Anne-Marie Lomenech

Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins.

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.

Leaf proteome analysis of eight Populus × euramericana genotypes: genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins.

Genetic variation of leaf proteome in drought response was investigated among eight Populus ×euramericana genotypes contrasting for their leaf carbon isotope discrimination (Δ), an estimate of intrinsic water‐use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86‐day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2‐DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast‐associated proteins either involved in the Calvin cycle or in the electron‐transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Δ variations were found for 45 reliably identified proteins. The abundance of ribulose‐1,5‐bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Δ regardless of the treatment, suggesting that a large intrinsic water‐use efficiency could be due to higher abundance of ribulose‐1,5‐bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Δ, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Δ.

Hunting down fungal secretomes using liquid-phase IEF prior to high resolution 2-DE.

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1‐DE and 2‐DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2‐DE. Compared with the previously published protocols for which only dozens of 2‐D spots were recovered from fungal secretome samples, up to approximately 2000 2‐D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, β‐glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid‐phase IEF to any fungal extracts.

Sensing of cell stress by human γδ TCR-dependent recognition of annexin A2

Significance Human γδ T lymphocytes have innate-like and adaptive-like functions and can circulate in blood or reside in tissues. They are activated by specific antigens recognized by their T-cell receptor and recognize infected and transformed cells, suggesting that cellular stress is involved in specific antigen expression. However, molecular characterization of stress-induced antigens remains elusive, hampering our understanding of the role of γδ T cells in cancer and infections. In the present study we identify annexin A2 as such stress-induced antigen known as a phospholipid-binding protein involved in tumorigenesis, redox potential regulation, and wound healing. Stress-mediated membrane exposure of annexin A2 could thus constitute a danger signal for γδ T cells to recognize various cell dysregulations and protect the host against cancer and infections. Human γδ T cells comprise a first line of defense through T-cell receptor (TCR) recognition of stressed cells. However, the molecular determinants and stress pathways involved in this recognition are largely unknown. Here we show that exposure of tumor cells to various stress situations led to tumor cell recognition by a Vγ8Vδ3 TCR. Using a strategy that we previously developed to identify antigenic ligands of γδ TCRs, annexin A2 was identified as the direct ligand of Vγ8Vδ3 TCR, and was found to be expressed on tumor cells upon the stress situations tested in a reactive oxygen species-dependent manner. Moreover, purified annexin A2 was able to stimulate the proliferation of a Vδ2neg γδ T-cell subset within peripheral blood mononuclear cells and other annexin A2-specific Vδ2neg γδ T-cell clones could be derived from peripheral blood mononuclear cells. We thus propose membrane exposure of annexin A2 as an oxidative stress signal for some Vδ2neg γδ T cells that could be involved in an adaptive stress surveillance.

Impact of foliar symptoms of "Esca proper" on proteins related to defense and oxidative stress of grape skins during ripening.

Esca is one of the major diseases affecting vineyards with direct impact on product yield; nevertheless, scientific studies concerning its impact on grape quality are scarce. As an attempt to better understand the mechanisms behind “Esca proper” development in grapes, this work focused on the identification of proteins whose expression is altered by the disease. 2‐DEs were performed on protein extracts from grape skins at different stages of maturity for two consecutive vintages. Grapes were collected in 2009 and in 2010 from plants that did not present signs of infection by Esca proper since the 2004 vintage and from plants that presented cast leaf symptoms at least once since 2004. For the first time, 13 proteins were shown to be influenced by Esca proper during the ripening process. Extensive bioinformatics analysis allowed the grouping of proteins involved in (i) stress tolerance and defense response, (ii) oxidative phosphorylation, (iii) oxidation–reduction processes in mitochondria, and (iv) oxidation–reduction processes in chloroplasts. Of these 13 proteins, cysteine synthase is the only one implicated in a metabolic pathway of oenological interest. This study shows how foliar symptoms of Esca proper may impact stress‐related pathways in grapes, which are characterized by modifications in the chain of oxidative phosphorylation and redox scavenging.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface.