Secretory Leukocyte Protease Inhibitor (SLPI) Is, like Its Homologue Trappin-2 (Pre-Elafin), a Transglutaminase Substrate

Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases

Steroid hormones content and proteomic analysis of canine follicular fluid during the preovulatory period

<p>Abstract</p> <p>Background</p> <p>Follicular fluid contains substances involved in follicle activity, cell differentiation and oocyte maturation. Studies of its components may contribute to better understanding of the mechanisms underlying follicular development and oocyte quality. The canine species is characterized by several ovarian activity features that are not extensively described such as preovulatory luteinization, oocyte ovulated at the GV stage (prophase 1) and poly-oocytic follicles. In this study, we examined the hypothesis that the preovulatory LH surge is associated with changes in steroid and protein content of canine follicular fluid prior to ovulation.</p> <p>Methods</p> <p>Follicular fluid samples were collected from canine ovaries during the preovulatory phase, before (pre-LH; n = 16 bitches) and after (post-LH; n = 16) the LH surge. Blood was simultaneously collected. Steroids were assayed by radioimmunoassay and proteomic analyses were carried out by 2D-PAGE and mass spectrometry.</p> <p>Results</p> <p>The concentrations of 17beta-estradiol and progesterone at the pre-LH stage were 737.2 +/- 43.5 ng/ml and 2630.1 +/- 287.2 ng/ml in follicular fluid vs. 53 +/- 4.1 pg/ml and 3.9 +/- 0.3 ng/ml in plasma, respectively. At that stage, significant positive correlations between follicular size and intra-follicular steroid concentrations were recorded. After the LH peak, the intrafollicular concentration of 17beta-estradiol decreased significantly (48.3 +/- 4.4 ng/ml; p < 0.001), whereas that of progesterone increased (11690.2 +/- 693.6 ng/ml; p < 0.001). Plasmatic concentration of 17beta-estradiol was not modified (49 +/- 9.6 pg/ml) after the LH peak, but that of progesterone significantly increased (9.8 +/- 0.63 ng/ml).</p> <p>Proteomic analysis of canine follicular fluid identified 38 protein spots, corresponding to 21 proteins, some of which are known to play roles in the ovarian physiology. The comparison of 2D-PAGE patterns of follicular fluids from the pre- and post-LH stages demonstrated 3 differentially stained single spot or groups of spots. One of them was identified as complement factor B. A comparison of follicular fluid and plasma protein patterns demonstrated a group of 4 spots that were more concentrated in plasma than in follicular fluid, and a single spot specific to follicular fluid. These proteins were identified as gelsolin and clusterin, respectively.</p> <p>Conclusion</p> <p>Our results provide the first demonstration of size-related changes in the steroid concentrations in canine follicular fluid associated with the LH surge. 2D protein mapping allowed identification of several proteins that may play a role in follicle physiology and ovarian activity at the preovulatory stage. This may help in the future to explain and to better understand the species specificities that are described in dogs.</p

2.5D Representations Combining in vivo 3D MRI and ex vivo 2D MSI Approaches to Study the Lipid Distribution in the Whole Sheep Brain

National audienceMass Spectrometry Imaging (MSI) provides easily high spatially resolved masses allowing characterization of endogenous lipids. These latter constitute about 70% of the composition of the white matter of the brain which can be implicated in developmental and/or cognitive troubles. In order to examine the molecular distribution of lipids in whole sheep brain, and especially in white/grey matter, we combined in vivo and ex vivo images, obtained in the same animals, using Magnetic Resonance Imaging (MRI) and MSI, respectively. In order to view the topology of the molecular species within the organ, we propose the construction of a 2.5 D representation where a single section imaged with 2D MSI is localized within the tissue volume obtained by 3D MRI. 3D T1-weighted MPRAGE images were acquired on two anesthetized sheep with a 3 Tesla MRI (Siemens, Verio ®). The parameters of acquisition for the MPRAGE were: TR 2500ms, TE 3.2ms, FA 12, NEX 1, matrix 384×384, FOV 192mm, 288 slices with a thickness of 0.5mm. In order to improve data quality, the 3D MRI volumes have been pre-processed using in-house algorithms using volume fitting and Markov random field methods. T1 3D planes corresponding to MSI planes were reconstructed using Osirix imaging software.Brains were collected after sacrifice and frozen at -80°C. Frontal and sagittal 14 µm brain sections were performed with a cryostat adapted to large sections (CM3050 S, Leica) and mounted onto conductive ITO-coated slides. The spray of α-cyano-4-hydroxycinnamic acid matrix was performed using an Image Prep device (Bruker). Spectra were acquired using an UltrafleXtrem MALDI-TOF instrument (Bruker) in the 200–1200 m/z range with a spatial resolution set at 125 µm. Raw spectra were analyzed with SCiLS Lab software to generate 2D ion density maps and segmentation maps (data partitioning). The tissue sections analyzed by MSI were stained with cresyl violet to manually delimitate neuronal nuclei and areas. This histological map was used to delineate the MRI and MSI 2D views and overlay them regardless the same brain areas used as fiducials. After, a 2.5 D representation was proposed to visualize the lipid distribution within the entire organ.In conclusion, in this study, frontal and sagittal whole sheep brain sections analyzed by MSI showed a clear difference in lipid distribution between different compartments of brain tissues, especially between grey and white matter, until the cerebral envelopment presenting circumvolution. Furthermore, the alignment of 2D MALDI-imaging with T1-weighted images showed that MSI can provide finer details on the structural connectivity of myelinated fiber tracts. Here, the 2.5 D representation combining MRI and MSI was presented as an alternative approach to 3D anatomical and molecular atlas providing a perfect topology of the molecular species within an organ. For the moment, 3D MSI of whole sheep brain is a challenge, while the 2.5 D construction demonstrated to be a capable tool for exploring molecular distributions throughout sample volumes.Nowadays, the reported results may serve as a starting point for further experiments associating MSI and dynamic and functional MRI, especially for the characterization of brain

Polyadenylation of a functional mRNA controls gene expression in Escherichia coli

Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine–6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5′ monophosphate and a 3′ stable hairpin is highly susceptible to poly(A)-dependent degradation

Data set for the proteomic inventory and quantitative analysis of chicken eggshell matrix proteins during the primary events of eggshell mineralization and the active growth phase of calcification

This research was funded by the French National Research Agency ANR (ANR-13-BSV6-0007-01, ANK-13-BSV6-0007-02 and ANK-13-BSV6-0007-05). The high resolution mass spectrometer was financed (SMHART project, 35069) by the European Regional Development Fund (ERDF), the Conseil Regional du Centre, the French National Institute for Agricultural Research (INRA) and the French National Institute of Health and Medical Research (Inserm), ARN acknowledges funding through Grants CGL2011-25906 (Ministerio de Economia, Spain).Chicken eggshell is a biomineral composed of 95% calcite calcium carbonate mineral and of 3.5% organic matrix proteins. The assembly of mineral and its structural organization is controlled by its organic matrix. In a recent study [1], we have used quantitative proteomic, bioinformatic and functional analyses to explore the distribution of 216 eggshell matrix proteins at four key stages of shell mineralization defined as: (1) widespread deposition of amorphous calcium carbonate (ACC), (2) ACC transformation into crystalline calcite aggregates, (3) formation of larger calcite crystal units and (4) rapid growth of calcite as columnar structure with preferential crystal orientation. The current article detailed the quantitative analysis performed at the four stages of shell mineralization to determine the proteins which are the most abundant. Additionally, we reported the enriched GO terms and described the presence of 35 antimicrobial proteins equally distributed at all stages to keep the egg free of bacteria and of 81 proteins, the function of which could not be ascribed.French National Research Agency (ANR) ANR-13-BSV6-0007-01 ANK-13-BSV6-0007-02 ANK-13-BSV6-0007-05European Union (EU) 35069Region Centre-Val de LoireFrench National Institute for Agricultural Research (INRA)Institut National de la Sante et de la Recherche Medicale (Inserm)Ministerio de Economia, Spain CGL2011-2590

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

International audienceBACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays

Protein expression reveals a molecular sexual identity of avian primordial germ cells at pre-gonadal stages

International audienceIn poultry, in vitro propagated primordial germ cells (PGCs) represent an important tool for the cryopreservation of avian genetic resources. However, several studies have highlighted sexual differences exhibited by PGCs during in vitro propagation, which may compromise their reproductive capacities. To understand this phenomenon, we compared the proteome of pregonadal migratory male (ZZ) and female (ZW) chicken PGCs propagated in vitro by quantitative proteomic analysis using a GeLC-MS/MS strategy. Many proteins were found to be differentially abundant in chicken male and female PGCs indicating their early sexual identity. Many of the proteins more highly expressed in male PGCs were encoded by genes localised to the Z sex chromosome. This suggests that the known lack of dosage compensation of the transcription of Z-linked genes between sexes persists at the protein level in PGCs, and that this may be a key factor of their autonomous sex differentiation. We also found that globally, protein differences do not closely correlate with transcript differences indicating a selective translational mechanism in PGCs. Male and female PGC expressed protein sets were associated with differential biological processes and contained proteins known to be biologically relevant for male and female germ cell development, respectively. We also discovered that female PGCs have a higher capacity to uptake proteins from the cell culture medium than male PGCs. This study presents the first evidence of an early predetermined sex specific cell fate of chicken PGCs and their sexual molecular specificities which will enable the development of more precise sex-specific in vitro culture conditions for the preservation of avian genetic resources

Phénotypage cellulaire et imagerie tissulaire pour la découverte de biomarqueurs : 2 approches originales au service de la protéomique et de la lipidomique

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Une nouvelle ère pour la protéomique

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