Myriam Ferro

Identification of the melatonin-binding site MT3 as the quinone reductase 2.

The regulation of the circadian rhythm is relayed from the central nervous system to the periphery by melatonin, a hormone synthesized at night in the pineal gland. Besides two melatonin G-coupled receptors, mt1 and MT2, the existence of a novel putative melatonin receptor,MT3 , was hypothesized from the observation of a binding site in both central and peripheral hamster tissues with an original binding profile and a very rapid kinetics of ligand exchange compared with mt1 and MT2. In this report, we present the purification of MT3 from Syrian hamster kidney and its identification as the hamster homologue of the human quinone reductase 2 (QR2, EC 1.6.99.2). Our purification strategy included the use of an affinity chromatography step which was crucial in purifying MT3 to homogeneity. The protein was sequenced by tandem mass spectrometry and shown to align with 95% identity with human QR2. After transfection of CHO-K1 cells with the human QR2 gene, not only did the QR2 enzymatic activity appear, but also the melatonin-binding sites with MT3 characteristics, both being below the limit of detection in the native cells. We further confronted inhibition data fromMT3 binding and QR2 enzymatic activity obtained from samples of Syrian hamster kidney or QR2-overexpressing Chinese hamster ovary cells, and observed an overall good correlation of the data. In summary, our results provide the identification of the melatonin-binding siteMT3 as the quinone reductase QR2and open perspectives as to the function of this enzyme, known so far mainly for its detoxifying properties.

AT_CHLORO, a Comprehensive Chloroplast Proteome Database with Subplastidial Localization and Curated Information on Envelope Proteins

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further validated as the compartmentation of well known pathways (for instance, photosynthesis and amino acid, fatty acid, or glycerolipid biosynthesis) within chloroplasts could be dissected. It also allowed revisiting the compartmentation of the chloroplast metabolism and functions.

Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters

A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a blast-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple α-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems.

Identification of New Intrinsic Proteins in Arabidopsis Plasma Membrane Proteome

Identification and characterization of anion channel genes in plants represent a goal for a better understanding of their central role in cell signaling, osmoregulation, nutrition, and metabolism. Though channel activities have been well characterized in plasma membrane by electrophysiology, the corresponding molecular entities are little documented. Indeed, the hydrophobic protein equipment of plant plasma membrane still remains largely unknown, though several proteomic approaches have been reported. To identify new putative transport systems, we developed a new proteomic strategy based on mass spectrometry analyses of a plasma membrane fraction enriched in hydrophobic proteins. We produced from Arabidopsis cell suspensions a highly purified plasma membrane fraction and characterized it in detail by immunological and enzymatic tests. Using complementary methods for the extraction of hydrophobic proteins and mass spectrometry analyses on mono-dimensional gels, about 100 proteins have been identified, 95% of which had never been found in previous proteomic studies. The inventory of the plasma membrane proteome generated by this approach contains numerous plasma membrane integral proteins, one-third displaying at least four transmembrane segments. The plasma membrane localization was confirmed for several proteins, therefore validating such proteomic strategy. An in silico analysis shows a correlation between the putative functions of the identified proteins and the expected roles for plasma membrane in transport, signaling, cellular traffic, and metabolism. This analysis also reveals 10 proteins that display structural properties compatible with transport functions and will constitute interesting targets for further functional studies.

Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis

During male germ cell postmeiotic maturation, dramatic chromatin reorganization occurs, which is driven by completely unknown mechanisms. For the first time, we describe a specific reprogramming of mouse pericentric heterochromatin. Initiated when histones undergo global acetylation in early elongating spermatids, this process leads to the establishment of new DNA packaging structures organizing the pericentric regions in condensing spermatids. Five new histone variants were discovered, which are expressed in late spermiogenic cells. Two of them, which we named H2AL1 and H2AL2, specifically mark the pericentric regions in condensing spermatids and participate in the formation of new nucleoprotein structures. Moreover, our investigations also suggest that TH2B, an already identified testis-specific H2B variant of unknown function, could provide a platform for the structural transitions accompanying the incorporation of these new histone variants.

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database (PPDB, Sun et al., 2009) presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. (2009) went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags (AMT) database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways (i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts) validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts (i.e. in envelope membranes, stroma, and/or thylakoids) that remains unclear until now.

Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins

As a complementary approach to genome projects, proteomic analyses have been set up to identify new gene products. One of the major challenges in proteomics concerns membrane proteins, especially the minor ones. A procedure based on the differential extraction of membrane proteins in chloroform/methanol mixtures, was tested on the two different chloroplast membrane systems: envolope and thylakoid membranes. Combining the use of classical sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and mass spectrometry analyses, this procedure enabled identification of hydrophobic proteins. The propensity of hydrophobic proteins to partition in chloroform/methanol mixtures was directly correlated with the number of amino acid residues/number of putative transmembrane regions (Res/TM ratio). Regardless of the particular case of some lipid‐interacting proteins, chloroform/methanol extractions allowed enrichment of hydrophobic proteins and exclusion of hydrophilic proteins from both membrane systems, thus demonstrating the versatility of the procedure.

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: With focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥2 peptides) identified by LC‐MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl‐lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol‐3‐phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.

Chloroplast proteomics highlights the subcellular compartmentation of lipid metabolism.

Recent advances in the proteomic field have allowed high throughput experiments to be conducted on chloroplast samples and the data are available in several databases such as the Plant Protein Database (PPDB), or the SubCellular Proteomic Database (SUBA). However, the accurate localization of many proteins that were identified in different subplastidial compartments often remains hypothetical, thus making quantitative proteomics important for going a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regard to their accurate localization within the chloroplast. Spectral counting, a semi-quantitative proteomic strategy based on accurate mass and time tags (AMT), was used to build up AT_CHLORO, a comprehensive chloroplast proteome database with curated subplastidial localization. In this review, we focus on about a hundred enzymes involved in fatty acid biosynthesis, export and metabolism (desaturation and oxylipin metabolism), in the synthesis of chloroplast-specific glycerolipids either with a eukaryotic or a prokaryotic structure. Two main chloroplast compartments play a major role in lipid biosynthesis: the initial steps of fatty acid biosynthesis take place in the stroma, then the envelope membranes concentrate most of the proteins involved in chloroplast glycerolipid metabolism.

PredAlgo, a new subcellular localization prediction tool dedicated to green algae

The unicellular green alga Chlamydomonas reinhardtii is a prime model for deciphering processes occurring in the intracellular compartments of the photosynthetic cell. Organelle-specific proteomic studies have started to delineate its various subproteomes, but sequence-based prediction software is necessary to assign proteins subcellular localizations at whole genome scale. Unfortunately, existing tools are oriented toward land plants and tend to mispredict the localization of nuclear-encoded algal proteins, predicting many chloroplast proteins as mitochondrion targeted. We thus developed a new tool called PredAlgo that predicts intracellular localization of those proteins to one of three intracellular compartments in green algae: the mitochondrion, the chloroplast, and the secretory pathway. At its core, a neural network, trained using carefully curated sets of C. reinhardtii proteins, divides the N-terminal sequence into overlapping 19-residue windows and scores the probability that they belong to a cleavable targeting sequence for one of the aforementioned organelles. A targeting prediction is then deduced for the protein, and a likely cleavage site is predicted based on the shape of the scoring function along the N-terminal sequence. When assessed on an independent benchmarking set of C. reinhardtii sequences, PredAlgo showed a highly improved discrimination capacity between chloroplast- and mitochondrion-localized proteins. Its predictions matched well the results of chloroplast proteomics studies. When tested on other green algae, it gave good results with Chlorophyceae and Trebouxiophyceae but tended to underpredict mitochondrial proteins in Prasinophyceae. Approximately 18% of the nuclear-encoded C. reinhardtii proteome was predicted to be targeted to the chloroplast and 15% to the mitochondrion.