Nieves Ibarrola

Systematic Interactome Mapping and Genetic Perturbation Analysis of a C. elegans TGF-β Signaling Network

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

Stimulation of the myelin basic protein gene expression by 9-cis-retinoic acid and thyroid hormone: activation in the context of its native promoter

Thyroid hormone plays an important role in brain development, in part by regulating myelination. Previous studies have shown that the myelin basic protein (MBP) promoter is activated by thyroid hormone (T3) via a T3-response element (T3RE) at position -186. Surprisingly, although MBP levels are initially decreased in hypothyroid neonates, they approach later control levels, in most brain regions, despite persistent hypothyroidism. We have studied the T3-independent transcriptional regulation of this gene, using transient transfection assays. We found that, in the absence of T3, the RXR ligand, 9-cis-retinoic acid (9cRA) was able to stimulate transcription of the MBP promoter in a dose-dependent manner. This activation was unaffected by the mutation or deletion of the T3RE and required DNA sequences located between positions -162/+60. Accordingly, this MBP promoter fragment bound RXR in vitro. The 9cRA-dependent activation of the MBP promoter required the presence of both, the DNA binding and the ligand-dependent transactivation domain (AF-2) in RXR. Furthermore, as T3, 9cRA was able to stimulate MBP expression in the CG-4 cell line after differentiation to oligodendrocytes and increased the number of cells expressing the MBP protein in primary rat optic nerve glial cell cultures.

Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

BackgroundTransforming growth factor-βs (TGF-βs), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation. Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors.ResultsThere are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules. We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (a ssociated m olecule with the SH 3 domain of STAM), an adapter shown to be involved in BMP signaling. Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in J un kinase a ctivation domain b inding protein and proteasomal subunits) domain at the carboxy-terminus. We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters. Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2. We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling. Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β.ConclusionsThis report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway. Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway.

Response to retreatment with Interferon-α plus ribavirin in chronic hepatitis C patients is independent of the NS5A gene nucleotide sequence

OBJECTIVE:Interferon-α plus ribavirin is an effective treatment for chronic hepatitis C patients. We evaluated whether the response to this combined therapy correlated with the presence of mutations in a region of 372 nucleotides within the NS5A gene.METHODS:Sixty-two patients, 42 nonresponders and 20 relapsers to a previous course of interferon-α, received 3 million units thrice weekly of interferon-α-2b and 1–1.2 g daily of ribavirin for 12 months. Basal biochemical and virological (HCV RNA and genotype) parameters were determined. Clinical examinations were carried out at 1, 2, 3, 6, and 12 months. In addition, nucleotide sequencing of the NS5A gene was determined for viral samples obtained from 38 of these patients at the baseline of the combined therapy, as well as in 15 of them before initiating the previous course of interferon as monotherapy.RESULTS:On finishing the 12 months, 36 patients (58.1%) had normal aminotransferases and 25 (40.3%) cleared viremia. Nucleotide sequencing indicated the same level of genetic variability within the group of responder and nonresponder patients all along the 124 amino acid residues of the NS5A gene studied. Neither the type of amino acid substitution nor the number of them was significantly different in one group relative to the other.CONCLUSIONS:Therapy with interferon-α-2b plus ribavirin was well tolerated, achieving an end-of-treatment response in 25 (40.3%) patients. Response did not correlate with the presence of mutations in the NS5A gene analyzed, including the interferon sensitivity determining region (ISDR) and its flanking sequences.

Sirtuin 1 is a key regulator of the interleukin-12 p70/interleukin-23 balance in human dendritic cells.

Background: Notch family transcriptional repressors explain the predominant production of IL-23 elicited by β-glucans. Results: Association of SIRT1 with the il12a promoter and an enhanced production of the sirtuin co-substrate NAD+ is described. Conclusion: SIRT1-mediated histone deacetylation regulates the accessibility of Rel family proteins to the il12a promoter. Significance: Blocking the interactions of the transcriptional machinery with acetyl-histone might modulate the Th1/Th17 balance. Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD+, and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response.

Thyroid hormone regulates the expression of the MAL proteolipid, a component of glycolipid-enriched membranes, in neonatal rat brain.

Detergent‐insoluble glycosphingolipid‐enriched membranes (DIGs) have been involved in the sorting and transport of specific proteins during oligodendrocyte maturation. The MAL (MAL, MVP17, VIP17) proteolipid, an integral membrane protein present in DIGs in mature oligodendrocytes, has been proposed as a component of the machinery for DIG‐mediated transport in a restricted pattern of cell types including myelinating cells. We have previously shown that thyroid hormone regulates the expression of the myelin protein genes coordinately, and have suggested a major role for thyroid hormone in the control of oligodendrocytes generation. Here we show that the expression of the MAL gene is down‐regulated by hypothyroidism and up‐regulated by hyperthyroidism in myelinated regions of the brain. In contrast, adult‐onset hypothyroidism has no effect on the steady‐state levels of MAL mRNA. Taken together, our results show that MAL expression during oligodendrocyte maturation is modulated by thyroid hormone, suggesting that this hormone could play an important role in the myelin biogenesis during neonatal development. J. Neurosci. Res. 52:584–590, 1998.

Determining the Plasmodium vivax VCG-1 strain blood stage proteome

Plasmodium vivax is the second most prevalent parasite species causing malaria in humans living in tropical and subtropical areas throughout the world. There have been few P. vivax proteomic studies to date and they have focused on using clinical isolates, given the technical difficulties concerning how to maintain an in vitro culture of this species. This study was thus focused on identifying the P. vivax VCG-1 strain proteome during its blood lifecycle through LC-MS/MS; this led to identifying 734 proteins, thus increasing the overall number reported for P. vivax to date. Some of them have previously been related to reticulocyte invasion, parasite virulence and growth and others are new molecules possibly playing a functional role during metabolic processes, as predicted by Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis. This is the first large-scale proteomic analysis of a P. vivax strain adapted to a non-human primate model showing the parasite protein repertoire during the blood lifecycle. Database searches facilitated the in silico prediction of proteins proposed for evaluation in further experimental assays regarding their potential as pharmacologic targets or as component of a totally efficient vaccine against malaria caused by P. vivax. BIOLOGICAL SIGNIFICANCE P. vivax malaria continues being a public health problem around world. Although considerable progress has been made in understanding genome- and transcriptome-related P. vivax biology, there are few proteome studies, currently representing only 8.5% of the predicted in silico proteome reported in public databases. A high-throughput proteomic assay was used for discovering new P. vivax intra-reticulocyte asexual stage molecules taken from parasites maintained in vivo in a primate model. The methodology avoided the main problem related to standardising an in vitro culture system to obtain enough samples for protein identification and annotation. This study provides a source of potential information contributing towards a basic understanding of P. vivax biology related to parasite proteins which are of significant importance for the malaria research community.

Common pitfalls in bioinformatics-based analyses: look before you leap

With the explosion of information in the nucleotide and protein sequencedatabases, biologists are increasinglyturning to web-based bioinformaticsprograms to analyze their molecules ofinterest. Some of these programs have an intuitive interface, whereas othersappear quite complex with severalparameters to choose from beforeanalysis. In either case, it is quite easy tocome to erroneous conclusions about thequestions that are being asked. This canresult from incorrect assumptions on the part of the biologist or because of alimitation of the program or the databasebeing used. In this article we will discusssome of the popular programs that can beused to make predictions in a scenariosuch as the discovery of a novel gene orwhen one is working on a less-characterized molecule. We will elaborateon some of the common pitfalls that canbe avoided if certain precautions aretaken during such analyses.

Multicenter experiment for quality control of peptide-centric LC–MS/MS analysis — A longitudinal performance assessment with nLC coupled to orbitrap MS analyzers ☆

Proteomic technologies based on mass spectrometry (MS) have greatly evolved in the past years, and nowadays it is possible to routinely identify thousands of peptides from complex biological samples in a single LC-MS/MS experiment. Despite the advancements in proteomic technologies, the scientific community still faces important challenges in terms of depth and reproducibility of proteomics analyses. Here, we present a multicenter study designed to evaluate long-term performance of LC-MS/MS platforms within the Spanish Proteomics Facilities Network (ProteoRed-ISCIII). The study was performed under well-established standard operating procedures, and demonstrated that it is possible to attain qualitative and quantitative reproducibility over time. Our study highlights the importance of deploying quality assessment metrics routinely in individual laboratories and in multi-laboratory studies. The mass spectrometry data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000205.This article is part of a Special Issue entitled: HUPO 2014.

Integration of Proteomics and Transcriptomics Data Sets for the Analysis of a Lymphoma B-Cell Line in the Context of the Chromosome-Centric Human Proteome Project

A comprehensive study of the molecular active landscape of human cells can be undertaken to integrate two different but complementary perspectives: transcriptomics, and proteomics. After the genome era, proteomics has emerged as a powerful tool to simultaneously identify and characterize the compendium of thousands of different proteins active in a cell. Thus, the Chromosome-centric Human Proteome Project (C-HPP) is promoting a full characterization of the human proteome combining high-throughput proteomics with the data derived from genome-wide expression profiling of protein-coding genes. Here we present a full proteomic profiling of a human lymphoma B-cell line (Ramos) performed using a nanoUPLC-LTQ-Orbitrap Velos proteomic platform, combined to an in-depth transcriptomic profiling of the same cell type. Data are available via ProteomeXchange with identifier PXD001933. Integration of the proteomic and transcriptomic data sets revealed a 94% overlap in the proteins identified by both -omics approaches. Moreover, functional enrichment analysis of the proteomic profiles showed an enrichment of several functions directly related to the biological and morphological characteristics of B-cells. In turn, about 30% of all protein-coding genes present in the whole human genome were identified as being expressed by the Ramos cells (stable average of 30% genes along all the chromosomes), revealing the size of the protein expression-set present in one specific human cell type. Additionally, the identification of missing proteins in our data sets has been reported, highlighting the power of the approach. Also, a comparison between neXtProt and UniProt database searches has been performed. In summary, our transcriptomic and proteomic experimental profiling provided a high coverage report of the expressed proteome from a human lymphoma B-cell type with a clear insight into the biological processes that characterized these cells. In this way, we demonstrated the usefulness of combining -omics for a comprehensive characterization of specific biological systems.