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Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1’s neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1–Kv1–MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localisation of these features, and gain broader insights into LGI1 neurobiology, we analysed the detailed localisation of LGI1, and the diversity of its protein interactome, in mouse brains using recombinant monoclonal LGI1-antibodies derived from encephalitis patients. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro- oligodendrocytic and astro-microglial protein complexes. Proteomic data support the hypothesis that destabilization of Kv1 / MAGUK complexes by autoantibodies could promote excitability, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

International audience; In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the Hop

International audience; Saprophytic fungi degrade lignocellulosic polymers present in plant cell walls using dedicated lignocellulosic enzymes such as ‘Carbohydrate Active enZYmes’ (CAZYmes) and peroxidases. These enzymes are secreted in the extracellular environment together with reactive oxygen species (ROS), shown to participate actively to vegetal biomass degradation. Numerous CAZYmes are routinely used in biotechnology approaches and understand their functioning is of crucial importance to improve and create new ways of biomass valorization. ROS can oxidize proteins on sensitive amino acids leading to post-translational modifications (PTMs), the consequences of which range from damaging effects to fine-tuned regulation of protein functions and fates. For example, the reversible oxidation of methionine into methionine sulfoxyde (MetO) can act as redox switch to regulate proteins involved in several cell functions. If the effects of ROS on numerous proteins of bacteria, plants or animals have been studied, almost nothing is known about fungal proteins and specifically during biomass degradation. Our study aims to identify and characterize proteins carrying MetO, that are secreted during biomass degradation in fungi. We chose Pycnoporus cinnabarinus as a model basidiomycete capable of producing a large arsenal of lignocellulosic enzymes. A chemoproteomic approach is being used with first the synthesis of an oxaziridine probe targeting redox-sensitive Met. The labeling of Met by this chemical probe has been validated in a model protein. Coupled to a mass spectrometry analysis, this probe will then allow us to identify proteins carrying redox-active Met. The effect of redox modification of Met will then be elucidated on recombinant proteins. This approach should help to uncover redox regulated CAZYmes.

Paraspeckles are nuclear membraneless structures composed of a long non-coding RNA, Nuclear-Enriched-Abundant-Transcript-1 and RNA binding proteins, which associate numerous mRNAs. It is therefore believed that their cellular function is to sequester in the nucleus their associated proteins and/or target mRNAs. However, little is known about the molecular determinant in mRNA targets that allow their association to paraspeckles except that inverted repeats of Alu sequences (IRAlu) present in 3’UTR of mRNAs may allow this association. While in a previous study we established the list of paraspeckle target RNAs in a rat pituitary cell line, we didn’t find however, inverted repeated SINEs, the rat equivalent of primate IRAlus in 3’UTR of these RNAs. By developing a candidate gene strategy, we selected a paraspeckle target gene, namely calreticulin mRNA, and we searched for other potential RNA recruitment elements in its 3’UTR, since 3’UTRs usually contain the sequence recognition for nuclear localization. We found a 15-nucleotide sequence, present as a tandem repeat in 3’UTR of this mRNA, which is involved in the nuclear retention by paraspeckles. While being not overrepresented in the 3’UTR of the paraspeckle target RNAs, this recruitment element was present in near 30% of all 3’UTR mRNAs. In addition, since an oligonucleotide containing this sequence binds the paraspeckle protein component HNRNPK, it is proposed that HNRNPK may constitute a bridging protein between paraspeckles and target mRNAs.

BackgroundParasitoid wasps have fascinating life cycles and play an important role in trophic networks, yet little is known about their genome content and function. Parasitoids that infect aphids are an important group with the potential for biocontrol, and infecting aphids requires overcoming both aphid defenses and their defensive endosymbionts.ResultsWe present thede novogenome assemblies, detailed annotation, and comparative analysis of two closely related parasitoid wasps that target pest aphids:Aphidius erviandLysiphlebus fabarum(Hymenoptera: Braconidae: Aphidiinae). The genomes are small (139 and 141 Mbp), highly syntenic, and the most AT-rich reported thus far for any arthropod (GC content: 25.8% and 23.8%). This nucleotide bias is accompanied by skewed codon usage, and is stronger in genes with adult-biased expression. AT-richness may be the consequence of reduced genome size, a near absence of DNA methylation, and age-specific energy demands. We identify expansions of F-box/Leucine-rich-repeat proteins, suggesting that diversification in this gene family may be associated with their broad host range or with countering defenses from aphids’ endosymbionts. The absence of some immune genes (Toll and Imd pathways) resembles similar losses in their aphid hosts, highlighting the potential impact of symbiosis on both aphids and their parasitoids.ConclusionsThese findings are of fundamental interest for insect evolution and beyond. This will provide a strong foundation for further functional studies including coevolution with respect to their hosts, the basis of successful infection, and biocontrol. Both genomes are available athttps://bipaa.genouest.org.

International audience; Synaptic vesicle proton V-ATPase is an essential component in synaptic vesicle function. Active acidification of synaptic vesicles, triggered by the V-ATPase, is necessary for neurotransmitter storage. Independently from its proton transport activity, an additional important function of the membrane-embedded sector of the V-ATPase has been uncovered over recent years. Subunits a and c of the membrane sector of this multi-molecular complex have been shown to interact with SNARE proteins and to be involved in modulating neurotransmitter release. The c-subunit interacts with the v-SNARE VAMP2 and facilitates neurotransmission. In this study, we used chromophore-assisted light inactivation and monitored the consequences on neuro-transmission on line in CA3 pyramidal neurons. We show that V-ATPase c-subunit V0c is a key element in modulating neurotransmission and that its specific inactivation rapidly inhibited neurotransmission.

Cold storage of fruit may induce the physiological disorder chilling injury (CI); however, the molecular basis of CI development remains largely unexplored. Simulated conditions of CI priming and suppression provided an interesting experimental system to study cold response in fruit. Peaches (cv. June Gold) at the commercial harvest (CH) or tree-ripe (TR) stages were immediately exposed to cold treatment (40 d, 0 °C) and an additional group of CH fruits were pre-conditioned 48 h at 20 °C prior to low-temperature exposure (pre-conditioning, PC). Following cold treatment, the ripening behaviour of the three groups of fruits was analysed (3 d, 20 °C). Parallel proteomic, metabolomic and targeted transcription comparisons were employed to characterize the response of fruit to CI expression. Physiological data indicated that PC suppressed CI symptoms and induced more ethylene biosynthesis than the other treatments. Differences in the protein and metabolic profiles were identified, both among treatments and before and after cold exposure. Transcriptional expression patterns of several genes were consistent with their protein abundance models. Interestingly, metabolomic and gene expression results revealed a possible role for valine and/or isoleucine in CI tolerance. Overall, this study provides new insights into molecular changes during fruit acclimation to cold environment.

Chronic inflammatory demyelination polyneuropathy is a heterogeneous and treatable immune-mediated disorder that lacks biomarkers to support diagnosis. Recent evidence indicates that paranodal proteins (contactin 1, contactin-associated protein 1, and neurofascin-155) are the targets of autoantibodies in subsets of patients showing distinct clinical presentations. Here, we identified neurofascin-186 and neurofascin-140 as the main targets of autoantibodies in five patients presenting IgG reactivity against the nodes of Ranvier. Four patients displayed predominantly IgG4 antibodies, and one patient presented IgG3 antibodies that activated the complement pathway in vitro. These patients present distinct clinical features compared to those with anti-neurofascin-155 IgG4. Most patients had a severe phenotype associated with conduction block or decreased distal motor amplitude. Four patients had a subacute-onset and sensory ataxia. Two patients presented with nephrotic syndromes and one patient with an IgG4-related retroperitoneal fibrosis. Intravenous immunoglobulin and corticosteroids were effective in three patients, and one patient remitted following rituximab treatment. Clinical remission was associated with autoantibody depletion and with recovery of conduction block and distal motor amplitude suggesting a nodo-paranodopathy. Our data demonstrate that the pathogenic mechanisms responsible for chronic inflammatory demyelination polyneuropathy are broad and may include dysfunctions at the nodes of Ranvier in a subgroup of patients.

The availability of a large variety of specific blockers, which inhibit different K(+) currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca(2+)-activated K(+) channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using (125)I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the (125)I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family.

Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

The interplay among polyamines (PAs) and reactive oxygen and nitrogen species (RNS and ROS) is emerging as a key issue in plant responses to salinity. To address this question, we analysed the impact of exogenous PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] on the oxidative and nitrosative status in citrus plants exposed to salinity. PAs partially reversed the NaCl-induced phenotypic and physiological disturbances. The expression of PA biosynthesis (ADC, SAMDC, SPDS and SPMS) and catabolism (DAO and PAO) genes was systematically up-regulated by PAs. In addition, PAs altered the oxidative status in salt-stressed plants as inferred by changes in ROS production and redox status accompanied by regulation of transcript expression and activities of various antioxidant enzymes. Furthermore, NaCl-induced up-regulation of NO-associated genes, such as NR, NADde, NOS-like and AOX, along with S-nitrosoglutathione reductase and nitrate reductase activities, was partially restored by PAs. Protein carbonylation and tyrosine nitration are depressed by specific PAs whereas protein S-nitrosylation was elicited by all PAs. Furthermore, we identified 271 S-nitrosylated proteins that were commonly or preferentially targeted by salinity and individual PAs. This work helps improve our knowledge on the plant's response to environmental challenge.

During the past decade mass spectrometry (MS) has become one of the major suitable techniques for peptide and protein analysis from animal venoms, not only to identify new toxins within these complex mixtures, but also for toxin structural studies. The goal of the experiments presented here was to test the ultimate limit for producing scorpion venom mass fingerprinting (VMF; i.e. a comprehensive list of all masses in presence). Thanks to recent developments in nano ultra high performance liquid chromatography (UHPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), we sought the experimental conditions for setting up an automated data acquisition workflow. As the main result, we demonstrate the possibility to easily achieve VMF having in hand only tens of nanogram of venom. ELISA on-line was also beneficial to monitor in the picogram range for toxin families using specific antisera raised against major toxins.

Alfalfa (Medicago sativa L.) yield is severely compromised by soil salinity, especially at the level of seedling establishment. This question was addressed by proteomics to decipher whether specific changes in protein accumulation correlate with germination performance of alfalfa seeds submitted to a salinity stress as obtained by imbibing seeds in the presence of NaCl. This study used alfalfa seeds submitted to an osmopriming invigoration treatment that proved very efficient in counteracting the negative effect of salinity stress on germination performance. Comparative proteomic analyses disclosed 94 proteins commonly characterizing the response of both the untreated control and osmoprimed seeds to the experimental salinity stress. Remarkably, many of them, representing 84 proteins, showed contrasting accumulation patterns when comparing the untreated control and osmoprimed seeds submitted to the same salt stress. Thus numerous changes observed in the proteome of the untreated control seeds imbibed in the presence of salt, and presumably accounting for the loss in seed vigour associated with salinity stress, can be substantially reversed in osmoprimed seeds undergoing this stress. These data therefore provide a biochemical understanding of the increase in seed vigour generally observed with primed seeds.

AGAP : équipe GE2pop; Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.

Venom composition of parasitoid wasps attracts increasing interest – notably molecules ensuring parasitism success on arthropod pests – but its variation within and among taxa is not yet understood. We have identified here the main venom proteins of two braconid wasps, Psyttalia lounsburyi (two strains from South Africa and Kenya) and P. concolor, olive fruit fly parasitoids that differ in host range. Among the shared abundant proteins, we found a GH1 β-glucosidase and a family of leucine-rich repeat (LRR) proteins. Olive is extremely rich in glycoside compounds that are hydrolyzed by β-glucosidases into defensive toxic products in response to phytophagous insect attacks. Assuming that Psyttalia host larvae sequester ingested glycosides, the injected venom GH1 β-glucosidase could induce the release of toxic compounds, thus participating in parasitism success by weakening the host. Venom LRR proteins are similar to truncated Toll-like receptors and may possibly scavenge the host immunity. The abundance of one of these LRR proteins in the venom of only one of the two P. lounsburyi strains evidences intraspecific variation in venom composition. Altogether, venom intra- and inter-specific variation in Psyttalia spp. were much lower than previously reported in the Leptopilina genus (Figitidae), suggesting it might depend upon the parasitoid taxa.

International audience; KEY MESSAGE: BABA or GABA induces salinity acclimation during citrus seeds germination via alternation of specific proteins (e.g., citrin). The impact of four elicitors, namely hydrogen peroxide (H2O2), β-amino butyric acid (BABA), γ-amino butyric acid (GABA) and hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS), in citrus seed germination under salinity (150 mM NaCl) was tested. The germination potential was adversely affected by NaCl-alone treatment. Pretreatment with H2O2 or the NaHS-H2S donor prior to salinity had no significant effect in germination process, however, BABA and GABA substantially improved seed acclimation to salinity, as evidenced by increased germination percentage and radicle length. Total soluble proteins of radicle and cotyledons were separated by 1DE SDS-PAGE and proteins zones were analyzed by mass spectrometry. In total, 27 and 3 proteins were identified in radicle and cotyledons, respectively. The identified proteins mainly include redox-regulated enzymes (i.e., glutathione S-transferase, dehydroascorbate reductase, Mn-superoxide dismutase, glutathione peroxidase), energy-related proteins (i.e., isocitrate lyase, malate synthase, pyruvate decarboxylase), stress proteins (i.e., stress-related protein, miraculin, thaumatin, disulfide isomerase), storage proteins (i.e., vicilin, Pis v 1 allergen 2S albumin) and transcriptional regulators (i.e., MarR family transcriptional regulator, MADS544 protein). Pretreatments with BABA or GABA altered the accumulation of protein zones exclusively corresponding to citrin, indicating that this protein may serve as a marker for salinity acclimation in citrus seeds.

Fruit development and ripening depends on highly coordinated phyto-hormonal activities. Although the role of synthetic cytokinin N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) in promoting fruit growth has been established, knowledge regarding the underlying mechanism is still lacking. Here, we characterize the effect of CPPU application 20 d after full bloom at pre- and post-harvest biology of kiwifruit (Actinidia deliciosa [A. Chev.] C.F. Liang et A.R. Ferguson var. deliciosa cv. ‘Hayward’). Data revealed that CPPU stimulates kiwifruit growth through the enlargement of small cells. During fruit development, the abundance of 16 proteins that are mainly related to defence was increased by CPPU while CPPU altered the expression of 19 polar metabolites in outer pericarp. Sugar homeostasis, cell wall modifications, TCA cycle and myo-inositol pathway were mostly affected by CPPU in kiwifruit during development. Upon postharvest ripening at 20 °C following 2 months of cold storage (0 °C), CPPU suppressed ethylene production and retained central placenta softening, indicating that CPPU induced tissue-dependent disturbances in climacteric ripening. Nineteen central placenta proteins and up to 15 metabolites of outer pericarp and central placenta tissues were affected by CPPU in ripened kiwifruits. These observations amplified our understanding in the regulation of fruit development and ripening by exogenously supplied cytokinins. Biological significance This study demonstrates that CPPU application, apart from fruit development, influenced also the kiwifruit climacteric ripening behaviour. An insight on the action of CPPU during kiwifruit development is provided, showing that it is partially based on a general stimulation of TCA cycle and myo-inositol pathway along with alternation in sugar and cell wall metabolism. Data also revealed that CPPU regulates ethylene biosynthesis and influences central placenta softening, indicating that this tissue may play a prominent role in kiwifruit ripening. Also, this work provides a first characterization of the ripening-affected central placenta proteins that offers insights into kiwifruit ripening. The current study provides a baseline of information for understanding the metabolic processes that are regulated by exogenous cytokinin during fruit development and ripening.

Reactive oxygen and nitrogen species are involved in a plethora of cellular responses in plants; however, our knowledge on the outcomes of oxidative and nitrosative signaling is still unclear. To better understand how oxidative and nitrosative signals are integrated to regulate cellular adjustments to external conditions, local and systemic responses were investigated in the roots and leaves of sour orange plants (Citrus aurantium L.) after root treatment with hydrogen peroxide (H(2) O(2) ) or sodium nitroprusside (a nitric oxide donor), followed by NaCl stress for 8 days. Phenotypic and physiological data showed that pre-exposure to these treatments induced an acclimation to subsequent salinity stress that was accompanied by both local and systemic H(2) O(2) and nitric oxide (NO) accumulation. Combined histochemical and fluorescent probe approaches showed the existence of a vascular-driven long-distance reactive oxygen species and NO signaling pathway. Transcriptional analysis of genes diagnostic for H(2) O(2) and NO signaling just after treatments or after 8 days of salt stress revealed tissue- and time-specific mechanisms controlling internal H(2) O(2) and NO homeostasis. Furthermore, evidence is presented showing that protein carbonylation, nitration and S-nitrosylation are involved in acclimation to salinity stress. In addition, this work enabled characterization of potential carbonylated, nitrated and nitrosylated proteins with distinct or overlapping signatures. This work provides a framework to better understand the oxidative and nitrosative priming network in citrus plants subjected to salinity conditions.

Endoparasitoid wasps inject venom proteins with their eggs to protect them from the host immune response and ensure successful parasitism. Here we report identification of Cu,Zn superoxide dismutase (SOD) transcripts for both intracellular SOD1 and extracellular SOD3 in the venom apparatus of two Leptopilina species, parasitoids of Drosophila. Leptopilina SODs show sequence and structure similarity to human SODs, but phylogenetic analyses indicate that the extracellular SODs are more related to cytoplasmic vertebrate SODs than to extracellular SODs, a feature shared by predicted insect extracellular SODs. We demonstrate that L. boulardi SOD3 is indeed secreted and active as monomeric glycosylated forms in venom. Our results also evidence quantitative variation in SOD3 venom contents between closely related parasitoid species, as sod3 is 100-fold less expressed in Leptopilina heterotoma venom apparatus and no protein and SOD activity are detected in its venom. Leptopilina recombinant SOD3s as well as a mammalian SOD in vitro inhibit the Drosophila phenoloxidase activity in a dose-dependent manner, demonstrating that SODs may interfere with the Drosophila melanization process and, therefore, with production of cytotoxic compounds. Although the recombinant L. boulardi SOD3 quantity needed to observe this effect precludes a systemic effect of the wasp venom SOD3, it is still consistent with a local action at oviposition. This work provides the first demonstration that insect extracellular SODs are indeed secreted and active in an insect fluid and can be used as virulence factors to counteract the host immune response, a strategy largely used by bacterial and fungal pathogens but also protozoan parasites during infection.

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H(2)O(2)) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mm H(2)O(2) on several oxidative features such as protein carbonylation, endogenous H(2)O(2) and lipid peroxidation levels. We report that H(2)O(2) treatment of the pea seeds increased their endogenous H(2)O(2) content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H(2)O(2) in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H(2)O(2) , whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H(2)O(2) in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels.

International audience; The venom of the North African scorpion Androctonus amoreuxi (Aam) was analyzed using a combination of gel filtration, C18 reverse phase HPLC together with mass spectrometry analysis and bioassays. Three novel Birtoxin-like (BTX-L) peptides of 58 amino acid residues comprising three disulfide bridges were isolated and chemically characterized. One peptide, AamBTX-L3, induced serious toxic symptoms in mice and was lethal at nanogram quantities using intracerebroventricular injection. The three BTX-L peptides were tested in competition experiments on rat brain synaptosomes against the (125)I-labeled "classical" α- and β-toxins of reference, as well as with the (125)I-KTX, a voltage-gated potassium channel blocker. Only AamBTX-L3 was able to prevent the equilibrium binding of the β-toxin (125)I-Css IV to its receptor site 4 with a IC(50) value of 189 nM. Even if previous electrophysiological data allowed the classification of other BTX-L peptides among the β-type toxins, this report clearly shows that AamBTX-L3 is pharmacologically a β-toxin, which recognizes the voltage-gated Na(+) (Na(v)) channels from central mammalian neurons. In order to uncover the residues functionally essential for interaction between the AamBTX-L3 with the putative receptor site of (125)I-Css IV on Na(v)1.2, molecular models of the three novel Aam BTX-L molecules were made and their surfaces were compared to the already described Css IV biologically interactive surfaces. A hypothesis is given that in BTX-L3, three residues found in the α-helix play a key role during target binding.

Hydrogen peroxide (H(2)O(2)) and nitric oxide (*NO) are key reactive species in signal transduction pathways leading to activation of plant defense against biotic or abiotic stress. Here, we investigated the effect of pre-treating citrus plants (Citrus aurantium L.) with either of these two molecules on plant acclimation to salinity and show that both pre-treatments strongly reduced the detrimental phenotypical and physiological effects accompanying this stress. A proteomic analysis disclosed 85 leaf proteins that underwent significant quantitative variations in plants directly exposed to salt stress. A large part of these changes was not observed with salt-stressed plants pre-treated with either H(2)O(2) or sodium nitroprusside (SNP; a *NO-releasing chemical). We also identified several proteins undergoing changes either in their oxidation (carbonylation; 40 proteins) and/or S-nitrosylation (49 proteins) status in response to salinity stress. Both H(2)O(2) and SNP pre-treatments before salinity stress alleviated salinity-induced protein carbonylation and shifted the accumulation levels of leaf S-nitrosylated proteins to those of unstressed control plants. Altogether, the results indicate an overlap between H(2)O(2)- and *NO-signaling pathways in acclimation to salinity and suggest that the oxidation and S-nitrosylation patterns of leaf proteins are specific molecular signatures of citrus plant vigour under stressful conditions.

Background Endoparasitoid wasps are important natural enemies of the widely distributed aphid pests and are mainly used as biological control agents. However, despite the increased interest on aphid interaction networks, only sparse information is available on the factors used by parasitoids to modulate the aphid physiology. Our aim was here to identify the major protein components of the venom injected at oviposition by Aphidius ervi to ensure successful development in its aphid host, Acyrthosiphon pisum. Results A combined large-scale transcriptomic and proteomic approach allowed us to identify 16 putative venom proteins among which three γ-glutamyl transpeptidases (γ-GTs) were by far the most abundant. Two of the γ-GTs most likely correspond to alleles of the same gene, with one of these alleles previously described as involved in host castration. The third γ-GT was only distantly related to the others and may not be functional owing to the presence of mutations in the active site. Among the other abundant proteins in the venom, several were unique to A. ervi such as the molecular chaperone endoplasmin possibly involved in protecting proteins during their secretion and transport in the host. Abundant transcripts encoding three secreted cystein-rich toxin-like peptides whose function remains to be explored were also identified. Conclusions Our data further support the role of γ-GTs as key players in A. ervi success on aphid hosts. However, they also evidence that this wasp venom is a complex fluid that contains diverse, more or less specific, protein components. Their characterization will undoubtedly help deciphering parasitoid-aphid and parasitoid-aphid-symbiont interactions. Finally, this study also shed light on the quick evolution of venom components through processes such as duplication and convergent recruitment of virulence factors between unrelated organisms. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-342) contains supplementary material, which is available to authorized users.

International audience; OBJECTIVE: We report the clinical and serologic features of Japanese patients with chronic inflammatory demyelinating polyneuropathy (CIDP) displaying anti-neurofascin-155 (NF155) immunoglobulin G4 (IgG4) antibodies.ăMETHODS: In sera from 533 patients with CIDP, anti-NF155 IgG4 antibodies were detected by ELISA. Binding of IgG antibodies to central and peripheral nerves was tested.ăRESULTS: Anti-NF155 IgG4 antibodies were identified in 38 patients (7%) with CIDP, but not in disease controls or normal participants. These patients were younger at onset as compared to 100 anti-NF155-negative patients with CIDP. Twenty-eight patients (74%) presented with sensory ataxia, 16 (42%) showed tremor, 5 (13%) presented with cerebellar ataxia associated with nystagmus, 3 (8%) had demyelinating lesions in the CNS, and 20 of 25 (80%) had poor response to IV immunoglobulin. The clinical features of the antibody-positive patients were statistically more frequent as compared to negative patients with CIDP (n = 100). Anti-NF155 IgG antibodies targeted similarly central and peripheral paranodes.ăCONCLUSION: Anti-NF155 IgG4 antibodies were associated with a subgroup of patients with CIDP showing a younger age at onset, ataxia, tremor, CNS demyelination, and a poor response to IV immunoglobulin. The autoantibodies may serve as a biomarker to improve patients' diagnosis and guide treatments.

*Summary At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 � C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.

Emerging evidence suggests that the gaseous molecules hydrogen sulfide (H2S) and nitric oxide (NO) enhances plant acclimation to stress; however, the underlying mechanism remains unclear. In this work, we explored if pretreatment of citrus roots with NaHS (a H2S donor) or sodium nitroprusside (SNP, a NO donor) for 2 days (d) could elicit long-lasting priming effects to subsequent exposure to PEG-associated drought stress for 21 d following a 5 d acclimation period. Detailed physiological study documented that both pretreatments primed plants against drought stress. Analysis of the level of nitrite, NOx, S-nitrosoglutahione reductase, Tyr-nitration and S-nitrosylation along with the expression of genes involved in NO-generation suggested that the nitrosative status of leaves and roots was altered by NaHS and SNP. Using a proteomic approach we characterized S-nitrosylated proteins in citrus leaves exposed to chemical treatments, including well known and novel S-nitrosylated targets. Mass spectrometry analysis also enabled the identification of 42 differentially expressed proteins in PEG alone-treated plants. Several PEG-responsive proteins were down-regulated, especially photosynthetic proteins. Finally, the identification of specific proteins that were regulated by NaHS and SNP under PEG conditions provides novel insight into long-term drought priming in plants and in a fruit crop such as citrus in particular.

The influence of salicylic acid (SA) on elicitation of defense mechanisms in Arabidopsis (Arabidopsis thaliana) seeds and seedlings was assessed by physiological measurements combined with global expression profiling (proteomics). Parallel experiments were carried out using the NahG transgenic plants expressing the bacterial gene encoding SA hydroxylase, which cannot accumulate the active form of this plant defense elicitor. SA markedly improved germination under salt stress. Proteomic analyses disclosed a specific accumulation of protein spots regulated by SA as inferred by silver-nitrate staining of two-dimensional gels, detection of carbonylated (oxidized) proteins, and neosynthesized proteins with [35S]-methionine. The combined results revealed several processes potentially affected by SA. This molecule enhanced the reinduction of the late maturation program during early stages of germination, thereby allowing the germinating seeds to reinforce their capacity to mount adaptive responses in environmental water stress. Other processes affected by SA concerned the quality of protein translation, the priming of seed metabolism, the synthesis of antioxidant enzymes, and the mobilization of seed storage proteins. All the observed effects are likely to improve seed vigor. Another aspect revealed by this study concerned the oxidative stress entailed by SA in germinating seeds, as inferred from a characterization of the carbonylated (oxidized) proteome. Finally, the proteomic data revealed a close interplay between abscisic signaling and SA elicitation of seed vigor.

Mass spectrometric analysis of a prion protein (PrP)-containing complex isolated from ram cauda epididymal fluid revealed a protein that showed homology to a carboxylesterase-like protein previously identified in cat urine (cauxin). Using anti-cauxin antibodies, immunoreactive bands were detected in corpus and cauda epididymal fluid from all mammals tested (ram, boar, mouse, and cat). In the ram, the protein was also present in seminal fluid but not found to be associated with sperm. The bands reacting with the anti-cauxin antibody coincided with those having esterase activity in a zymographic assay and its levels paralleled the esterase activity of native epididymal fluids. A partial nucleotide sequence of 1143 bp, corresponding to 380 amino acids, was obtained by RT-PCR amplification from total RNA from the corpus epididymis (zone 6). The deduced protein sequence shows a high degree of homology (up to 90%) with the different cauxin proteins found in databases but only up to 60% with other known carboxylesterases. By PCR, strong mRNA expression was found in the corpus and cauda epididymis, while the testis, kidney, and caput epididymis had low expression. No mRNA was detected in the lung, heart, or liver. These data demonstrate that an epididymal form of the cauxin enzyme is secreted into mammalian epididymal fluid. In the ram, it is associated with a high molecular-weight PrP-associated complex and may be responsible for the majority of the esterase activity in the cauda epididymal fluid of this species.

The present report describes how the soluble germinal angiotensin I-converting enzyme (gACE) appears in the epididymal fluid, where it has been identified in some laboratory rodents and domestic ungulates. We showed that this gACE results from an active proteolytic process that releases the enzyme's extracellular domain from sperm in a precise spatiotemporal location during epididymal transit and that this process involves serine protease activity. Using polyclonal antibodies against the C-terminal intracellular sequence of ACE, a fragment of approximately 10 kDa was detected on the sperm extract only in the epididymal region, where the gACE release occurs. The fluid enzyme was purified, and the cleavage site was determined by mass spectrometry to be between Arg622 and Leu623 of the mature sheep gACE sequence (equivalent to Arg627 and Arg1203 of the human mature gACE and somatic ACE sequences, respectively). Thereafter, the C-terminal Arg was removed, leaving Ala621 as a C-terminal. Using an in vitro assay, gACE cleavage from sperm was strongly increased by the presence of epididymal fluid from the release zone, and this increase was inhibited specifically by the serine protease-inhibitor AEBSF but not by para-aminobenzamidine. None of the other inhibitors tested, such as metallo- or cystein-protease inhibitors, had a similar effect on release. It was also found that this process did not involve changes in gACE phosphorylation.

Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.