowth and precipitation experiments confirm the singularity of A. digitifera OM molecules as mineralizers. Moreover, their comparison indicates that only specific molecules are involved in the polymorphism control and suggests that when the whole extracted materials are used the OM’s main effect is on the control of particles’ shape and morphology.
The precipitation of calcium carbonate was carried out in the presence of the intraskeletal organic matrix (OM) extracted from Mediterranean corals. They were diverse in growth form and trophic ...strategy, Balanophyllia europaea and Leptopsammia pruvotisolitary corals, only the first zooxanthellate coraland Cladocora caespitosa and Astroides calyculariscolonial corals, only the first zooxanthellate coral. The results showed that, although the OM marked differences among species, the diverse influence over the calcium carbonate precipitation was evident only for B. europaea. This OM was the most prone to favor the precipitation of aragonite in the absence of magnesium ions, according to overgrowth and solution precipitation experiments. In artificial seawater, where magnesium ions were present, this OM, as well the one from A. calycularis, precipitated mainly a form of amorphous calcium carbonate different from that obtained with SOM from L. pruvoti or C. caespitosa. The amorphous calcium carbonate from B. europaea was the most stable upon heating up to 100 °C and was the one that mainly converted into aragonite instead of magnesium calcite after heating at 300 °C. All this indicated a higher control of B. europaea OM over the calcium carbonate polymorphism than the other species. The influence of SOMs over precipitate morphology turned out to be also species related. In conclusion, this comparative study has shown that the influence of OM on in vitro precipitation of calcium carbonate was not related to the coral ecology, solitary vs colonial and zooxanthellate vs nonzooxanthellate, and suggested that the coral control over biomineralization process was species specific and encoded in coral genes.
CP12 is a protein of 8.7 kDa that contributes to Calvin cycle regulation by acting as a scaffold element in the formation of a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase ...(GAPDH) and phosphoribulokinase (PRK) in photosynthetic organisms. NMR studies of recombinant CP12 (isoform 2) of Arabidopsis thaliana show that CP12-2 is poorly structured. CP12-2 is monomeric in solution and contains four cysteines, which can form two intramolecular disulfides with midpoint redox potentials of –326 and –352 mV, respectively, at pH 7.9. Site-specific mutants indicate that the C-terminal disulfide is involved in the interaction between CP12-2 and GAPDH (isoform A4), whereas the N-terminal disulfide is involved in the interaction between this binary complex and PRK. In the presence of NAD, oxidized CP12-2 interacts with A4-GAPDH (KD = 0.18 μm) to form a binary complex of 170 kDa with (A4-GAPDH)-(CP12-2)2 stoichiometry, as determined by isothermal titration calorimetry and multiangle light scattering analysis. PRK is a dimer and by interacting with this binary complex (KD = 0.17 μm) leads to a 498-kDa ternary complex constituted by two binary complexes and two PRK dimers, i.e. ((A4-GAPDH)-(CP12-2)2-(PRK))2. Thermodynamic parameters indicate that assembly of both binary and ternary complexes is exoergonic although penalized by a decrease in entropy that suggests an induced folding of CP12-2 upon binding to partner proteins. The redox dependence of events leading to supramolecular complexes is consistent with a role of CP12 in coordinating the reversible inactivation of chloroplast enzymes A4-GAPDH and PRK during darkness in photosynthetic tissues.
The Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) can form under oxidizing conditions a supramolecular complex with the regulatory protein CP12. ...Both GAPDH and PRK activities are inhibited within the complex, but they can be fully restored by reduced thioredoxins (TRXs). We have investigated the interactions of eight different chloroplast thioredoxin isoforms (TRX f1, m1, m2, m3, m4, y1, y2, x) with GAPDH (A4, B4, and B8 isoforms), PRK and CP12 (isoform 2), all from Arabidopsis thaliana. In the complex, both A4-GAPDH and PRK were promptly activated by TRX f1, or more slowly by TRXs m1 and m2, but all other TRXs were ineffective. Free PRK was regulated by TRX f1, m1, or m2, while B4- and B8-GAPDH were absolutely specific for TRX f1. Interestingly, reductive activation of PRK caged in the complex was much faster than reductive activation of free oxidized PRK, and activation of A4-GAPDH in the complex was much faster (and less demanding in terms of reducing potential) than activation of free oxidized B4- or B8-GAPDH. It is proposed that CP12-assembled supramolecular complex may represent a reservoir of inhibited enzymes ready to be released in fully active conformation following reduction and dissociation of the complex by TRXs upon the shift from dark to low light. On the contrary, autonomous redox-modulation of GAPDH (B-containing isoforms) would be more suited to conditions of very active photosynthesis.
•AIR12 is a cytochrome b, GPI-anchored to the external face of the plasma membrane.•AIR12 interacts with redox-active compounds of the apoplast.•Botrytis cinerea and wounding induce AIR12 expression ...in Arabidopsis.•Arabidopsis AIR12-knock out mutants are resistant to B. cinerea.•AIR12 is a cell wall-related susceptibility factor for B. cinerea infection.
AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. Recombinant AIR12 from Arabidopsis accepted electrons from ascorbate or superoxide, and donated electrons to either monodehydroascorbate or oxygen. AIR12 was found associated in vivo to the plasma membrane. Though linked to the membrane by a glycophosphatidylinositol anchor, AIR12 is a hydrophilic and glycosylated protein predicted to be fully exposed to the apoplast. The expression pattern of AIR12 in Arabidopsis is developmentally regulated and correlated to sites of controlled cell separation (e.g. micropilar endosperm during germination, epidermal cells surrounding the emerging lateral root) and cells around wounds. Arabidopsis (Landsberg erecta-0) mutants with altered levels of AIR12 did not show any obvious phenotype. However, AIR12-overexpressing plants accumulated ROS (superoxide, hydrogen peroxide) and lipid peroxides in leaves, indicating that AIR12 may alter the redox state of the apoplast under particular conditions. On the other hand, AIR12-knock out plants displayed a strongly decreased susceptibility to Botrytis cinerea infection, which in turn induced AIR12 expression in susceptible wild type plants. Altogether, the results suggest that AIR12 plays a role in the regulation of the apoplastic redox state and in the response to necrotrophic pathogens. Possible relationships between these functions are discussed.
In animal cells, many proteins have been shown to undergo glutathionylation under conditions of oxidative stress. By contrast, very little is known about this post‐translational modification in ...plants. In the present work, we showed, using mass spectrometry, that the recombinant chloroplast A4‐glyceraldehyde‐3‐phosphate dehydrogenase (A4‐GAPDH) from Arabidopsis thaliana is glutathionylated with either oxidized glutathione or reduced glutathione and H2O2. The formation of a mixed disulfide between glutathione and A4‐GAPDH resulted in the inhibition of enzyme activity. A4‐GAPDH was also inhibited by oxidants such as H2O2. However, the effect of glutathionylation was reversed by reductants, whereas oxidation resulted in irreversible enzyme inactivation. On the other hand, the major isoform of photosynthetic GAPDH of higher plants (i.e. the AnBn‐GAPDH isozyme in either A2B2 or A8B8 conformation) was sensitive to oxidants but did not seem to undergo glutathionylation significantly. GAPDH catalysis is based on Cys149 forming a covalent intermediate with the substrate 1,3‐bisphosphoglycerate. In the presence of 1,3‐bisphosphoglycerate, A4‐GAPDH was fully protected from either oxidation or glutathionylation. Site‐directed mutagenesis of Cys153, the only cysteine located in close proximity to the GAPDH active‐site Cys149, did not affect enzyme inhibition by glutathionylation or oxidation. Catalytic Cys149 is thus suggested to be the target of both glutathionylation and thiol oxidation. Glutathionylation could be an important mechanism of regulation and protection of chloroplast A4‐GAPDH from irreversible oxidation under stress.
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