M. Luz Valero

Excretory/secretory proteome of the adult stage of Echinostoma caproni

The excretory/secretory proteome of Echinostoma caproni (Trematoda: Echinostomatidae) adults collected from experimentally infected mice was investigated using a proteomic approach. We performed a shot-gun liquid chromatography/tandem mass spectrometry for the separation and identification of tryptic peptides from the excretory/secretory products of E. caproni adult worms. Database search was performed using MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems). A total of 39 parasite proteins were accurately identified. Strikingly, metabolic enzymes, and particularly glycolytic enzymes, constituted the largest protein family in the excretory/secretory proteome of E. caproni adult worms. Moreover, representative proteins involved in parasite structure, response against stress, chaperones, calcium-binding, and signal transduction were also identified. This work extends our knowledge of host–parasite relationships in the E. caproni-rodent model that is extensively used to analyze the factors determining the intestinal helminth rejection. Consequently, information on many proteins may be useful to better understand the molecular basis that determines the survival of this parasite in the definitive host.

Combination of snap freezing, differential pH two-dimensional reverse-phase high-performance liquid chromatography, and iTRAQ technology for the peptidomic analysis of the effect of prolyl oligopeptidase inhibition in the rat brain.

In vitro, prolyl oligopeptidase (POP) cleaves proline-containing bioactive peptides such as substance P, gonadotropin-releasing hormone, thyrotropin-releasing hormone, arginine-vasopressin, and neurotensin. Based on specific in vivo inhibition, POP has been suggested to be involved in cognitive and psychiatric processes but the identity of its physiological substrates has remained inconclusive. We have combined (a) sample snap-freezing and boiling buffer extraction, to limit protein degradation and reduce sample complexity; (b) pH two-dimensional liquid reverse-phase chromatography to enhance resolution; and (c) iTRAQ isobaric labeling to identify the rat brain peptides whose levels were differentially changed due to in vivo POP inhibition. In the hypothalamus, all the substrates found were part of precursors of secreted peptides such as copeptin, PACAP-related peptide, somatostatin, and proSAAS derived peptides, while in the cerebellum the peptides were derived from carcinoma-amplified sequence 1 homolog and calmodulin. In the striatum, somatostatin precursor derived peptide, fragments from E3-SUMO protein ligase RanBP2, and the subunit 5A of cytochrome c oxidase were increased. When analyzing the peptides that were significantly reduced by POP inhibition we found fragments from large protein complexes but, exclusively in the cerebellum, bioactive peptides such as cerebellin and fibrinopeptides A and B were detected.

Proteomic analysis of phosphorylated nuclear proteins underscores novel roles for rapid actions of retinoic acid in the regulation of mRNA splicing and translation.

Retinoic acid (RA) signaling is mediated by the retinoic acid receptor (RAR), belonging to the nuclear hormone receptor superfamily. In addition to its classical transcriptional actions, RAR also mediates rapid transcription-independent (nongenomic) actions, consisting in the activation of signal transduction pathways, as the phosphatidyl-inositol-3-kinase or the ERK MAPK-signaling pathways. RA-induced rapid transcription-independent actions play a role in different physiological contexts. As an effort toward understanding the functions of those rapid actions on signaling elicited by RA, we have identified nuclear proteins the phosphorylation state of which is rapidly modified by RA treatment in neuroblastoma cells, using a proteomic approach. Our results show that RA treatment led to changes in the phosphorylation patterns in two families of proteins: 1) those related to chromatin dynamics in relation to transcriptional activation, and 2) those related to mRNA processing and, in particular, mRNA splicing. We show that treatment of neuroblastoma cells with RA leads to alteration of the regulation of pre-mRNA splicing and mRNA translation. Thus, our results underscore novel functions for the rapid signaling elicited by RAR in the regulation of mRNA processing. We conclude that RA activation of signaling pathways can indeed regulate mRNA processing as part of a cellular response orchestrated by the nuclear receptor RAR.

Proteomic analysis of Strongyloides stercoralis L3 larvae

Strongyloidiasis can be perpetuated by autoinfection with the filariform larvae L3, causing asymptomatic chronic infections and creating a population of carriers, affecting not only developing countries. So far, very little is known about the proteins that interact with the human host, and few proteins from the infective Strongyloides stercoralis L3 have been characterized. Here, we report results obtained from a proteomic analysis of the proteins from S. stercoralis L3 larvae obtained from patients. Since the genome of S. stercoralis is not yet available, we used proteomic analysis to identify 26 different proteins, 13 of them released by short digestion with trypsin, which could represent surface-associated proteins. The present work extends our knowledge of host-parasite interactions by identifying proteins that could be of interest in the development of diagnostic tools, vaccines, or treatments for a neglected disease like strongyloidiasis.

The transcriptome of Echinostoma caproni adults: Further characterization of the secretome and identification of new potential drug targets

UNLABELLED Echinostomes are cosmopolitan parasites that infect a large number of different warm-blooded hosts, both in nature and in the laboratory. They also constitute an important group of food-borne trematodes of public health importance mainly in Southeast Asia and the Far East. In addition, echinostomes are an ideal model to study several aspects of intestinal helminth biology, since they present a number of advantages. For example, echinostomes are large worms whose life cycle is relatively easy to maintain in the laboratory. Recently, several studies documented their great value in the study of intestinal helminth-vertebrate host relationship. Detailed knowledge of their genome, transcriptome and proteome is likely to have an important impact on the development of control strategies for intestinal helminths. We present the first transcriptome of the adult stage of Echinostoma caproni using 454 sequencing coupled to a semi-automated bioinformatic analyses. 557,236 raw sequence reads were assembled into 28,577 contiguous sequences using iAssembler. 23,296 putative proteins were characterized based on homology, gene ontology and/or biochemical pathways. Comparisons of the transcriptome of E. caproni with those of other trematodes revealed similarities in the transcription pattern of molecules inferred to have key roles in parasite-host interactions. Enzymatic proteins like kinases and peptidases were abundant. Of the 3415 predicted excretory/secretory proteins compiled (including non-classical secretory proteins), 180 different proteins were confirmed by proteomic analysis. Potential drug targets were also identified. BIOLOGICAL SIGNIFICANCE In this study the first transcriptome of the adult stage of E. caproni is presented and compared to those of other trematodes revealing similarities in transcription for molecules inferred to have key roles in parasite-host interactions. 3415 predicted excretory/secretory proteins were compiled, being 180 different proteins confirmed by proteomic analysis. The current transcriptome data increased by nine times the number of previous protein identifications. In addition, potential drug targets for this parasite were identified. The present dataset should provide a solid foundation for future fundamental genomic, proteomic, and metabolomic explorations of E. caproni, as well as a basis for applied outcomes, such as the development of novel methods of intervention against this model organism and related parasites.

Development of a Human Extracellular Matrix for Applications Related with Stem Cells and Tissue Engineering

The recent progress in stem cell biology has created new approaches for their study as well as their application to the treatment of human diseases [1–3]. The success of stem-cell based technologies in the clinical setting [4, 5] has emphasized the need to improve the standards of quality for all phases of cell therapy, particularly the development of culture methods that circumvent products of animal origin since these might provoke infections or immune rejection following transplantation in patients. Indeed, since Martin et al. (2005) [6] demonstrated that hESC cultured with animal or serum products retained non-human sialic acid which was immunogenic when transplanted in humans, the establishment of animal-free conditions to support the maintenance and differentiation of human stem cells has been a major goal of the field of regenerative medicine [7]. Thus, chemically-defined culture systems that are devoid of non-human substances will greatly facilitate the use of stem cells in regenerative strategies. The concept of a niche is crucial for the organization of stem cells. A niche is consider as a subset of tissue cells and extracellular substrates (matrix and soluble factors) that can support stem cells and control their self-renewal in vivo [8]. Extracellular matrices help to structure niches spatially and modulate the concentration of adhesive and signalling molecules locally. The ECM is a molecular complex that contains collagens and other glycoproteins, hyaluronic acid, proteoglycans, glycosaminoglycans (GAGs), and elastins. Additionally, the ECM harbours growth factors or cytokines to protect against degradation [9]. ECM components are responsible for adhesion during the majority of cell interactions and are implicated in the maintenance of embryonic induction during development as well as stem cell differentiation in vitro [10]. Thus, local changes in ECM can dramatically modulate the proliferation and migration of stem cells and may participate in the specification of lineages. hESC have provided invaluable tools for gaining insight into the developmental origins of human tissues. However, to realize the full biological and clinical potential of hESC, certain problems related with the routine culture of these cells must be solved. Mouse embryonic fibroblasts (MEFs) and murine derivatives such as Matrigel are widely used in the maintenance and differentiation of hESC. Recently, considerable effort has been dedicated to the elimination of animal-derived reagents from the culture of hESC and in parallel, to the control of cell growth parameters by avoiding human feeder cells. For example, in 2006 Ludwig et al. reported the use of conditioned media and high doses of FGF to maintain the undifferentiated state in hESC cultured on plastic, but some abnormalities were detected at passage 20 under these conditions [11, 12]. The use of human feeders complicates the growth and molecular analysis of both pluripotency and differentiation since experimental data may reflect the combined effects of hESC and feeder cells in the culture. Given these considerations, the use of an ECM of human or synthetic origin would provide many advantages. Indeed, there have been attempts [13, 14] to produce such a tool but the results have not been satisfactory because the products were unable to maintain hESC in the undifferentiated state over time. Matrix proteins have been used as coating for in vitro cultures of human stem cells but they have usually been applied as undefined protein mixtures [11] of animal origin [15] with undefined media [13], serum [16] or a synthetic mixture [17, 18]. However, most of these human biological reagents are expensive to manufacture and thus, are cost-prohibitive for many laboratories. Decellularization procedures have been used traditionally to isolate ECM from cells in culture, tissues or organs [19]. The goal of decellularization protocols is to efficiently remove cellular and nuclear material while minimizing any adverse effect on the composition, biological activity and mechanical and structural integrity of the remaining ECM [20, 21]. Decellularized human scaffolds have facilitated the remodelling of various tissues in both animal models and humans [4, 22]. However, any biochemical procedure employed to remove cells may also alter the native three-dimensional architecture of the ECM and thus, a balance must be achieved between chemical and physical treatments during the decellularization process [4, 5, 23]. Given the potential importance of hESC in translational research and regenerative medicine, the aim of the present study was to develop a simple, efficient protocol for the production of a human ECM that is both safe and economical. Here we report that hypotonic lysis of human foreskin fibroblasts (HFF) generates a human ECM that retains protein components which are essential for attachment and cell-cell interaction. This hffECM was capable of maintaining the pluripotency of hESC and supporting their differentiation when used with the appropriate medium. Therefore, our results reveal hffECM as a novel tool which may facilitate the clinical application of hESC-based technologies.

Unveiling novel interactions of histone chaperone Asf1 linked to TREX-2 factors Sus1 and Thp1

Anti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes. These include the assembly and disassembly of histones H3 and H4 from chromatin during replication, transcription, and DNA repair. In addition, Asf1 is required for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109. Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the functional links of Asf1 with other cellular machineries, we employed mass spectrometry coupled to tandem affinity purification (TAP) to investigate novel physical interactions of Asf1. Under different TAP-MS analysis conditions, we describe a new repertoire of Asf1 physical interactions and novel Asf1 post-translational modifications as ubiquitination, methylation and acetylation that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and Nup57, which are all involved in transcription coupled to mRNA export in eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export and GAL1 transcription are not affected in asf1Δ a strong genetic interaction exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1 and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56 acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we provide evidence for a role of Asf1 in histone H2B ubiquitination. This work proposes a functional link between Asf1 and TREX-2 components in histone metabolism at the vicinity of the nuclear pore complex.

Tandem affinity purification of histones, coupled to mass spectrometry, identifies associated proteins and new sites of post-translational modification in Saccharomyces cerevisiae

Histones and their post-translational modifications contribute to regulating fundamental biological processes in all eukaryotic cells. We have applied a conventional tandem affinity purification strategy to histones H3 and H4 of the yeast Saccharomyces cerevisiae. Mass spectrometry analysis of the co-purified proteins revealed multiple associated proteins, including core histones, which indicates that tagged histones may be incorporated to the nucleosome particle. Among the many other co-isolated proteins there are histone chaperones, elements of chromatin remodeling, of nucleosome assembly/disassembly, and of histone modification complexes. The histone chaperone Rtt106p, two members of chromatin assembly FACT complex and Psh1p, an ubiquitin ligase, were the most abundant proteins obtained with both H3-TAP and H4-TAP, regardless of the cell extraction medium stringency. Our mass spectrometry analyses have also revealed numerous novel post-translational modifications, including 30 new chemical modifications in histones, mainly by ubiquitination. We have discovered not only new sites of ubiquitination but that, besides lysine, also serine and threonine residues are targets of ubiquitination on yeast histones. Our results show the standard tandem affinity purification procedure is suitable for application to yeast histones, in order to isolate and characterize histone-binding proteins and post-translational modifications, avoiding the bias caused by histone purification from a chromatin-enriched fraction.

Zygocotyle lunata: Proteomic analysis of the adult stage

The somatic extract of Zygocotyle lunata (Trematoda: Paramphistomidae) adults collected from experimentally infected mice was investigated using a proteomic approach to separate and identify tryptic peptides from the somatic extract of Z. lunata adult worms. A shot-gun liquid chromatography/tandem mass spectrometry procedure was used. We used the MASCOT search engine (Matrix-Science) and ProteinPilot software v2.0 (Applied Biosystems) for the database search. A total of 36 proteins were accurately identified from the worms. The largest protein family consisted of metabolic enzymes. Structural, motor and receptor binding proteins and proteins related to oxygen transport were identified in the somatic extract of Z. lunata. This is the first study that attempts to identify the proteome of Z. lunata. However, more work is needed to improve our knowledge of trematodiasis in general and more specifically to have a better understanding about host-parasite relationships in infections with paramphistomes.

Data for the identification of proteins and post-translational modifications of proteins associated to histones H3 and H4 in S. cerevisiae, using tandem affinity purification coupled with mass spectrometry

Tandem affinity purification method (TAP) allows the efficient purification of native protein complexes which incorporate a target protein fused with the TAP tag. Purified multiprotein complexes can then be subjected to diverse types of proteomic analyses. Here we describe the data acquired after applying the TAP strategy on histones H3 and H4 coupled with mass spectrometry to identify associated proteins and protein post-translational modifications in the budding yeast, Saccharomyces cerevisiae. The mass spectrometry dataset described here consists of 14 files generated from four different analyses in a 5600 Triple TOF (Sciex) by information‐dependent acquisition (IDA) LC–MS/MS. The above files contain information about protein identification, protein relative abundance, and PTMs identification. The instrumental raw data from these files has been also uploaded to the ProteomeXchange Consortium via the PRIDE partner repository, with the dataset identifier PRIDE: PXD002671 and http://dx.doi.org/10.6019/PXD002671. These data are discussed and interpreted in http://dx.doi.org/10.1016/j.jprot.2016.01.004. Valero et al. (2016) [1].