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ABSTRACT: Many physiological processes are regulated by dynamic protein interaction networks whose characterization provides valuable information on cell biology. Several strategies can be used to analyze protein-protein interactions. Among them, affinity purification combined with mass spectrometry (AP-MS) is arguably the most widely employed technique, not only owing to its high throughput and sensitivity but also because it can answer critical questions such as where, when, and how protein-protein interactions occur. In AP-MS workflows, both the target protein and its interacting partners are isolated before being identified by MS. The main challenge of this approach is to distinguish bona fide binders from background contaminants. This review focuses on the different strategies designed to circumvent this limitation. In this regard, the combination of quantitative proteomics and affinity purification emerges as one of the most powerful, yet relatively simple, strategies to characterize protein-protein interactions. © IUBMB Life, 65(1):9-16, 2013.
International Union of Biochemistry and Molecular Biology Life 01/2013; 65(1):9-16. · 3.51 Impact Factor
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ABSTRACT: SILAC is a widely accepted approach for quantitative proteomics in which proteins are labeled with stable isotopes during cell culture. A major drawback of this technique is the metabolic conversion of labeled amino acids that may hamper accurate quantification. A paradigmatic example of this phenomenon is the generation of labeled proline from arginine, known to occur in a good number of biological models. We propose a novel methodology to identify and quantitate metabolic conversions as well as to evaluate labeling efficiency in SILAC experiments. In this approach, labeled proteins are reduced to amino acids by acid hydrolysis before LC-MS/MS analysis. Since it is carried out at the amino acid level, tracking the fate of the isotope label is straightforward and can be performed for each amino acid independently. After applying this method to mammalian cells, grown in the presence of heavy arginine and lysine, labeling efficiency and amino acid conversions could be accurately evaluated. Only undesirable labeling of proline was found to occur at a significant extent, varying greatly among cell lines. Finally, increasing proline concentration in the growing medium was shown to be effective at preventing arginine conversion without any noticeable side effect.
Talanta 04/2011; 84(2):430-6. · 3.79 Impact Factor
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ABSTRACT: An evaluation of the ICPL (isotope-coded protein labeling) non-isobaric labeling technique was performed using two different biological models. Two samples containing phage T4 capsids were mixed in a 1:1 ratio after being labeled with the light or heavy versions of the ICPL reagent. The analysis of this proteome demonstrated the feasibility of this approach for differential quantitative proteomics and was employed to optimize the experimental parameters of the ICPL workflow. ICPL-mediated analysis of two more complex proteomes, those of a Salmonella enterica serovar Typhimurium virulent strain and an isogenic attenuated mutant, and its comparison with the results obtained in a 2D-PAGE "classical" approach confirmed that ICPL is a valuable alternative to other labeling techniques currently in use. In addition, our results suggest that labeling at the peptide level instead of following the standard ICPL workflow should increase both the number of proteins quantified and the reliability of the quantification.
Talanta 02/2010; 80(4):1496-502. · 3.79 Impact Factor
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ABSTRACT: A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of tripeptidyl peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.
European Journal of Immunology 04/2008; 38(3):631-9. · 5.10 Impact Factor
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ABSTRACT: Human leucocyte antigen (HLA)-B27 is strongly associated with spondyloarthropathies, including reactive arthritis. Several Gram-negative bacteria, such as Salmonella typhimurium, can trigger this disease. It has been suggested that peptides derived from bacterial proteins and presented by HLA-B27 to cytotoxic T lymphocytes might show molecular mimicry with autologous peptides, leading to T-cell cross-reaction and autoimmunity. Antigen presentation in Salmonella-infected cells could be modulated by changes in the composition of the proteasome, which is the major proteolytic system that generates major histocompatibility complex class I ligands. In this study we analysed whether the composition or activity of the 20S proteasome was altered upon infection of lymphoid cells by S. typhimurium. Two-dimensional gel electrophoresis failed to show any differences between the composition of 20S proteasomes from cells infected with S. typhimurium for 24 hr, relative to non-infected cells. In addition, digestions of oxidized insulin B-chain with purified 20S proteasomes from non-infected and infected cells generated the same products, indicating that the proteasomal cleavage specificity was not altered upon infection. These data indicate that infection of lymphoid cells by S. typhimurium fails to induce formation of immunoproteasomes or otherwise alter the proteolytic specificity of the 20S proteasome.
Immunology 10/2007; 122(1):131-9. · 3.32 Impact Factor
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ABSTRACT: Many of the constitutive peptide ligands of HLA-B27, a molecule strongly associated with spondyloarthritis, are proteasome-independent. Stable isotope tagging, mass spectrometry, and epoxomicin-mediated inhibition were used to determine their percentage, structural features, and parental proteins. Of 104 molecular species examined, 29.8% were proteasome-independent, paralleling the level of HLA-B27 re-expression in the presence of epoxomicin after acid stripping. Proteasome-dependent and -independent ligands differed little in peptide motifs, flanking sequences, and cellular localization of the parental proteins. In contrast, whereas the former set arose from proteins whose size and isoelectric point distribution largely reflected those in the human proteome, proteasome-independent ligands, other than a few matching signal sequences, were almost totally derived from small (about 6-16.5 kDa) and basic proteins, which account for only 6.6% of the human proteome. Thus, a non-proteasomal proteolytic pathway with strong preference for small proteins is responsible for a significant fraction of the HLA-B27-bound peptide repertoire.
Molecular & Cellular Proteomics 06/2007; 6(5):923-38. · 7.40 Impact Factor
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ABSTRACT: B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.
The Journal of Immunology 12/2006; 177(10):7015-23. · 5.79 Impact Factor
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ABSTRACT: HLA-B*2707 is associated with ankylosing spondylitis in most populations. Like the non-associated allotypes B*2706 and B*2709, it lacks Asp116 and shows preference for peptides with nonpolar C-terminal residues. The relationships between the peptide specificity of B*2707 and those of the disease-associated B*2705 and the non-associated subtypes were analyzed by determining the overlap between the corresponding peptide repertoires, the sequence of shared and differential ligands, and by comparing allospecific T cell epitopes with peptide sharing. The B*2707-bound repertoire was as different from that of B*2705 as from those of B*2706, B*2709, or the two latter subtypes from each other. Differences between B*2707 and B*2705 were based on their C-terminal residue specificity and a subtle modulation at other positions. Differential usage of secondary anchor residues explained the disparity between the B*2707-, B*2706-, and B*2709-bound repertoires. Similar differences in residue usage were found between B*2707 and both B*2704 and B*2706, as expected from the high peptide overlap between the two latter subtypes. T cell cross-reaction paralleled peptide sharing, suggesting that many shared ligands conserve their alloantigenic features on distinct subtypes. Our results indicate that association of HLA-B27 subtypes with ankylosing spondylitis does not correlate with higher peptide sharing among disease-associated subtypes or with obvious peptide motifs.
European Journal of Immunology 08/2006; 36(7):1867-81. · 5.10 Impact Factor
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ABSTRACT: Tapasin (Tpn) is a chaperone of the endoplasmic reticulum involved in peptide loading to MHC class I proteins. The influence of mouse Tpn (mTpn) on the HLA-B*2705-bound peptide repertoire was analyzed to characterize the species specificity of this chaperone. B*2705 was expressed on Tpn-deficient human 721.220 cells cotransfected with human (hTpn) or mTpn. The heterodimer to beta(2)-microglobulin-free H chain ratio on the cell surface was reduced with mTpn, suggesting lower B*2705 stability. The B*2705-bound peptide repertoires loaded with hTpn or mTpn shared 94-97% identity, although significant differences in peptide amount were observed in 16-17% of the shared ligands. About 3-6% of peptides were bound only with either hTpn or mTpn. Nonamers differentially bound with mTpn had less suitable anchor residues and bound B*2705 less efficiently in vitro than those loaded only with hTpn or shared nonamers. Decamers showed a different pattern: those found only with mTpn had similarly suitable residues as shared decamers and bound B*2705 with high efficiency. Peptides differentially presented by B*2705 on human or mouse cells showed an analogous pattern of residue suitability, suggesting that the effect of mTpn on B*2705 loading is comparable in both cell types. Thus, mTpn has quantitative and qualitative effects on the B*2705-bound peptide repertoire, impairing presentation of some suitable ligands and allowing others with suboptimal anchor residues and lower affinity to be presented. Our results favor a size-dependent peptide editing role of Tpn for HLA-B*2705 that is species-dependent and suboptimally performed, at least for nonamers, by mTpn.
The Journal of Immunology 07/2005; 174(12):7833-44. · 5.79 Impact Factor
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ABSTRACT: Expression of HLA-B27 in murine cells has been used to establish animal models for human spondyloarthritis and for antigen presentation studies, but the effects of xenogeneic HLA-B27 expression on peptide presentation are little known. The issue was addressed in this study. HLA-B27-bound peptide repertoires from human and murine cells overlapped by 75-85%, indicating that many endogenous HLA-B27 ligands are generated and presented in both species. Of 20 differentially presented peptides that were sequenced, only 40% arose from obvious inter-species protein polymorphism, suggesting that differences in antigen processing-loading accounted for many species-specific ligands. Digestion of synthetic substrates with human and murine 20 S proteasomes revealed cleavage differences that accounted for or correlated with differential expression of particular peptides. One HLA-B27 ligand found only in human cells was similarly generated in vitro by human and murine proteasomes. Differential presentation correlated with significantly decreased amounts of this ligand in human tapasin-deficient cells reconstituted with murine tapasin, indicating that species-specific interactions between HLA-B27, tapasin, and/or other proteins in the peptide-loading complex influenced presentation of this peptide. Our results indicate that differences in proteasomal specificity and in interactions involving tapasin determine differential processing and presentation of a significant number of HLA-B27 ligands in human and murine cells.
Journal of Biological Chemistry 12/2003; 278(47):46461-72. · 4.77 Impact Factor
