Alberto Paradela

Quantitative and Qualitative Influences of Tapasin on the Class I Peptide Repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Advances in the Analysis of Protein Phosphorylation

Phosphorylation is one of the most relevant and ubiquitous post-translational modifications. Despite its relevance, the analysis of protein phosphorylation has been revealed as one of the most challenging tasks due to its highly dynamic nature and low stoichiometry. However, the development and introduction of new analytical methods are modifying rapidly and substantially this field. Especially important has been the introduction of more sensitive and specific methods for phosphoprotein and phosphopeptide purification as well as the use of more sensitive and accurate MS-based analytical methods. The integration of both approaches has enabled large-scale phosphoproteome studies to be performed, an unimaginable task few years ago. Additionally, methods originally developed for differential proteomics have been adapted making the study of the highly dynamic nature of protein phosphorylation feasible. This review aims at offering an overview on the most frequently used methods in phosphoprotein and phosphopeptide enrichment as well as on the most recent MS-based analysis strategies. Current strategies for quantitative phosphoproteomics and the study of the dynamics of protein phosphorylation are highlighted.

Differential Association of HLA-B*2705 and B*2709 to Ankylosing Spondylitis Correlates with Limited Peptide Subsets but Not with Altered Cell Surface Stability

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to β2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.

Modulation at multiple anchor positions of the peptide specificity of HLA–B27 subtypes differentially associated with ankylosing spondylitis

OBJECTIVE To investigate the rules governing peptide binding to HLA-B*2705, and to B*2704 and B*2706, which are 2 subtypes differentially associated with ankylosing spondylitis. METHODS Poly-Ala analogs carrying the HLA-B27 motif Arg-2, and substitutions at anchor positions P1, P3, or Pomega, were used to determine a binding score for each residue at each position. Binding was assessed in a quantitative epitope stabilization assay, where the cell surface expression of HLA-B27 was measured by flow cytometry as a function of peptide concentration. RESULTS Peptide anchor residues contributed additively to B*2705 binding. About 15% of the natural B*2705 ligands used a deficient P3 or Pomega anchor, but never both, indicating that detrimental anchoring at one of these positions is always compensated by a good anchor at the other one. About 50% of the B*2705 ligands used suboptimal P1 residues. However, this was compensated with optimal P3 and/or Pomega anchoring. Peptides that were longer than decamers used good anchor residues at the 3 positions, suggesting more stringent binding requirements. B*2704 and B*2706 differed in their residue specificity at P1, P3, and Pomega. The rules derived for B*2705 also applied to the known ligands of these 2 subtypes. CONCLUSION The B*2705, B*2704, and B*2706 peptide repertoires are limited by the allowed residue combinations described in this study. The differential association of B*2704 and B*2706 with spondylarthropathy correlates with differences in their peptide specificity at multiple anchor positions. However, it is now possible to predict the peptide features that determine this differential binding to both subtypes.

Limited Diversity of Peptides Related to an Alloreactive T Cell Epitope in the HLA-B27-Bound Peptide Repertoire Results from Restrictions at Multiple Steps Along the Processing-Loading Pathway

The influence of various factors along the processing-loading pathway in limiting the diversity of HLA-B27-bound peptides around a core protein sequence was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY peptides are natural B*2705 ligands. The octamer is an allospecific CTL epitope. Digestion of a 27-mer fragment of C5 revealed that both ligands are generated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8–11 residues containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for the absence of four of the six peptides. The two remaining differed from the natural octamer or nonamer ligands only by an additional N-terminal Ser residue. Their stability in complex with B*2705 was lower than the respective natural ligands, raising the possibility that N-terminal trimming might have favored a shift toward the more stable peptides. The results suggest that the B*2705-bound peptide repertoire has a highly restricted diversity around a core alloantigenic sequence. This is not explained by a single bottleneck feature, but by multiple factors, including proteasomal generation, TAP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not excluded, were not required to explain the absence in vivo of the particular peptide set in this study.

The presence of sinapyl lignin in Ginkgo biloba cell cultures changes our views of the evolution of lignin biosynthesis

Suspension cell cultures (SCCs) from one of the oldest seed plants, Ginkgo biloba, show unpredictable alterations in the nature of the lignins, such as is the recruitment of sinapyl alcohol for lignin biosynthesis, compared with the woody tissues of the same species, which lack syringyl (S) lignins. These results show that, in this gymnosperm, the genes involved in sinapyl alcohol biosynthesis are latent and that their regulatory regions respond, by initiating gene expression, to the developmental signals and the environmental clues, which condition its in vitro culture. G. biloba SCCs not only synthesize S lignins but also their extracellular proteome contains both class III peroxidases capable of oxidizing sinapyl alcohol and enzymes involved in H2O2 production, observation which suggests that the peroxidase branch for the oxidative coupling of sinapyl alcohol units into lignins is operative. The incomplete knowledge of the G. biloba peroxidase-encoding genes led us to purify, characterize and partially sequence the peroxidase responsible for monolignol oxidation. When the major peroxidase from G. biloba SCCs (GbPrx) was purified to homogeneity, it showed absorption maxima in the visible region at 414 (Soret band), and at 543 and 570 nm, which calls to mind those shown by low-spin ferric peroxidases. However, the results also showed that the paraperoxidase-like character of GbPrx is not an obstacle for oxidizing the three monolignols compared with high-spin ferric peroxidases. Taken together, these results mean that the time at which the evolutionary gain of the segment of the route that leads to the biosynthesis of S lignins took place in seed plants needs to be revised.

New structural and functional defects in polyphosphate deficient bacteria: A cellular and proteomic study

BackgroundInorganic polyphosphate (polyP), a polymer of tens or hundreds of phosphate residues linked by ATP-like bonds, is found in all organisms and performs a wide variety of functions. PolyP is synthesized in bacterial cells by the actions of polyphosphate kinases (PPK1 and PPK2) and degraded by exopolyphosphatase (PPX). Bacterial cells with polyP deficiencies due to knocking out the ppk1 gene are affected in many structural and important cellular functions such as motility, quorum sensing, biofilm formation and virulence among others. The cause of this pleiotropy is not entirely understood.ResultsThe overexpression of exopolyphosphatase in bacteria mimicked some pleitropic defects found in ppk1 mutants. By using this approach we found new structural and functional defects in the polyP-accumulating bacteria Pseudomonas sp. B4, which are most likely due to differences in the polyP-removal strategy. Colony morphology phenotype, lipopolysaccharide (LPS) structure changes and cellular division malfunction were observed. Finally, we used comparative proteomics in order to elucidate the cellular adjustments that occurred during polyP deficiency in this bacterium and found some clues that helped to understand the structural and functional defects observed.ConclusionsThe results obtained suggest that during polyP deficiency energy metabolism and particularly nucleoside triphosphate (NTP) formation were affected and that bacterial cells overcame this problem by increasing the flux of energy-generating metabolic pathways such as tricarboxilic acid (TCA) cycle, β-oxidation and oxidative phosphorylation and by reducing energy-consuming ones such as active transporters and amino acid biosynthesis. Furthermore, our results suggest that a general stress response also took place in the cell during polyP deficiency.

Generalized Method for Probability-based Peptide and Protein Identification from Tandem Mass Spectrometry Data and Sequence Database Searching

Tandem mass spectrometry-based proteomics is currently in great demand of computational methods that facilitate the elimination of likely false positives in peptide and protein identification. In the last few years, a number of new peptide identification programs have been described, but scores or other significance measures reported by these programs cannot always be directly translated into an easy to interpret error rate measurement such as the false discovery rate. In this work we used generalized lambda distributions to model frequency distributions of database search scores computed by MASCOT, X!TANDEM with k-score plug-in, OMSSA, and InsPecT. From these distributions, we could successfully estimate p values and false discovery rates with high accuracy. From the set of peptide assignments reported by any of these engines, we also defined a generic protein scoring scheme that enabled accurate estimation of protein-level p values by simulation of random score distributions that was also found to yield good estimates of protein-level false discovery rate. The performance of these methods was evaluated by searching four freely available data sets ranging from 40,000 to 285,000 MS/MS spectra.

B*2707 differs in peptide specificity from B*2705 and B*2704 as much as from HLA‐B27 subtypes not associated to spondyloarthritis

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.

Guidelines for reporting quantitative mass spectrometry based experiments in proteomics

UNLABELLED Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). BIOLOGICAL SIGNIFICANCE The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and Quality Control.