Database construction and Peptide identification strategies for proteogenomic studies on sequenced genomes.

Since the advent of high-throughput DNA sequencing technologies, the ever-increasing rate at which genomes have been published has generated new challenges notably at the level of genome annotation. Even if gene predictors and annotation softwares are more and more efficient, the ultimate validation is still in the observation of predicted gene product( s). Mass-spectrometry based proteomics provides the necessary high throughput technology to show evidences of protein presence and, from the identified sequences, confirmation or invalidation of predicted annotations. We review here different strategies used to perform a MS-based proteogenomics experiment with a bottom-up approach. We start from the strengths and weaknesses of the different database construction strategies, based on different genomic information (whole genome, ORF, cDNA, EST or RNA-Seq data), which are then used for matching mass spectra to peptides and proteins. We also review the important points to be considered for a correct statistical assessment of the peptide identifications. Finally, we provide references for tools used to map and visualize the peptide identifications back to the original genomic information.

Highly efficient DNA incorporation of intratumourally injected [125I]iododeoxyuridine under thymidine synthesis blocking in human glioblastoma xenografts.

Intratumoural (i.t.) injection of radio-iododeoxyuridine (IdUrd), a thymidine (dThd) analogue, is envisaged for targeted Auger electron- or beta-radiation therapy of glioblastoma. Here, biodistribution of [(125)I]IdUrd was evaluated 5 hr after i.t. injection in subcutaneous human glioblastoma xenografts LN229 after different intravenous (i.v.) pretreatments with fluorodeoxyuridine (FdUrd). FdUrd is known to block de novo dThd synthesis, thus favouring DNA incorporation of radio-IdUrd. Results showed that pretreatment with 2 mg/kg FdUrd i.v. in 2 fractions 0.5 hr and 1 hr before injection of radio-IdUrd resulted in a mean tumour uptake of 19.8% of injected dose (% ID), representing 65.3% ID/g for tumours of approx. 0.35 g. Tumour uptake of radio-IdUrd in non-pretreated mice was only 4.1% ID. Very low uptake was observed in normal nondividing and dividing tissues with a maximum concentration of 2.9% ID/g measured in spleen. Pretreatment with a higher dose of FdUrd of 10 mg/kg prolonged the increased tumour uptake of radio-IdUrd up to 5 hr. A competition experiment was performed in FdUrd pretreated mice using i.t. co-injection of excess dThd that resulted in very low tumour retention of [(125)I]IdUrd. DNA isolation experiments showed that in the mean >95% of tumour (125)I activity was incorporated in DNA. In conclusion, these results show that close to 20% ID of radio-IdUrd injected i.t. was incorporated in tumour DNA after i.v. pretreatment with clinically relevant doses of FdUrd and that this approach may be further exploited for diffusion and therapy studies with Auger electron- and/or beta-radiation-emitting radio-IdUrd.

An improved synthesis of dansylated α-galactosylceramide and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells.

A highly efficient synthesis of the biologically important fluorescent probe dansyl α-GalCer is presented. Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatography, respectively, and the use of a high yielding α-selective glycosylation reaction between the phytosphingosine lipid and a galactosyl iodide donor. The ability of dansyl α-GalCer to activate iNKT cells and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.

Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Preferential synthesis of prostaglandin D2 by neurons and prostaglandin E2 by fibroblasts and nonneuronal cells in chick dorsal root ganglia.

To determine the type and the relative amount of prostaglandins (PGs) synthesized by various neural tissues, homogenates of meninges, dorsal root ganglia (DRG) capsules, decapsulated DRG, and unsheathed sciatic nerves were incubated with [1-14C]arachidonic acid. Homogenates of cultured cells (meningeal cells, fibroblasts, and nonneuronal or neuronal DRG cells) were used to specify the cells producing particular PGs. The highest synthetic capacity was found in fibroblast-rich tissues (meninges and DRG capsules) and in cultures of meningeal cells or fibroblasts. Two major cyclooxygenase products were formed: [14C]PGE2 and an unusual 14C-labeled compound, Y. The accumulation of compound Y, corresponding probably to 15-hydroperoxy PGE2, was completely impaired by addition of exogenous GSH, which conversely enhanced the synthesis of [14C]PGE2 and promoted the formation of [14C]PGD2. In contrast, decapsulated DRG or unsheathed sciatic nerves displayed a 10-20 times lower capacity to synthesize PGs than fibroblast-rich tissues and produced mainly [14C]PGE2 and [14C]PGD2. In this case, [14C]PGE2 or [14C]PGD2 synthesis was neither enhanced nor promoted by addition of exogenous GSH. Neuron-enriched DRG cell cultures allowed us to specify that [14C]PGD2 is the major prostanoid produced by primary sensory neurons as compared with nonneuronal DRG cells. Because PGD2 synthesis in DRG and more specifically in DRG neurons does not depend on exogenous GSH and differs from PGD2 synthesis in fibroblast-rich tissues, it is concluded that at least two distinct enzymatic processes contribute to PGD2 formation in the nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Physiological state and rate protein synthesis of phytoplankton off the coast of Peru as measured by sulfur-35 incorporation

Analiza el estado de la fisiología del fitoplancton de las aguas costeras cercanas a Perú

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation.

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA-viral peptide interaction as the major factor modulating durable control of HIV infection.

The role of proteolytic processing and the stable signal peptide in expression of the Old World arenavirus envelope glycoprotein ectodomain.

Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.

The predictive value of preoperative B-type natriuretic peptide in children undergooing heart surgery.

Introduction: Low cardiac output syndrome is frequent in childrenafter heart surgery for congenital heart disease and may result in pooroutcome and increased morbidity. In the adult population, preoperativebrain natriuretic peptide (BNP) was shown to be predictive of postoperative complications. In children, the value of preoperative BNP onpostoperative outcome is not so clear. The aim of this study was todetermine the predictive value of preoperative BNP on postoperativeoutcome and low cardiac output syndrome in children after heartsurgery for congenital heart disease.Methods: We examined, retrospectively, the postoperative course of97 pediatric patients (mean age 3.7 years, range 0-14 years old) whounderwent heart surgery in a tertiary care pediatric intensive caresetting. NTproBNP was measured preoperatively in all patients(median 412 pg/ml, range 12-35'000 pg/ml). Patients were divided intothree groups according to their NTproBNP levels (group 1: 0-300 pg/ml, group 2: 300-600 pg/ml, group 3: >600 pg/ml) and then,correlations with postoperative outcomes were examined.Results: We found that patients with a high preoperative BNP requiredmore frequently prolonged (>2 days) mechanical ventilation (33%vs 40% vs 61%, p = 0.045) and stayed more frequently longer than6 days in the intensive care unit (42% vs 50% vs 71%, p = 0.03).However, high preoperative BNP was not correlated with occurrenceof low cardiac output syndrome.Conclusion: Preoperative BNP cannot be used, in children, as areliable and sole predictor of postoperative low cardiac outputsyndrome. However it may help identify, before surgery, those patientsat risk of having a difficult postoperative course.

The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.

RHAMM-R3 peptide vaccination in patients with acute myeloid leukemia, myelodysplastic syndrome, and multiple myeloma elicits immunologic and clinical responses.

The receptor for hyaluronic acid-mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8(+) T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8(+)/HLA-A2/RHAMM R3 tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells in accordance with an increase of R3-specific CD8(+) T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.

How T cell receptors interact with peptide-MHCs: A multiple steered molecular dynamics study.

The T-cell receptor (TCR) interaction with antigenic peptides (p) presented by the major histocompatibility complex (MHC) molecule is a key determinant of immune response. In addition, TCR-pMHC interactions offer examples of features more generally pertaining to protein-protein recognition: subtle specificity and cross-reactivity. Despite their importance, molecular details determining the TCR-pMHC binding remain unsolved. However, molecular simulation provides the opportunity to investigate some of these aspects. In this study, we perform extensive equilibrium and steered molecular dynamics simulations to study the unbinding of three TCR-pMHC complexes. As a function of the dissociation reaction coordinate, we are able to obtain converged H-bond counts and energy decompositions at different levels of detail, ranging from the full proteins, to separate residues and water molecules, down to single atoms at the interface. Many observed features do not support a previously proposed two-step model for TCR recognition. Our results also provide keys to interpret experimental point-mutation results. We highlight the role of water both in terms of interface resolvation and of water molecules trapped in the bound complex. Importantly, we illustrate how two TCRs with similar reactivity and structures can have essentially different binding strategies. Proteins 2011; © 2011 Wiley-Liss, Inc.

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness.

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ∼1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ∼1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.