Christine Schaeffer

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named ‘Candidatus Fodinabacter communificans.’ Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase–PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.

Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy

Ubiquitin‐dependent processes can be antagonized by substrate‐specific deubiquitination enzymes involved in many cellular functions. In this study, we show that the yeast Ubp3–Bre5 deubiquitination complex interacts with both the chaperone‐like Cdc48, a major actor of the ubiquitin and proteasome system, and Ufd3, a ubiquitin‐binding cofactor of Cdc48. We observed that these partners are required for the Ubp3–Bre5‐dependent and starvation‐induced selective degradation of yeast mature ribosomes, also called ribophagy. By contrast, proteasome‐dependent degradation does not participate in this process. Our data favour the idea that these factors cooperate to recognize and deubiquitinate specific substrates of ribophagy before their vacuolar degradation.

The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies.

Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs. First a top-down approach was used on intact and reduced MAbs by liquid chromatography coupled to an electrospray ionization-time of flight mass spectrometer (LC-ESI-TOF), which provided fast and accurate profiles of MAbs glycosylation patterns for routine controls. Secondly, after digestion of the antibody with the peptide N-glycosidase F (PNGase F) enzyme, released N-linked glycans were directly analyzed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) without any prior derivatization, which gave precise details on the structure of the most abundant glycoforms. Finally, a bottom-up approach on tryptic glycopeptides using a nanoLC-Chip-MS/MS ion trap (IT) system equipped with a graphitized carbon column was investigated. Data were compared to those obtained with a more classical C18 reversed phase column showing that this last method is 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 Approach To Identify Champagne Wine Proteins as Modified by Botrytis cinerea Infection

The presence of the fungal pathogen, Botrytis cinerea, in the vineyard causes reductions in both quality and quantity of grapes and wine. Because proteins are involved in the foam stabilization of sparkling wines, we have undertaken, for the first time, a thorough proteomic analysis of two champagne base wines prepared with either healthy or botrytized Chardonnay grapes, using two-dimensional electrophoresis (2DE) coupled with immunodetection and tandem mass spectrometry. Most of the identified proteins were from grape origin: invertase and pathogenesis-related (PR) proteins. The disappearance of numerous grape proteins was observed in the botrytized wine, suggesting that they were probably degraded or even repressed or the result of a differential expression of grape proteins upon fungal infection. On the other hand, two pectinolytic enzymes secreted by B. cinerea were found in the botrytized wine.

Proteomics and Glycomics Analyses of N-Glycosylated Structures Involved in Toxoplasma gondii-Host Cell Interactions

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man5–8(GlcNAc)2) and paucimannosidic (Man3–4(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Study of Anoxic and Oxic Cholesterol Metabolism by Sterolibacterium denitrificans

The initial enzymes and genes involved in the anoxic metabolism of cholesterol were studied in the denitrifying bacterium Sterolibacterium denitrificans Chol-1S(T). The second enzyme of the proposed pathway, cholest-4-en-3-one-Delta1-dehydrogenase (AcmB), was partially purified. Based on amino acid sequence analysis, a gene probe was derived to screen a cosmid library of chromosomal DNA for the acmB gene. A positive clone comprising a 43-kbp DNA insert was sequenced. In addition to the acmB gene, the DNA fragment harbored the acmA gene, which encodes the first enzyme of the pathway, cholesterol dehydrogenase/isomerase. The acmA gene was overexpressed, and the recombinant dehydrogenase/isomerase was purified. This enzyme catalyzes the predicted transformation of cholesterol to cholest-4-en-3-one. S. denitrificans cells grown aerobically with cholesterol exhibited the same pattern of soluble proteins and cell extracts formed the same 14C-labeled products from [14C]cholesterol as cells that were grown under anoxic, denitrifying conditions. This is especially remarkable for the late products that are formed by anaerobic hydroxylation of the cholesterol side chain with water as the oxygen donor. Hence, this facultative anaerobic bacterium may use the anoxic pathway lacking any oxygenase-dependent reaction also under oxic conditions. This confers metabolic flexibility to such facultative anaerobic bacteria.

The nuclear hormone receptor PPARγ counteracts vascular calcification by inhibiting Wnt5a signalling in vascular smooth muscle cells

Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.

Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells.

Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2‐D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))‐EGC and (11.5 dpc)‐EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)‐EGC, and 36 (11.5 dpc)‐EGC spots were identified by MALDI‐TOF‐MS and/or nano‐LC‐MS/MS. This approach led to the identification of two isoforms (with and without N‐terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2‐D gel‐MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N‐methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

Tandem use of X-ray crystallography and mass spectrometry to obtain ab initio the complete and exact amino acids sequence of HPBP, a human 38-kDa apolipoprotein

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N‐terminal residues, are systematically absent from eukaryotic genome databases. As a consequence, HPBP amino acids sequence had to be first assigned from the electronic density map. Then, an original approach combining X‐ray crystallography and mass spectrometry provides the complete and a priori exact sequence of the 38‐kDa HPBP. This first complete sequence of a eukaryotic DING protein will be helpful to study HPBP and the entire DING protein family. Proteins 2008.

Taxonomic and functional prokaryote diversity in mildly arsenic-contaminated sediments

Arsenic-resistant prokaryote diversity is far from being exhaustively explored. In this study, the arsenic-adapted prokaryotic community present in a moderately arsenic-contaminated site near Sainte-Marie-aux-Mines (France) was characterized, using metaproteomic and 16S rRNA-encoding gene amplification. High prokaryotic diversity was observed, with a majority of Proteobacteria, Acidobacteria and Bacteroidetes, and a large archaeal community comprising Euryarchaeaota and Thaumarchaeota. Metaproteomic analysis revealed that Proteobacteria, Planctomycetes and Cyanobacteria are among the active bacteria in this ecosystem. Taken together, these results highlight the unsuspected high diversity of the arsenic-adapted prokaryotic community, with some phyla never having been described in highly arsenic-exposed sites.