Stéphane Claverol

Proteomics of Plant Detergent-resistant Membranes

A large body of evidence from the past decade supports the existence, in membrane from animal and yeast cells, of functional microdomains that play important roles in protein sorting, signal transduction, or infection by pathogens. Recent reports demonstrated the presence, in plants, of detergent-resistant fractions isolated from plasma membrane. Analysis of the lipidic composition of this fraction revealed its enrichment in sphingolipids and sterols and depletion in phospho- and glycerolipids as previously observed for animal microdomains. One-dimensional gel electrophoresis experiments indicated that these detergent-resistant fractions are able to recruit a specific set of plasma membrane proteins and exclude others. In the present study, we used mass spectrometry to give an extensive description of a tobacco plasma membrane fraction resistant to solubilization with Triton X-100. This led to the identification of 145 proteins whose functional and physicochemical characteristics were analyzed in silico. Parameters such as isoelectric point, molecular weight, number and length of transmembrane segments, or global hydrophobicity were analyzed and compared with the data available concerning plant plasma membrane proteins. Post-translational modifications, such as myristoylation, palmitoylation, or presence of a glycosylphosphatidylinositol anchor, were examined in relation to the presence of the corresponding proteins in these microdomains. From a functional point of view, this analysis indicated that if a primary function of the plasma membrane, such as transport, seems under-represented in the detergent-resistant fraction, others undergo a significant increase of their relative importance. Among these are signaling and response to biotic and abiotic stress, cellular trafficking, and cell wall metabolism. This suggests that these domains are likely to constitute, as in animal cells, signaling platforms involved in these physiological functions.

The Production of a New MAGE-3 Peptide Presented to Cytolytic T Lymphocytes by HLA-B40 Requires the Immunoproteasome

By stimulating human CD8+ T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3–expressing tumor cells only when they were first treated with IFN-γ. Since this treatment is known to induce the exchange of the three catalytic subunits of the proteasome to form the immunoproteasome, this result suggested that the processing of this MAGE-3 peptide required the immunoproteasome. Transfection experiments showed that the substitution of β5i (LMP7) for β5 is necessary and sufficient for producing the peptide, whereas a mutated form of β5i (LMP7) lacking the catalytically active site was ineffective. Mass spectrometric analyses of in vitro digestions of a long precursor peptide with either proteasome type showed that the immunoproteasome produced the antigenic peptide more efficiently, whereas the standard proteasome more efficiently introduced cleavages destroying the antigenic peptide. This is the first example of a tumor-specific antigen exclusively presented by tumor cells expressing the immunoproteasome.

Destructive cleavage of antigenic peptides either by the immunoproteasome or by the standard proteasome results in differential antigen presentation.

The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100209–217 (ITDQVPSFV) and peptide tyrosinase369–377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2336–344 (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.

Mapping and Structural Dissection of Human 20 S Proteasome Using Proteomic Approaches

The proteasome, a proteolytic complex present in all eukaryotic cells, is part of the ATP-dependent ubiquitin/proteasome pathway. It plays a critical role in the regulation of many physiological processes. The 20 S proteasome, the catalytic core of the 26 S proteasome, is made of four stacked rings of seven subunits each (α7β7β7α7). Here we studied the human 20 S proteasome using proteomics. This led to the establishment of a fine subunit reference map and to the identification of post-translational modifications. We found that the human 20 S proteasome, purified from erythrocytes, exhibited a high degree of structural heterogeneity, characterized by the presence of multiple isoforms for most of the α and β subunits, including the catalytic ones, resulting in a total of at least 32 visible spots after Coomassie Blue staining. The different isoforms of a given subunit displayed shifted pI values, suggesting that they likely resulted from post-translational modifications. We then took advantage of the efficiency of complementary mass spectrometric approaches to investigate further these protein modifications at the structural level. In particular, we focused our efforts on the α7 subunit and characterized its N-acetylation and its phosphorylation site localized on Ser250.

Characterization of Protein Variants and Post-Translational Modifications: ESI-MSn Analyses of Intact Proteins Eluted from Polyacrylamide Gels

We have developed a strategy to characterize protein isoforms, resulting from single-point mutations and post-translational modifications. This strategy is based on polyacrylamide gel electrophoresis separation of protein isoforms, mass spectrometry (MS) and MSn analyses of intact proteins, and tandem MS analyses of proteolytic peptides. We extracted protein isoforms from polyacrylamide gels by passive elution using SDS, followed by nanoscale hydrophilic phase chromatography for SDS removal. We performed electrospray ionization MS analyses of the intact proteins to determine their molecular mass, allowing us to draw hypotheses on the nature of the modification. In the case of labile post-translational modifications, like phosphorylations and glycosylations, we conducted electrospray ionization MSn analyses of the intact proteins to confirm their presence. Finally, after digestion of the proteins in solution, we performed tandem MS analyses of the modified peptides to locate the modifications. Using this strategy, we have determined the molecular mass of 5–10 pmol of a protein up to circa 50 kDa loaded on a gel with a 0.01% mass accuracy. The efficiency of this approach for the characterization of protein variants and post-translational modifications is illustrated with the study of a mixture of κ-casein isoforms, for which we were able to identify the two major variants and their phosphorylation site and glycosylation motif. We believe that this strategy, which combines two-dimensional gel electrophoresis and mass spectrometric analyses of gel-eluted intact proteins using a benchtop ion trap mass spectrometer, represents a promising approach in proteomics.

Two-dimensional Blue Native/SDS Gel Electrophoresis of Multiprotein Complexes from Helicobacter pylori

The study of protein interactions constitutes an important domain to understand the physiology and pathogenesis of microorganisms. The two-dimensional blue native/SDS-PAGE was initially reported to analyze membrane protein complexes. In this study, both cytoplasmic and membrane complexes of a bacterium, the strain J99 of the gastric pathogen Helicobacter pylori, were analyzed by this method. It was possible to identify 34 different proteins grouped in 13 multiprotein complexes, 11 from the cytoplasm and two from the membrane, either previously reported partially or totally in the literature. Besides complexes involved in H. pylori physiology, this method allowed the description of interactions involving known pathogenic factors such as (i) urease with the heat shock protein GroEL or with the putative ketol-acid reductoisomerase IlvC and (ii) the cag pathogenicity island CagA protein with the DNA gyrase GyrA as well as insight on the partners of TsaA, a peroxide reductase/stress-dependent molecular chaperone. The two-dimensional blue native/SDS-PAGE combined with mass spectrometry is a potential tool to study the differences in complexes isolated in various situations and also to study the interactions between bacterial and eucaryotic cell proteins.

Analysis of the Flavobacterium psychrophilum outer-membrane subproteome and identification of new antigenic targets for vaccine by immunomics

Flavobacterium psychrophilum is an important infectious Gram-negative bacterium causing cold-water disease (CWD) and rainbow trout fry syndrome. Outer-membrane proteins (OMPs) are key molecules with regard to the interface between the cell and its environment. Therefore, we sought to define the outer-membrane (OM) subproteome of F. psychrophilum in order to gain insight into the biology and pathogenesis of this bacterium and to identify the dominant antigens targeted by the rainbow trout (Oncorhynchus mykiss) immune system during infection. First, OMs were prepared from a cell-envelope suspension by differential Sarkosyl (sodium lauryl sarcosinate) solubility. We then isolated the OMPs and identified 36 proteins from 34 spots resolved by two-dimensional electrophoresis and LC-MS/MS. An immunoproteomic approach using antibodies from CWD-convalescent rainbow trout was then used to identify 25 immunoreactive F. psychrophilum antigens that may be relevant in pathogenesis and diagnosis. These included the previously characterized surface-exposed OMPs OmpA, OmpH/P18 and FspA, as well as newly described antigenic proteins. This study provides a number of novel candidate proteins for developing vaccine(s) against flavobacteriosis infection in aquaculture.

Proteomic analysis of an imatinib-resistant K562 cell line highlights opposing roles of heat shock cognate 70 and heat shock 70 proteins in resistance.

Understanding the molecular basis of resistance to imatinib, a tyrosine kinase inhibitor used as front‐line therapy in chronic myeloid leukemia, remains a challenge for successful treatment. In an attempt to identify new mechanisms of resistance, we performed a comparative proteomic analysis of an imatinib‐resistant cell line generated from the erythroblastic cell line K562 (K562‐r) for which no known mechanism of resistance has been detected. Bidimensional gel electrophoresis was carried out to compare the protein expression pattern of imatinib‐sensitive and of imatinib‐resistant K562 cells. Among the 400 matched spots on five pairs of gels, only 14 spots had a significantly increased or decreased expression leading to the identification of 24 proteins identified as scaffold proteins, metabolic enzymes, DNA translation and maturation, and chaperon proteins. Among the chaperon family, only Hsp70 and Hsc70 are overexpressed in K562‐r, results confirmed by Western blotting. We recently reported the participation of Hsp70 overexpression in imatinib resistance whereas a role for Hsc70 has yet to be determined. Hsc70 is not involved in imatinib resistance as the inhibition of its expression by siRNA does not restore sensitivity to imatinib. In contrast, the induced decreased expression of Hsc70 was accompanied by a greater overexpression of Hsp70. This proteomic study therefore suggests opposing roles of Hsp70 and Hsc70 in imatinib resistance.

Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography

Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe³⁺ and TiO₂ (respectively IMAC and MOAC media), using a simple mixture of peptides from casein-albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe³⁺ and TiO₂ were pH dependant. In the case of liver extract, selectivity increased from 12-18% to 58-60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe³⁺ column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe³⁺ was associated with strong Fe³⁺ leaching. Furthermore, repetitive use of IMAC-Fe³⁺ with the 0.5 M NH₄OH solution required for phosphopeptide elution induced Fe₂O₃ accumulation in the column. By comparison, MOAC columns packed with TiO₂ support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole‐genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm‐specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.