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Carbon dots are emerging nanoparticles (NPs) with tremendous applications, especially in the biomedical field. Herein is reported the first quantitative proteomic analysis of the protein corona formed on CDs with different surface charge properties. Four CDs were synthesized from citric acid and various amine group-containing passivation reagents, resulting in cationic NPs with increasing zeta (ζ)-potential and density of positive charges. After CD contact with serum, we show that protein corona identity is influenced by CD surface charge properties, which in turn impacts CD uptake and viability loss in macrophages. In particular, CDs with high ζ-potential (+30 mV) and charge density (2 μmol mg

In humans, hyperglycemia is associated with protein glycation, which may contribute to aging. Strikingly, birds usually outlive mammals of the same body mass, while exhibiting high plasma glucose levels. However, how birds succeed in escaping pro-aging effects of glycation remains unknown. Using a specific mass spectrometry-based approach in captive zebra finches of known age, we recorded high glycaemia values but no glycated hemoglobin form was found. Still, we showed that zebra finch hemoglobin can be glycated in vitro, albeit only to a limited extent compared to its human homologue. This may be due to peculiar structural features, as supported by the unusual presence of three different tetramer populations with balanced proportions and a still bound cofactor that could be inositol pentaphosphate. High levels of the glycated forms of zebra finch plasma serotransferrin, carbonic anhydrase 2, and albumin were measured. Glucose, age or body mass correlations with either plasma glycated proteins or hemoglobin isoforms suggest that those variables may be future molecular tools of choice to monitor glycation and its link with individual fitness. Our molecular advance may help determine how evolution succeeded in associating flying ability, high blood glucose and long lifespan in birds.

International audience; Plasmodione (PD) is a potent antimalarial redox-active drug acting at low nM range concentrations on different malaria parasite stages. In this study, in order to determine the precise PD protein interactome in parasites, we developed a class of (pro-)activity-based protein profiling probes (ABPP) as precursors of photoreactive benzophenonelike probes based on the skeleton of PD metabolites (PDO) generated in a cascade of redox reactions. Under UV-photoirradiation, we clearly demonstrate that benzylic oxidation of 3-benzylmenadione 11 produces the 3-benzoylmenadione probe 7, allowing investigation of the proof-of-concept of the ABPP strategy with 3-benzoylmenadiones 7-10. The synthesized 3-benzoylmenadiones, probe 7 with an alkyne group or probe 9 with-NO2 in para position of the benzoyl chain, were found to be the most efficient photoreactive and clickable probes. In the presence of various H-donor partners, the UV-irradiation of the photo-reactive ABPP probes generates different adducts, the expected 'benzophenone-like' adducts (pathway 1) in addition to 'benzoxanthone' adducts (via two other pathways, 2 and 3). Using both human and Plasmodium falciparum glutathione reductases three protein ligand binding sites were identified following photolabeling with probes 7 or 9. The photoreduction of 3-benzoylmenadiones (PDO and probe 9) promoting the formation of both the corresponding benzoxanthone and the derived enone could be replaced by the glutathione reductase-catalyzed reduction step. In particular, the electrophilic character of the benzoxanthone was evidenced by its ability to alkylate heme, as a relevant event supporting the antimalarial mode of action of PD. This work provides a proof-of-principle that (pro-)ABPP probes can generate benzophenonelike metabolites enabling optimized activity-based protein profiling conditions that will be instrumental to analyse the interactome of early-lead antiplasmodial 3-benzylmenadiones displaying an original and innovative mode of action.

Alpha4A-tubulin is the predominant α-tubulin isotype in platelets. Mutations in α4A-tubulin cause abnormal platelet biogenesis and marginal band formation in mice and in a patient, establishing an essential role of this tubulin isotype., During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in Tuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Summary Single-cell transcriptomics (scRNA-seq) has revolutionized the understanding of the spatial architecture of tissue structure and function. Advancing the “transcript-centric” view of scRNA-seq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyze post-translational modifications. Here, by combining spatial cell sorting with transcriptomics and quantitative proteomics/phosphoproteomics, we established the spatially resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signaling mechanisms. Phosphorylation of receptor tyrosine kinases was detected preferentially in the central vein area, resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation identified Tie receptor signaling as a selective and specific regulator of vascular Wnt activity orchestrating angiocrine signaling, thereby controlling hepatocyte function during liver regeneration. Taken together, the study has yielded fundamental insight into the spatial organization of liver endothelial cell signaling. Spatial sorting may be employed as a universally adaptable strategy for multiomic analyses of scRNA-seq-defined cellular (sub)-populations., Graphical abstract, Highlights • ScRNA-seq-guided spatial sort enables multiomic dissection of the liver vasculature • Liver sinusoidal endothelial cells have a hybrid vascular-lymphatic phenotype • Tyrosine phosphorylation of endothelial cell molecules is enriched on central vein • Endothelial Tie1 shapes hepatic Wnt signal zonation and promotes liver regeneration, Inverso, Shi et al. generate a multiomic encyclopedia of liver endothelial cells (L-ECs) with spatial resolution of transcriptome, proteome, and phosphoproteome. The study provides insight into liver vascular zonation and a template for scRNA-seq-data-guided spatial proteome and phosphoproteome analyses.

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of β-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Elicitors are known to trigger plant defenses in response to biotic stress, but do not systematically lead to effective resistance to pathogens. The reasons explaining such differences remain misunderstood. Therefore, elicitation and induced resistance (IR) were investigated through the comparison of two modified β-1,3 glucans applied on grapevine (Vitis vinifera) leaves before and after inoculation with Plasmopara viticola, the causal agent of downy mildew. The sulfated (PS3) and the shortened (H13) forms of laminarin are both known to elicit defense responses whereas only PS3 induces resistance against downy mildew. The analysis of the 2-DE gel electrophoresis revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Our results point out that the PS3-induced resistance is associated with the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could be considered as useful markers of induced resistance. Significance One strategy to reduce the application of fungicides is the use of elicitors which induce plant defense responses. Nonetheless, the elicitors do not systematically lead to resistance against pathogens. The lack of correlation between plant defense activation and induced resistance (IR) requires the investigation of what makes the specificity of elicitor-IR. In this study, the two β-glucans elicitors, sulfated (PS3) and short (H13) laminarins, were used in the grapevine/Plasmopara viticola interaction since only the first one leads to resistance against downy mildew. To disclose IR specificity, proteomic approach has been employed to compare the two treatments before and after P. viticola inoculation. The analysis of the 2-DE revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Significant increase of the number of proteins regulated by PS3, relative to both H13 and time-points, is correlated with the resistance process establishment. Our results point that the PS3-induced resistance requires the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could constitute useful markers of PS3 induced resistance.

The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance–conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ∼50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT.

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation inTuba4a-mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Introduction: Gla-rich protein (GRP) is a vitamin K-dependent protein (VKDP) acting as a calcification inhibitor and anti-inflammatory agent in cardiovascular and articular systems, and THP1 monocyte/macrophage cells [1,2]. Calcification and inflammation processes are known to be involved in the etiology of several calcification-related chronic inflammatory diseases such as atherosclerosis, CKD and osteoarthritis, in a complex bi-directional interplay that drives disease progression. Here, we developed an innovative system to produce human c-carboxylated GRP (cGRP), and a nanotechnology strategy based on GRP loading into extracellular vesicles (EVs) as a gold standard delivery system for GRP in therapeutic applications. Materials and methods: Human GRP protein was co-expressed with c-carboxylase enzyme (GGCX), vitamin K oxidoreductase (GGCX) and furin, in the insect cell baculovirus system in the presence of vitamin K. GRP released in the cell culture media was characterized by mass spectrometry based techniques and Western blot analysis. EVs released by the insect cells overexpressing GRP were isolated by ultracentrifugation, and characterized for GRP content through TEM-immunogold staining, Western blot, ELISA, qPCR. Functional assays using isolated EVs containing GRP were performed in primary vascular smooth muscle cells (VSMCs) and THP1 monocyte/macrophage cells, for anti-mineralizing and anti-inflammatory screening.Results: GRP released in the cell culture media when co-expressed with GGCX, VKOR and furin in the presence of vitamin K, is processed at the pro-peptide and contain Gla residues. EVs released by the insect cells in this system were shown to be loaded with GRP protein and mRNA, and capable of reducing ECM calcium deposition of calcifying VSMCs and the production of TNFa in THP1 monocyte/macrophage cells stimulated with LPS. Discussion and conclusions: While the successful production of human cGRP constitutes a major achievement, this innovative methodology will open new opportunities for the production of other biological active VKDPs. Furthermore, EVs loaded with GRP were shown to have anti-mineralizing and anti-inflammatory properties, with promising therapeutic potentialities for calcification-related chronic inflammatory diseases. Portuguese Foundation for Science and Technology (EU/PID1003201) info:eu-repo/semantics/publishedVersion

International audience; The chloroquine resistance transporter PfCRT of the human malaria parasite Plasmodium falciparum confers resistance to the former first-line antimalarial drug chloroquine, and it modulates the responsiveness to a wide range of quinoline and quinoline-like compounds. PfCRT is post-translationally modified by phosphorylation, palmitoylation, and, possibly, ubiquitination. However, the impact of these post-translational modifications on P. falciparum biology and, in particular, the drug resistance–conferring activity of PfCRT has remained elusive. Here, we confirm phosphorylation at Ser-33 and Ser-411 of PfCRT of the chloroquine-resistant P. falciparum strain Dd2 and show that kinase inhibitors can sensitize drug responsiveness. Using CRISPR/Cas9 genome editing to generate genetically engineered PfCRT variants in the parasite, we further show that substituting Ser-33 with alanine reduced chloroquine and quinine resistance by ∼50% compared with the parental P. falciparum strain Dd2, whereas the phosphomimetic amino acid aspartic acid could fully and glutamic acid could partially reconstitute the level of chloroquine/quinine resistance. Transport studies conducted in the parasite and in PfCRT-expressing Xenopus laevis oocytes linked phosphomimetic substitution at Ser-33 to increased transport velocity. Our data are consistent with phosphorylation of Ser-33 relieving an autoinhibitory intramolecular interaction within PfCRT, leading to a stimulated drug transport activity. Our findings shed additional light on the function of PfCRT and suggest that chloroquine could be reevaluated as an antimalarial drug by targeting the kinase in P. falciparum that phosphorylates Ser-33 of PfCRT.

Significance Using an interactomic approach, we have identified the nuclear receptor REV-ERBα as a O-GlcNAc transferase (OGT) protein partner. REV-ERBα protects cytoplasmic OGT from proteasomal degradation and facilitates cytosolic and nuclear protein O-GlcNAcylation while REV-ERα ligands decreased cytoplasmic OGT activity. REV-ERBα thus exerts pleiotropic activities through OGT, coordinating signal transduction, epigenomic programming, and transcriptional response in the liver., The nuclear receptor REV-ERBα integrates the circadian clock with hepatic glucose and lipid metabolism by nucleating transcriptional comodulators at genomic regulatory regions. An interactomic approach identified O-GlcNAc transferase (OGT) as a REV-ERBα−interacting protein. By shielding cytoplasmic OGT from proteasomal degradation and favoring OGT activity in the nucleus, REV-ERBα cyclically increased O-GlcNAcylation of multiple cytoplasmic and nuclear proteins as a function of its rhythmically regulated expression, while REV-ERBα ligands mostly affected cytoplasmic OGT activity. We illustrate this finding by showing that REV-ERBα controls OGT-dependent activities of the cytoplasmic protein kinase AKT, an essential relay in insulin signaling, and of ten-of-eleven translocation (TET) enzymes in the nucleus. AKT phosphorylation was inversely correlated to REV-ERBα expression. REV-ERBα enhanced TET activity and DNA hydroxymethylated cytosine (5hmC) levels in the vicinity of REV-ERBα genomic binding sites. As an example, we show that the REV-ERBα/OGT complex modulates SREBP-1c gene expression throughout the fasting/feeding periods by first repressing AKT phosphorylation and by epigenomically priming the Srebf1 promoter for a further rapid response to insulin. Conclusion: REV-ERBα regulates cytoplasmic and nuclear OGT-controlled processes that integrate at the hepatic SREBF1 locus to control basal and insulin-induced expression of the temporally and nutritionally regulated lipogenic SREBP-1c transcript.

International audience; The high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and proteogenomics fields. The present study describes the free N-terminome analysis of human mitochondria-enriched samples using trimethoxyphenyl phosphonium (TMPP) labelling approaches. Owing to the extent of protein import and cleavage for mitochondrial proteins, determining the new N-termini generated after translocation/processing events for mitochondrial proteins is crucial to understand the transformation of precursors to mature proteins. The doublet N-terminal oriented proteomics (dN-TOP) strategy based on a double light/heavy TMPP labelling has been optimized in order to improve and automate the workflow for efficient, fast and reliable high throughput N-terminome analysis. A total of 2714 proteins were identified and 897 N-terminal peptides were characterized (424 N-α-acetylated and 473 TMPP-labelled peptides). These results allowed the precise identification of the N-terminus of 693 unique proteins corresponding to 26% of all identified proteins. Overall, 120 already annotated processing cleavage sites were confirmed while 302 new cleavage sites were characterized. The accumulation of experimental evidence of mature N-termini should allow increasing the knowledge of processing mechanisms and consequently also enhance cleavage sites prediction algorithms. Complete datasets have been deposited to the ProteomeXchange Consortium with identifiers PXD001521, PXD001522 and PXD001523 (http://proteomecentral.proteomexchange.org/dataset/PXD001521, http://proteomecentral.proteomexchange.org/dataset/PXD0001522 and http://proteomecentral.proteomexchange.org/dataset/PXD001523, respectively).

The bioproduction of recombinant monoclonal antibodies results in complex mixtures of a main isoform and numerous macro- and microvariants. Monoclonal antibodies therefore present different levels of heterogeneities ranging from primary sequence variants to post-translational modifications. Among these heterogeneities, the truncation and fragmentation of the primary amino-acid sequence result in shorter or cleaved polypeptide chains while the incomplete processing of the signal peptide produces N-terminal elongated polypeptide chains. Here, we present an in-gel protein N-terminal chemical derivatization method using (N-succinimidyloxycarbonylmethyl)-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This chemical tag enhances the detection by mass spectrometry of the N-terminal positions of proteins and allows their unambiguous assignment without altering the identification of internal digestion peptides. This method adds just one step to the classical peptide mapping workflow. Using this in-gel N-TOP (N-terminal oriented proteomics) strategy, the N-terminal sequence heterogeneities of several monoclonal antibodies, among which are minor unexpected proteoforms, were successfully detected and characterized.

SPEIPMINRAUBCNRS; "Omics-" for a mapping of grapevine response to elicitors and identification of induced resistance markers . 12. European Fondation for Plant Pathology (EFPP) & 10. French Society for Plant Pathology Conference ‘Deepen Knwoledge in Plant Pathology for Innovative Agro-Ecology’

The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.

The study of the N-terminome and the precise identification of proteolytic processing events are key in biology. Dedicated methodologies have been developed as the comprehensive characterization of the N-terminome can hardly be achieved by standard proteomics methods. In this context, we have set up a trimethoxyphenyl phosphonium (TMPP) labeling approach that allows the characterization of both N-terminal and internal digestion peptides in a single experiment. This latter point is a major advantage of our strategy as most N-terminomics methods rely on the enrichment of N-terminal peptides and thus exclude internal peptides.We have implemented a double heavy/light TMPP labeling and an automated data validation workflow that make our doublet N-terminal oriented proteomics (dN-TOP) strategy efficient for high-throughput N-terminome analysis.

International audience; Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in the presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc-treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.

Botryosphaeria dieback is a fungal grapevine trunk disease that currently represents a threat for viticulture worldwide because of the important economical losses due to reduced yield of affected plants and their premature death. Neofusicoccum parvum and Diplodia seriata are among the causal agents. Vine green stems were artificially infected with N. parvum or D. seriata at the onset of three different phenological stages (G stage (separated clusters), flowering and veraison). Highest mean lesion lengths were recorded at flowering. Major proteome changes associated to artificial infections during the three different phenological stages were also reported using two dimensional gel electrophoresis (2D)-based analysis. Twenty (G stage), 15 (flowering) and 13 (veraison) differentially expressed protein spots were subjected to nanoLC-MS/MS and a total of 247, 54 and 25 proteins were respectively identified. At flowering, a weaker response to the infection was likely activated as compared to the other stages, and some defense-related proteins were even down regulated (e.g., superoxide dismutase, major latex-like protein, and pathogenesis related protein 10). Globally, the flowering period seemed to represent the period of highest sensitivity of grapevine to Botryosphaeria dieback agent infection, possibly being related to the high metabolic activity in the inflorescences.

Background information Commitment to splicing occurs co-transcriptionally, but a major unanswered question is the extent to which various modifications of chromatin, the template for transcription in vivo, contribute to the regulation of splicing. Results Here, we perform genome-wide analyses showing that inhibition of specific marks – H2B ubiquitylation, H3K4 methylation and H3K36 methylation – perturbs splicing in budding yeast, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analysis on intron-containing genes indicated that H2B ubiquitylation, but not Set1-, Set2- or Dot1-dependent H3 methylation, stimulates recruitment of the early splicing factors, namely U1 and U2 snRNPs, onto nascent RNAs. Conclusions These results suggest that histone modifications impact splicing of distinct subsets of genes using distinct pathways.

We report a new slow-moving δ chain hemoglobin (Hb) variant, named Hb A2-Konz [δ50(D1)Ser → Thr; HBD: c.151T > A]. It was detected during simultaneous measurement of Hb A1C and Hb A2 by high resolution cation exchange high performance liquid chromatography (HPLC) using a PolyCATA column. Hb A2-Konz comprised 0.8% of total Hb. This new variant was identified by peptide mapping using nanoliquid chromatography electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS) as a serine to threonine substitution at δ50(D1), indicating that the variant was due to a single base change at codon 51 (TCT > ACT) of the δ-globin gene. The new mutant is clinically silent but could lead to a misdiagnosis of β-thalassemia (β-thal) based on the level of Hb A2.

Exquisite chemoselectivity for cysteine has been found for a novel class of remarkably hydrolytically stable reagents, 3-arylpropiolonitriles (APN). The efficacy of the APN-mediated tagging was benchmarked against other cysteine-selective methodologies in a model study on a series of traceable amino acid derivatives. The selectivity of the methodology was further explored on peptide mixtures obtained by trypsin digestion of lysozyme. Additionally, the superior stability of APN-cysteine conjugates in aqueous media, human plasma, and living cells makes this new thiol-click reaction a promising methodology for applications in bioconjugation.

Plasmodium falciparum extensively modifies its chosen host cell, the mature human erythrocyte. This remodelling is carried out by parasite-encoded proteins that are exported into the host cell. To gain access to the human red blood cell, these proteins must cross the parasitophorous vacuole, a membrane bound compartment surrounding the parasite that is generated during the invasion process. Many exported proteins carry a so-called PEXEL/HT signal that directs their transport. We recently reported the unexpected finding of a species-restricted parasite-encoded Hsp70, termed PfHsp70x, which is exported into the host erythrocyte cytosol. PfHsp70x lacks a classical PEXEL/HT motif, and its transport appears to be mediated by a 7 amino acid motif directly following the hydrophobic N-terminal secretory signal. In this report, we analyse this short targeting sequence in detail. Surprisingly, both a reversed and scrambled version of the motif retained the capacity to confer protein export. Site directed mutagenesis of glutamate residues within this region leads to a block of protein trafficking within the lumen of the PV. In contrast to PEXEL-containing proteins, the targeting signal is not cleaved, but appears to be acetylated. Furthermore we show that, like other exported proteins, trafficking of PfHsp70x requires the vacuolar translocon, PTEX.

International audience; Mutations in hemoglobin can cause a wide range of pheno-typic outcomes, including anemia due to protein instability and red cell lysis. Uncovering the biochemical basis for these phenotypes can provide new insights into hemoglobin structure and function as well as identify new therapeutic opportunities. We report here a new hemoglobin ␣ chain variant in a female patient with mild anemia, whose father also carries the trait and is from the Turkish city of Kirklareli. Both the patient and her father had a His-58(E7) 3 Leu mutation in ␣1. Surprisingly, the patient's father is not anemic, but he is a smoker with high levels of HbCO (ϳ16%). To understand these phenotypes, we examined recombinant human Hb (rHb) Kirklareli containing the ␣ H58L replacement. Mutant ␣ subunits containing Leu-58(E7) autoxidize ϳ8 times and lose hemin ϳ200 times more rapidly than native ␣ subunits, causing the oxygenated form of rHb Kirklareli to denature very rapidly under physiological conditions. The crystal structure of rHb Kirklareli shows that the ␣ H58L replacement creates a completely apolar active site, which prevents electrostatic stabilization of bound O 2 , promotes autoxidation, and enhances hemin dissociation by inhibiting water coordination to the Fe(III) atom. At the same time, the mutant ␣ subunit has an ϳ80,000-fold higher affinity for CO than O 2 , causing it to rapidly take up and retain carbon monoxide , which prevents denaturation both in vitro and in vivo and explains the phenotypic differences between the father, who is a smoker, and his daughter.

Vacuolar invertase is a key enzyme of sugar metabolism in grape berries. A full characterisation of this highly N-glycosylated protein is required to help understand its biological and biochemical significance in grapes. We have developed a mass spectrometry (MS)-based glycoproteomic approach wherein deglycosylated peptides are analysed by LC-MS/MS, while intact glycopeptides are characterised using a dedicated MS method to determine the attachment sites and micro-heterogeneity. For grape invertase, in parallel with deglycosylated peptides analysis, different enzymatic digestions were performed and glycopeptide detection was improved by enrichment method, nanoLC-MS and oxonium glycan ions. This MS-based glycoproteomic approach demonstrates that vacuolar invertase is glycosylated at all twelve potential N-glycosylation sites. Glycosylation is heterogeneous, with twelve glycoforms identified at six of the sites. The identification of several types of N-glycans is a major result to correlate with the surface and foaming properties of wine, the solubility, allergenicity, and protease resistance of wine proteins.

Membrane trafficking pathways play critical roles in Apicomplexa, a phylum of protozoan parasites that cause life-threatening diseases worldwide. Here we report the first retromer-trafficking interactome in Toxoplasma gondii. This retromer complex includes a trimer Vps35–Vps26–Vps29 core complex that serves as a hub for the endosome-like compartment and parasite-specific proteins. Conditional ablation of TgVps35 reveals that the retromer complex is crucial for the biogenesis of secretory organelles and for maintaining parasite morphology. We identify TgHP12 as a parasite-specific and retromer-associated protein with functions unrelated to secretory organelle formation. Furthermore, the major facilitator superfamily homologue named TgHP03, which is a multiple spanning and ligand transmembrane transporter, is maintained at the parasite membrane by retromer-mediated endocytic recycling. Thus, our findings highlight that both evolutionarily conserved and unconventional proteins act in concert in T. gondii by controlling retrograde transport that is essential for parasite integrity and host infection.

International audience; Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles.TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.

The importance of hemoglobin A

Summary Microbial or endogenous molecular patterns as well as pathogen functional features can activate innate immune systems. Whereas detection of infection by pattern recognition receptors has been investigated in details, sensing of virulence factors activities remains less characterized. In Drosophila, genetic evidences indicate that the serine protease Persephone belongs to a danger pathway activated by abnormal proteolytic activities to induce Toll signaling. However, neither the activation mechanism of this pathway nor its specificity has been determined. Here, we identify a unique region in the pro-domain of Persephone that functions as bait for exogenous proteases independently of their origin, type, or specificity. Cleavage in this bait region constitutes the first step of a sequential activation and licenses the subsequent maturation of Persephone to the endogenous cysteine cathepsin 26-29-p. Our results establish Persephone itself as an immune receptor able to sense a broad range of microbes through virulence factor activities rather than molecular patterns., Graphical Abstract, Highlights • All pathogen-secreted proteases activate the danger-sensing arm of the Toll pathway • The protease Persephone is the immune sensor for microbial proteolytic activities • A sensitive region in Persephone zymogen functions as a bait for exogenous proteases • Bait-region hydrolysis primes maturation of Persephone by the host cathepsin 26-29-p, Innate immune systems are activated by microbial molecular patterns or pathogen functional features. Issa et al. show that the Drosophila Toll pathway senses pathogen proteases through a hydrolysis-sensitive region localized in the Persephone pro-domain. Cleavage of this bait region primes maturation of Persephone and activation of the pathway by the host cathepsin 26-29-p.

In the present study, we applied the combination of one-dimensional gel electrophoresis, immunoblot and nanoliquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) to identify potential immunogenic proteins of Toxoplasma gondii tachyzoites that can be used for the development of reliable assays in the serodiagnosis of acquired toxoplasmosis in immunocompetent subjects. For this purpose, we developed an immunoblot using soluble and membrane extracts of GT1 Toxoplasma gondii tachyzoites and tested 194 positive and 100 negative sera obtained from pregnant women. Five bands of soluble antigens (98 kDa, 36 kDa, 33 kDa, 32 kDa and 21 kDa) and 4 bands of membrane antigens (41 kDa, 35 kDa, 32 kDa and 30 kDa) were selected as the most valuable in terms of sensitivity and specificity. Among these bands, only 2 bands of soluble antigen (33 kDa and 32 kDa) and 2 bands of membrane antigen (32 kDa and 30 kDa) showed a specificity ≥ 90%. After mass spectrometry and bioinformatics analysis, 7 proteins were identified as potential markers for serodiagnosis of toxoplasmosis. These proteins are: SRS34A, GRA7, GRA1, DG32, MIC5, ROP5 and Toxofilin. These proteins showed a 86% to 100% homology with proteins of both VEG and ME49 strains of T. gondii and a 58% to 87% homology with Hammondia hammondi; and can be considered as attractive candidates for the development of an immunochromatography test that can be used for the rapid diagnosis of toxoplasmosis and as a confirmatory test when routine techniques give equivocal results.

Commitment to splicing occurs co-transcriptionally, but a major unanswered question is the extent to which various modifications of chromatin, the template for transcription in vivo, contribute to the regulation of splicing.Here, we perform genome-wide analyses showing that inhibition of specific marks - H2B ubiquitylation, H3K4 methylation and H3K36 methylation - perturbs splicing in budding yeast, with each modification exerting gene-specific effects. Furthermore, semi-quantitative mass spectrometry on purified nuclear mRNPs and chromatin immunoprecipitation analysis on intron-containing genes indicated that H2B ubiquitylation, but not Set1-, Set2- or Dot1-dependent H3 methylation, stimulates recruitment of the early splicing factors, namely U1 and U2 snRNPs, onto nascent RNAs.These results suggest that histone modifications impact splicing of distinct subsets of genes using distinct pathways.

Platelets are produced from mature megakaryocytes (MK) following a profound cellular reorganization. This includes the assembly of microtubules (MT) into a unique submenbranous coiled structure, the marginal band (MB). This process is thought to depend on a specific αβ-tubulin isotype repertoire. The MK-restricted-β1-tubulin, the predominant isoform of the MB, is already known to be important for platelet biogenesis but the implication of other isotypes is currently unknown. Our goal was to establish the αβ-tubulin repertoire in platelets and during megakaryopoiesis and to evaluate the implication of selected isotypes in platelet formation. To establish an exhaustive list of the tubulin isotypes, we used combination of RT PCR and proteomic analyses to quantify the expression of each isotype in human platelets and in human MK differentiated in culture from CD34+ hematopoietic progenitors. Information gained on the hierarchical combination of tubulin isoforms in the course of platelet biogenesis has been extended at the functional level to investigate both their role in marginal band formation and platelet functions β6-, β5- and α1c-tubulin transcripts were already present in CD34+ cells and decreased during the final stages of megakaryopoiesis. On the other hand, β1-, α4A- and α8-tubulin transcripts were only observed later during MK differentiation and in platelets. Quantitative LC-SRM mass spectrometry confirmed the predominant expression of β1 and α4A-isotypes in platelets. A functional role of the newly identified α4a-tubulin was supported by the thrombocytopenia and enlarged platelets with a decreased number of MT coils (1-3) comprising less-acetylated tubulin in mice carrying a point mutation in tuba4a. Additionally, a tendency to increased responses to several agonists was observed in these platelets. This study reveals new information on the evolution of the tubulin isotype repertoire in platelet formation pointing to a role of less-widely expressed α-isotypes. Disclosures No relevant conflicts of interest to declare.

Messinian marls from evaporitic cycle IV of the Gessoso-solfifera formation (Italy) are known to contain in high abundance the diaromatic carotenoid isorenieratene of green sulfur bacterial (Chlorobiaceae) origin along with a second diaromatic carotenoid. The previous tentative assignment of the latter as a cis diastereomer of isorenieratene has now been confirmed; this indicates that double bond isomerization occurs during early diagenesis and provides further evidence for the pathway proposed previously to link isorenieratene to a number of aromatic isoprenoid hydrocarbon biomarkers. Stable carbon isotopic data confirm the Chlorobiaceae origin of the intact all-trans isorenieratene isomer and indicate that the isomerization is accompanied by little or no kinetic isotope effect. Comparison of the δ13C values of each of the isolated carotenoids (measured as isorenieratane after hydrogenation) with those of their sulfide-bound counterpart in the polar fraction of the extracts (also measured as isorenieratane) indicates that sulfurization resulted in little depletion in 13C.

Stepwise chemical degradation involving cleavage of ester bonds (KOH/MeOH), sulfide and remaining ester bonds (Li/EtNH2), ether bonds (HI/LiAlH4) and sub-units linked to aromatic moieties (RuO4) has been carried out on the kerogens of two immature sulfur-rich marls (IV-1.4 and 1.8, TOC ca. 1.5%) from evaporitic cycle IV of the Gesosso-solfifera Formation (Messinian, Vena del Gesso, Italy). Up to 80% of the organic matter was converted to solvent-soluble material, with the greatest proportion released by Li/EtNH2. The majority by far of the extracts comprises polar macromolecular material which is thought to correspond to high molecular weight sub-units of the kerogen. Quantification of the small amounts (

In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

In the field of therapeutic recombinant proteins, monoclonal antibodies (mAbs) have achieved a rising success with more than 30 mAbs that have reached the market in the past 20 years. From a structural standpoint, one of the most important posttranslational modifications affecting antibodies is by far glycosylation. Furthermore, glycosylation of mAbs directly impacts on their biological activity and safety and therefore needs to be well characterized. Glycoprotein analysis requires high-resolution separation techniques that can provide detailed structural analysis able to discriminate between glycoforms of various abundances. This chapter describes a protocol for nanoLC-Chip-MS/MS analysis of a proteolytic digest of the heavy chain of a recombinant mAb. The use of graphitized carbon column instead of classical C18 reversed-phase material is shown to be well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.

In the field of therapeutic recombinant proteins, monoclonal antibodies (mAbs) have achieved a rising success with more than 30 mAbs that have reached the market in the past 20 years. From a structural standpoint, one of the most important posttranslational modifications affecting antibodies is by far glycosylation. Furthermore, glycosylation of mAbs directly impacts on their biological activity and safety and therefore needs to be well characterized. Glycoprotein analysis requires high-resolution separation techniques that can provide detailed structural analysis able to discriminate between glycoforms of various abundances. This chapter describes a protocol for nanoLC-Chip-MS/MS analysis of a proteolytic digest of the heavy chain of a recombinant mAb. The use of graphitized carbon column instead of classical C18 reversed-phase material is shown to be well suited to detect low abundant glycoforms and to provide in one shot information regarding both the oligosaccharide structure and the amino acid sequence of its peptide moiety.

Proteomic analysis of the stigmatic exudate of Lilium longiflorum and Olea europaea led to the identification of 51 and 57 proteins, respectively, most of which are described for the first time in this secreted fluid. These results indicate that the stigmatic exudate is an extracellular environment metabolically active, participating in at least 80 different biological processes and 97 molecular functions. The stigma exudate showed a markedly catabolic profile and appeared to possess the enzyme machinery necessary to degrade large polysaccharides and lipids secreted by papillae to smaller units, allowing their incorporation into the pollen tube during pollination. It may also regulate pollen-tube growth in the pistil through the selective degradation of tube-wall components. Furthermore, some secreted proteins were involved in pollen-tube adhesion and orientation, as well as in programmed cell death of the papillae cells in response to either compatible pollination or incompatible pollen rejection. Finally, the results also revealed a putative cross-talk between genetic programmes regulating stress/defence and pollination responses in the stigma., This work was supported by the Spanish Ministry of Science and Innovation (ERDF-cofinanced projects AGL2008-00517 and PIE-200840I186) and Junta de Andalucía (ERDF-cofinanced project P2010-CVI5767). This work was also funded by the CNRS, the ‘Agence National de la Recherche’ (ANR) and the ‘Region Alsace’. JDR thanks the MICIIN for providing FPU grant funding.

International audience; Gene regulation in apicomplexan parasites, a phylum containing important protozoan parasites such as Plasmodium and Toxoplasma, is poorly understood. The life cycle of T. gondii is complex, with multiple proliferation and differentiation steps of which tachyzoite proliferation is the most relevant to pathogenesis in humans and animals. Tachyzoites express invasion and virulence factors that are crucial for their survival and manipulation of host cell functions. The expression of those factors is tightly controlled during the tachyzoite cell cycle to permit their correct packaging in newly formed apical secretory organelles named micronemes and rhoptries in the daughter cells. However, little is known about the factors that control the expression of genes encoding the virulence factors present in these parasite-specific secretory organelles. We report that the plant-like nuclear factor TgAP2XI-5 targets more than 300 gene promoters and actively controls the transcription of these genes. Most of these target genes, including those that are essential for parasite virulence, showed a peak of expression in S and M phases of the cell cycle. Furthermore, we identified the cis-regulatory element recognized by TgAP2XI-5 and demonstrated its ability to actively drive gene transcription. Our results demonstrated that TgAP2XI-5 is a novel DNA sequence-specific transcription factor associated with promoter activation. TgAP2XI-5 may regulate gene transcription of crucial virulence factors in T. gondii.

Apicomplexan parasites are responsible for some of the most deadly parasitic diseases affecting humans and livestock. There is an urgent need for new medicines that will target apicomplexan-specific pathways. We characterized a Toxoplasma gondii C2H2 zinc finger protein, named TgZNF2, which is conserved among eukaryotes. We constructed an inducible KO strain (iKO-TgZNF2) for this gene where the tgznf2 gene expression is repressed in the presence of a tetracycline analog (ATc). We showed that the iKO-TgZNF2 parasites are unable to proliferate after depletion of the TgZNF2 protein. Complementation with a full length copy of the gene restores the phenotype Moreover, the homolog of this protein in the related apicomplexan Plasmodium falciparum was shown to efficiently rescue the phenotype, suggesting that this pathway is likely conserved among apicomplexan parasites. We demonstrated that the iKO-mutant lacking TgZNF2 are arrested during the cell cycle during the G1 phase. We identified potential protein partners of this protein among which are spliceosomal complex and mRNA nuclear export components. We confirmed that TgZNF2 is able to bind in vivo to transcripts but splicing is not perturbed in the ATc-treated parasites. Instead, we demonstrated that TgZNF2 depletion leads to the sequestration of polyA+ mRNAs in the nucleus while ribosomal RNAs are not affected. We discovered a conserved protein with specific apicomplexan functional properties that is essential for the survival of T. gondii. TgZNF2 may be crucial to ensure the correct polyA+ mRNA nuclear export, a function that is conserved in P. falciparum.