Carine Puppo

Glutathionylation in the photosynthetic model organism Chlamydomonas reinhardtii: a proteomic survey

Protein glutathionylation is a redox post-translational modification occurring under oxidative stress conditions and playing a major role in cell regulation and signaling. This modification has been mainly studied in nonphotosynthetic organisms, whereas much less is known in photosynthetic organisms despite their important exposure to oxidative stress caused by changes in environmental conditions. We report a large scale proteomic analysis using biotinylated glutathione and streptavidin affinity chromatography that allowed identification of 225 glutathionylated proteins in the eukaryotic unicellular green alga Chlamydomonas reinhardtii. Moreover, 56 sites of glutathionylation were also identified after peptide affinity purification and tandem mass spectrometry. The targets identified belong to a wide range of biological processes and pathways, among which the Calvin-Benson cycle appears to be a major target. The glutathionylation of four enzymes of this cycle, phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, ribose-5-phosphate isomerase, and phosphoglycerate kinase was confirmed by Western blot and activity measurements. The results suggest that glutathionylation could constitute a major mechanism of regulation of the Calvin-Benson cycle under oxidative stress conditions.

Tyrosine‐Targeted Spin Labeling and EPR Spectroscopy: An Alternative Strategy for Studying Structural Transitions in Proteins

Such a difficulty was recently encountered in the study of a small and flexible chloroplast protein CP12 from the green alga Chlamydomonas reinhardtii by spin-labeling EPR spectroscopy. In this organism, CP12 contains four cysteine residues involved in two disulfide bridges in its oxidized state. Although the introduction of spin labels at the two cysteine residues of the C-terminal disulfide bridge enabled us to identify a new role of the partner protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), it precluded the direct study of the complex formation GAPDH/CP12. [4] To overcome this difficulty, grafting of the nitroxide probe to residues other than cysteines is required. One strategy using a genetically encoded unnatural amino acid has been recently proposed. [5] The incorporation of such unnatural amino acids relies, however, on a rather complex strategy involving an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid. As such a strategy is difficult to set up, we propose an alternative method consisting of selectively targeting residues other than cysteines with a nitroxide probe. Bioconjugation of small molecules to protein residues is very challenging, and several reactions have recently been proposed to target specific residues selectively. [6] In particular, efforts have been paid to modify the aromatic amino acid side chains of tryptophan [7] and tyrosine. [8] Among them, a threecomponent Mannich-type reaction has been developed that allows the modification of tyrosine under mild, biocompatible, and metal-free conditions. [8a] Inspired by these recent studies, we present the selective grafting of a nitroxide probe to tyrosine by using the Mannich-type reaction on CP12, a protein bearing only one natural tyrosine residue. This unique tyrosine residue, located at position 78 in the sequence of a total of 80 amino acids, makes this protein an ideal candidate for demonstrating the feasibility of tyrosine-targeted spin

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

Glyceraldehyde‐3‐phosphate dehydrogenase is regulated by ferredoxin‐NADP reductase in the diatom Asterionella formosa

Diatoms are a widespread and ecologically important group of heterokont algae that contribute c. 20% to global productivity. Previous work has shown that regulation of their key Calvin cycle enzymes differs from that of the Plantae, and that in crude extracts, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be inhibited by nicotinamide adenine dinucleotide phosphate reduced (NADPH) under oxidizing conditions. The freshwater diatom, Asterionella formosa, was studied using enzyme kinetics, chromatography, surface plasmon resonance, mass spectrometry and sequence analysis to determine the mechanism behind this GAPDH inhibition. GAPDH interacted with ferredoxin-nicotinamide adenine dinucleotide phosphate (NADP) reductase (FNR) from the primary phase of photosynthesis, and the small chloroplast protein, CP12. Sequences of copurified GAPDH and FNR were highly homologous with published sequences. However, the widespread ternary complex among GAPDH, phosphoribulokinase and CP12 was absent. Activity measurements under oxidizing conditions showed that NADPH can inhibit GAPDH-CP12 in the presence of FNR, explaining the earlier observed inhibition within crude extracts. Diatom plastids have a distinctive metabolism, including the lack of the oxidative pentose phosphate pathway, and so cannot produce NADPH in the dark. The observed down-regulation of GAPDH in the dark may allow NADPH to be rerouted towards other reductive processes contributing to their ecological success.

Responses of the marine diatom Thalassiosira pseudonana to changes in CO2 concentration: a proteomic approach

The concentration of CO2 in many aquatic systems is variable, often lower than the KM of the primary carboxylating enzyme Rubisco, and in order to photosynthesize efficiently, many algae operate a facultative CO2 concentrating mechanism (CCM). Here we measured the responses of a marine diatom, Thalassiosira pseudonana, to high and low concentrations of CO2 at the level of transcripts, proteins and enzyme activity. Low CO2 caused many metabolic pathways to be remodeled. Carbon acquisition enzymes, primarily carbonic anhydrase, stress, degradation and signaling proteins were more abundant while proteins associated with nitrogen metabolism, energy production and chaperones were less abundant. A protein with similarities to the Ca2+/ calmodulin dependent protein kinase II_association domain, having a chloroplast targeting sequence, was only present at low CO2. This protein might be a specific response to CO2 limitation since a previous study showed that other stresses caused its reduction. The protein sequence was found in other marine diatoms and may play an important role in their response to low CO2 concentration.

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa

Abstract We report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replication.

Absence of residual structure in the intrinsically disordered regulatory protein CP12 in its reduced state

The redox switch protein CP12 is a key player of the regulation of the Benson-Calvin cycle. Its oxidation state is controlled by the formation/dissociation of two intramolecular disulphide bridges during the day/night cycle. CP12 was known to be globally intrinsically disordered on a large scale in its reduced state, while being partly ordered in the oxidised state. By combining Nuclear Magnetic Resonance and Small Angle X-ray Scattering experiments, we showed that, contrary to secondary structure or disorder predictions, reduced CP12 is fully disordered, with no transient or local residual structure likely to be precursor of the structures identified in the oxidised active state and/or in the bound state with GAPDH or PRK. These results highlight the diversity of the mechanisms of regulation of conditionally disordered redox switches, and question the stability of oxidised CP12 scaffold.

Regulation of glyceraldehyde-3-phosphate dehydrogenase in the eustigmatophyte Pseudocharaciopsis ovalis is intermediate between a chlorophyte and a diatom

The regulation of NADPH-dependent GAPDH was analysed in the chromalveolate (eustigmatophyte) Pseudocharaciopsis ovalis and compared with the well-studied chlorophyte Chlamydomonas reinhardtii and with another chromalveolate (diatom), Asterionella formosa. Optimal pH for GAPDH activity in P. ovalis and C. reinhardtii ranged between 8 and 9, but in A. formosa ranged between 6.2 and 8.1. Assuming dark pH values of about 7 in the plastids of all three species, GAPDH would be down-regulated in the dark in C. reinhardtii and P. ovalis, but fully active in A. formosa. The time required for half-maximal GAPDH activity on transfer to reducing conditions, was significantly different in each species: 1.4, 4.0 and 5.9 min in A. formosa, P. ovalis and C. reinhardtii respectively. Under oxidized conditions in P. ovalis and A. formosa, NADPH caused a large inhibition in GAPDH activity even at very low concentrations (10 to 20 µM) unlike in C. reinhardtii. This inhibition was relieved by addition of a reducing agent suggesting that NADPH can control GAPDH activity under dark-light transitions. A small increase of GAPDH activity with NADP at concentrations higher than 0.5 mM was observed with P. ovalis and C. reinhardtii, while a greater than 1.5-fold stimulation was observed in A. formosa. Regulation of GAPDH in P. ovalis was intermediate between the diatom and the chlorophyte and the possible evolutionary reasons for this are discussed.

Cryptic disorder out of disorder: Encounter between conditionally disordered CP12 and glyceraldehyde-3-phosphate dehydrogenase

Among intrinsically disordered proteins, conditionally disordered proteins undergo dramatic structural disorder rearrangements upon environmental changes and/or post-translational modifications that directly modulate their function. Quantifying the dynamics of these fluctuating proteins is extremely challenging but paramount to understanding the regulation of their function. The chloroplast protein CP12 is a model of such proteins and acts as a redox switch by formation/disruption of its two disulfide bridges. It regulates the Calvin cycle by forming, in oxidized conditions, a supramolecular complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and then phosphoribulokinase. In this complex, both enzymes are inactive. The highly dynamic nature of CP12 has so far hindered structural characterization explaining its mode of action. Thanks to a synergistic combination of small-angle X-ray scattering, nuclear magnetic resonance and circular dichroism that drove the molecular modeling of structural ensembles, we deciphered the structural behavior of Chlamydomonas reinhardtii oxidized CP12 alone and in the presence of GAPDH. Contrary to sequence-based structural predictions, the N-terminal region is unstable, oscillates at the ms timescale between helical and random conformations, and is connected through a disordered linker to its C-terminus, which forms a stable helical turn. Upon binding to GAPDH, oxidized CP12 undergoes an induced unfolding of its N-terminus. This phenomenon called cryptic disorder contributes to decrease the entropy cost and explains CP12 unusual high affinity for its partners.

Exploring the microbiome of the “star” freshwater diatom Asterionella formosa in a laboratory context: The laboratory microbiome of Asterionella formosa

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.