2014 :



Evolution of the RAS Superfamily of GTPases

    Ana M. Rojas and Alfonso Valencia

The Ras superfamily of small GTPases illustrates a large functional diversification in the context of a preserved structural framework and a prototypic GTP-binding site. The Ras superfamily of small GTP-binding proteins is essential to regulate the cellular organization and signaling in cells. Members of this superfamily contain a structurally and mechanistically preserved GTP-binding core, with considerable functional and sequence divergence. In this chapter we review the evolutionary structure of the superfamily at the organism and sequence level, presenting a representative tree that reflects the history of the Ras superfamily including crucial evolutionary time points and detailed trees for the Rho, Ras, Rab, Arf, and Ran families. Based on this information we discuss some of the complex relationships between the evolution of proteins and the acquisition of distinctive cellular functions.


             Ras Superfamily Small G proteins: Biology and Mechanisms 1 [Springer Link]


The Pseudo GTPase CENP-M drives human kinetochore assembly.

Basilico, F.*, Maffini, S*., Weir. J.R*., Prumbaum, D., Rojas, A.M, Zimniak, Z., De Antoni, A., M, Jeganathan, S., Voss, B., van Gerwen, S., Krenn, V., Massimiliano, L., Valencia, A., Vetter, I.R., Herzog, F., Raunser, S., Pasqualato, S & Musacchio, A.

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore-centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers.


              eLIFE . [Pubmed]


The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma.

    Ambrosio MR, Navari M, Di Lisio L, Eduardo Andrés León, Onnis A, Gazaneo S, Mundo L, Ulivieri C, Gomez G, Lazzi S, Piris MA, Leoncini L, De Falco G.

Burkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression using its own encoded microRNAs. METHODS: Using microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNA. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases. RESULTS: We showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Ralpha and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression. CONCLUSIONS: Our preliminary r

              Infect Agent Cancer . [Pubmed]


Lipoprotein(a) levels in Familial Hipercholesterolaemia: an important predictor for cardiovascular disease independent of the type of LDL-receptor mutation.

    Alonso R, Eduardo Andrés León, Mata N, Fuentes-Jiménez F, Badimón L, López-Miranda J, Padró T, Muñiz O, Díaz-Díaz JL, Mauri M, Ordovás JM, Mata P; SAFEHEART investigators

To determine the relationship between lipoprotein(a) [Lp(a)] and cardiovascular disease (CVD) in a large cohort of heterozygous familial hypercholesterolemia (FH) patients. Lipoprotein(a) is considered a cardiovascular risk factor. Nevertheless, the role of Lp(a) as a predictor of CVD in FH has been a controversial issue. Cross-sectional analysis of 1960 FH and 957 non-FH relatives recruited at SAFEHEART, a long-term observational cohort study of a molecularly well-defined FH population. Lipoprotein(a) concentrations were measured in plasma using an immunoturbidimetric method. Patients with FH, especially those with CVD had higher Lp(a) plasma levels compared with their unaffected relatives (p<0.001). Lipoprotein(a) is an independent predictor of CVD in men and women with FH. Cardiovascular risk is higher in those patients with lipoprotein(a) > 50 mg/dL carrying a receptor-negative mutation in LDLR gene compared with other less severe mutations.

              JACC . [Pubmed]


Molecular Evolution of Multiple-Level Control of Heme Biosynthesis Pathway in Animal Kingdom.

    Wen-Shyong Tzou mail, Ying Chu,Tzung-Yi Lin, Chin-Hwa Hu, Tun-Wen Pai, Hsin-Fu Liu, Han-Jia Lin, Ildefonso Cases, Rojas, A.M, Mayka Sanchez, Zong-Ye You, Ming-Wei Hsu.

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5′ untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

              PLOS one. [Pubmed]



Emergence and evolutionary analysis of the human DDR network: implications in comparative genomics and downstream analyses.

    Arcas, A., Fernandez-Capetillo, O., Cases, I Rojas, A.M

The DNA Damage response is a crucial signaling network that preserves the integrity of the genome. This network is an ensemble of distinct but often overlapping sub-networks, where different components fulfill distinct functions in precise spatial and temporal scenarios. To understand how these elements have been assembled together in humans, we performed comparative genomic analyses in 47 selected species to trace back their emergence using systematic phylogenetic analyses and estimated gene ages. The emergence of the contribution of post-translational modifications to the complex regulation of DDR was also investigated. This is the first time a systematic analysis has focused on the evolution of DDR sub-networks as a whole. Our results indicate that a DDR core, mostly constructed around metabolic activities, appeared soon after the emergence of eukaryotes, and that additional regulatory capacities appeared later through complex evolutionary process. Potential key post-translational modifications were also in place then, with interacting pairs preferentially appearing at the same evolutionary time, although modifications often led to the subsequent acquisition of new targets afterwards. We also found extensive gene loss in essential modules of the regulatory network in fungi, plants and arthropods, important for their validation as model organisms for DDR studies.              

Molecular Biology and Evolution. [Pubmed]



2013 :

Serine/Threonine Kinases and E2 -ubiquitin conjugating enzymes in Planctomycetes: unexpected findings.

    Arcas, A., Cases, I., Rojas, A.M

The regulation of signal transduction by phosphorylation and ubiquitination is essential to ensure the correct behavior of eukaryotic cells. We searched for protein families involved in such signaling in several eukaryotic species and in a limited set of prokaryotes, where two members of the Planctomycetes phylum were included as they exhibit eukaryote-like features (Gemmata obscuriglobus and Pirellula staleyi). We identified sequences homologous to eukaryotic serine/threonine kinases (STKs) and E2-ubiquitin conjugating enzymes in the two Planctomycetes species. To extend these analyses to the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum, we performed comparative analyses using domains from kinases, phosphatases and GTPases that serve as signaling signatures, and we analyzed their distributions. We found substantial differences in kinome densities with regards to other prokaryote clades and among the groups in the Planctomycetes/Verrucomicrobia/Chlamydia super-phylum. In addition, we identified the presence of classic eukaryotic E2-conjugating ubiquitin proteins in prokaryotes, these having previously believed to exist only in eukaryotes. Our phylogenetic analyses of the STKs signature domains and E2-enzymes suggest the existence of horizontal gene transfer.

              Antonie van Leeuwenhoek Journal of Microbiology. [Pubmed]



Two Novel Missense Mutations in Iron Transport Protein Transferrin Causing Hypochromic Microcytic An aemia and Haemosiderosis: molecular characterisation and structural implications.

Athiyarath R, Arora N, Fuster F, Schwarzenbacher R, Ahmed R, George B, Chand y M, Srivastava A, Rojas A.M, Sánchez M, Edison

Transferrin (TF) is a monomeric glycoprotein that facilitates the transport of ferric iron (Fe(III)) between the sites of absorption, storage and utilization. Atransferrinaemia or hypotransferrinaemia is an extremely rare autosomal recessive disease (OMIM#209300, ORPHA1195) characterized by severe deficiency of serum TF, the ligand for TF receptor which enables cellular iron uptake into various tissues. To date, only 14 cases have been reported, half of which have been characterized at the molecular level (Beutler et al2000; Chen et al2009). In this report we describe two novel missense mutations D647V and R609W (Uniprot: TRFE_HUMAN; P02787) in the transferrin gene (TF) found in two patients from two Indian non-consanguineous families.


                  J. Brit Haematol. [Pubmed]




MicroRNA-based molecular classification of non-BRCA1/2 hereditary breast tumours.

M Tanic,
Eduardo Andrés León, S M Rodriguez-Pinilla, I Marquez-Rodas, M Cebollero-Presmanes, V Fernandez, A Osorio, J Benítez and B Martinez-Delgado

Hereditary breast cancer comprises 5–10% of all breast cancers. Mutations in two high-risk susceptibility genes, BRCA1 and BRCA2, along with rare intermediate-risk genes and common low-penetrance alleles identified, altogether explain no more than 45% of the high-risk breast cancer families, although the majority of cases are unaccounted for and are designated as BRCAX tumours. Micro RNAs have called great attention for classification of different cancer types and have been implicated in a range of important biological processes and are deregulated in cancer pathogenesis.


                  British Journal of Cancer [Pubmed]



A common structural scaffold in CTD phosphatases that supports distinct catalytic mechanisms.

    Pons,T., Paramonov, I., Boullosa, C., Ibañez, K., Rojas, A.M, & Valencia, A .

The phosphorylation and dephosphorylation of the carboxyl-terminal domain (CTD) of the largest RNA polymerase II (RNAPII) subunit is a critical regulatory checkpoint for transcription and mRNA processing. This CTD is unique to eukaryotic organisms and it contains multiple tandem-repeats with the consensus sequence. Traditionally, CTD phosphatases that use metal-ion-independent (cysteine-based) and metal-ion-assisted (aspartate-based) catalytic mechanisms have been considered to belong to two independent groups. However, using structural comparisons we have identified a common structural scaffold in these two groups of CTD phosphatases. This common scaffold accommodates different catalytic processes with the same substrate specificity, in this case phospho-serine/threonine residues flanked by prolines. Furthermore, this scaffold provides a structural connection between two groups of protein tyrosine phosphatases (PTPs): Cys-based (classes I, II, and III) and Asp-based (class IV) PTPs. Redundancy in catalytic mechanisms is not infrequent and may arise in specific biological settings. To better understand the activity of the CTD phosphatases, we combined our structural analyses with data on CTD phosphatase expression in different human and mouse tissues. The results suggest that aspartate- and cysteine-based CTD-dephosphorylation acts in concert during cellular stress, when high levels of reactive oxygen species can inhibit the nucleophilic function of the catalytic cysteine, as occurs in mental and neurodegenerative disorders like schizophrenia, Alzheimer's and Parkinson's diseases. Moreover, these findings have significant implications for the study of the RNAPII-CTD dephosphorylation in eukaryotes. 





A proteomic characterization of factors enriched on nascent DNA molecules.

    Lopez-Contreras, AJ., Ruppen, I.,Nieto-Soler, M., Murga, M., Rodriguez, S., Remeseiro, S., Rodrigo, S.,& Rojas, A.M, Mendez, J., Muñoz, J, Fernandez-Capetillo, O.

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance.


                Cell Reports .[Pubmed].



ChiTaRS: a database of human, mouse and fruit fly chimeric transcripts and RNA-sequencing data.

    Frenkel-Morgenstern M, Gorohovski A, Lacroix V, Rogers M, Ibanez K, Boullosa C, Eduardo Andrés León, Ben-Hur A, Valencia A.

Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS database of Chimeric Transcripts and RNA-Sequencing data (http://chitars.bioinfo.cnio.es/) collects more than 16 000 chimeric RNAs from humans, mice and fruit flies, 233 chimeras confirmed by RNA-seq reads and ∼2000 cancer breakpoints. The database indicates the expression and tissue specificity of these chimeras, as confirmed by RNA-seq data, and it includes mass spectrometry results for some human entries at their junctions. Moreover, the database has advanced features to analyze junction consistency and to rank chimeras based on the evidence of repeated junction sites. Finally, 'Junction Search' screens through the RNA-seq reads found at the chimeras' junction sites to identify putative junctions in novel sequences entered by users. Thus, ChiTaRS is an extensive catalog of human, mouse and fruit fly chimeras that will extend our understanding of the evolution of chimeric transcripts in eukaryotes and can be advantageous in the analysis of human cancer breakpoints.


                Nucleic Acids Research .[Pubmed].




2012 : 

Uncovering the molecular machinery of the human spindle - an integration of wet and dry systems biology.

    Rojas, A.M, Santamaria, A.*,Malik, R.*, Jensen, T.S.*, Koerner, R., Morilla, I., de Juan,D., Krallinger, M., Hansen, D.A., Hoffmann,R., Lees,J., Reid, A., Yeats, C., Wehner, A., Elowe, S., Clegg. A.B., Brunak.,S., Nigg,E.A., Orengo, C., Valencia, A., Ranea., J.A.G.

Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS database of Chimeric Transcripts and RNA-Sequencing data (http://chitars.bioinfo.cnio.es/) collects more than 16 000 chimeric RNAs from humans, mice and fruit flies, 233 chimeras confirmed by RNA-seq reads and ∼2000 cancer breakpoints. The database indicates the expression and tissue specificity of these chimeras, as confirmed by RNA-seq data, and it includes mass spectrometry results for some human entries at their junctions. Moreover, the database has advanced features to analyze junction consistency and to rank chimeras based on the evidence of repeated junction sites. Finally, 'Junction Search' screens through the RNA-seq reads found at the chimeras' junction sites to identify putative junctions in novel sequences entered by users. Thus, ChiTaRS is an extensive catalog of human, mouse and fruit fly chimeras that will extend our understanding of the evolution of chimeric transcripts in eukaryotes and can be advantageous in the analysis of human cancer breakpoints.


                PLOS ONE .[Pubmed].



Epstein-Barr virus microRNAs repress BCL6 expression in diffuse large B-cell lymphoma.

    Martín-Pérez D, Vargiu P, Montes-Moreno S, Eduardo Andrés León, , Rodríguez-Pinilla SM, Lisio LD, Martínez N, Rodríguez R, Mollejo M, Castellvi J, Pisano DG, Sánchez-Beato M, Piris MA.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of all newly diagnosed lymphomas. DLBCL is considered a heterogeneous disease, with some specific clinicopathological variants of DLBCLs being associated with the presence of the EBV.1 EBV is a lymphotropic virus that has been implicated in the development of several lymphoid malignancies, mainly Burkitt lymphoma (BL) and Hodgkin's lymphoma and with low prevalence in DLBCL.1 BCL6 is a key transcriptional repressor during normal B-cell differentiation that has been shown to repress NF-kB in some DLBCLs.2 In some B-cell lymphomas, BCL6 expression was inversely correlated with LMP1 expression, and some evidences suggest that LMP1 can cause downregulation of BCL6(ref. 3), but other possible mechanisms have not been studied. We have found a strong inverse correlation between BCL6 protein expression and EBV infection (P<0.001, Figures 1a and b) in a series of 149 DLBCL samples, where only one out of 34 EBV-positive cases (2.94%) expressed BCL6, although 87 out of 115 EBV-negative cases expressed BCL6 (75.65%). However, this correlation was independent of LMP1 because 54% of EBER-positive samples were LMP1-negative (Spearman, P-value: 0.18). Little is known about the mechanisms that cause the absence of BCL6 in EBV-positive DLBCL; however, the possibility that EBV-encoded miRNAs could contribute to BCL6 repression has never been explored. 


                Leukemia .[Pubmed].



The Ras protein superfamily: Evolutionary tree and role of conserved amino acids.

    Rojas, A.M, Fuentes, G., Rausell, A., Valencia, A.

The Ras superfamily is a fascinating example of functional diversification in the context of a preserved structural framework and a prototypic GTP binding site. Thanks to the availability of complete genome sequences of species representing important evolutionary branch points, we have analyzed the composition and organization of this superfamily at a greater level than was previously possible. Phylogenetic analysis of gene families at the organism and sequence level revealed complex relationships between the evolution of this protein superfamily sequence and the acquisition of distinct cellular functions. Together with advances in computational methods and structural studies, the sequence information has helped to identify features important for the recognition of molecular partners and the functional specialization of different members of the Ras superfamily.


                The Journal of Cell Biology .[Pubmed].



2011 : 

Sumoylation regulates nuclear localization of repressor DREAM.

    Palczewska, M, Casafont I, Ghimire K, Rojas, A.M, Valencia A, Lafarga M, Mellstrom B, Naranjo JR./span>

DREAM is a Ca(2+)-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons


                Biochim Biophys Acta .[Pubmed].



B Lymphocyte Commitment Program Is Driven by the Proto-oncogene c-myc.

   Vallespinós, M., Fernandez, D., Rodríguez, L., Alvaro-Blanco, J., Baena, E., Ortiz, M., Dukovska, D., Martinez, D., Rojas, A.M, Campanero, M., Moreno de Alborán, I.

c-Myc, a member of the Myc family of transcription factors, is involved in numerous biological functions including the regulation of cell proliferation, differentiation, and apoptosis in various cell types. Of all of its functions, the role of c-Myc in cell differentiation is one of the least understood. We addressed the role of c-Myc in B lymphocyte differentiation. We found that c-Myc is essential from early stages of B lymphocyte differentiation in vivo and regulates this process by providing B cell identity via direct transcriptional regulation of the ebf-1 gene. Our data show that c-Myc influences early B lymphocyte differentiation by promoting activation of B cell identity genes, thus linking this transcription factor to the EBF-1/Pax-5 pathway. 


                The Journal of Immunology .[Pubmed].



2010 :

An isoform-specific PDZ-binding motif targets type I PIP5 kinase beta to the uropod and controls polarization of netrophil-like HL60 cells.

    Mañes, S, Fuentes, G., Peregil, RM. Rojas, A.M, Lacalle, RA

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)-beta participates in establishing polarity during leukocyte chemotaxis. Its final 83 amino acids localize PIP5KIbeta to the uropod of chemotaxing neutrophils and T cells, and interact with ezrin-radixin-moesin (ERM) proteins and EBP50 (4.1-ERM-binding phosphoprotein 50), a scaffold protein with 2 PDZ (PSD-95, disc large, ZO-1) domains. The structural motifs at the PIP5KIbeta C terminus that confer signaling specificity are, nonetheless, unknown. We show that the last 4 residues of PIP5KIbeta constitute an atypical PDZ-binding motif, which steers PIP5KIbeta to the uropod by binding to both EBP50 PDZ domains. Molecular modeling and mutagenesis indicated that PDZ-binding motif is necessary for PIP5KIbeta localization and for chemoattractant-induced neutrophil polarization. Polarity in cells that express PIP5KIbeta mutants lacking the PDZ-binding motif was restored by overexpression of PIP5KIbeta, but not of PIP5KIgamma_i2, another isoform that localizes to the neutrophil uropod. Our results identify an isoform-specific PDZ-binding motif in PIP5KIbeta, which confers specificity for PIP5KIbeta signaling at the uropod during leukocyte chemotaxis.


                FASEB Journal .[Pubmed].



Inference of functional relations in predicted protein networks with a machine learning approach..

   García-Jiménez, B., Juan, D., Ezkurdia, I., Eduardo Andrés León, Valencia, A

Molecular biology is currently facing the challenging task of functionally characterizing the proteome. The large number of possible protein-protein interactions and complexes, the variety of environmental conditions and cellular states in which these interactions can be reorganized, and the multiple ways in which a protein can influence the function of others, requires the development of experimental and computational approaches to analyze and predict functional associations between proteins as part of their activity in the interactome.


                PLOS ONE .[Pubmed].



2009 :

Databases of protein-protein interactions and their uses in drug discovery.

    Fuentes, G., Oyarzabal, J., Rojas, A.M,

Proteins rarely act alone. With the development of experimental technologies, researchers have started to map the interactions of proteins in different organisms. The data gathered constitute potential frameworks for system-based drug discovery. The possibility of targeting protein-protein interactions with specific drugs raises expectations of huge impact in the therapeutics field, despite the large quantity of research still necessary to delineate the complete interactome of a single cell. This review summarizes some concepts relating to protein-protein interfaces in relation to drug discovery, and discusses studies aiming to develop protein-protein interaction modulators through the combination of in silico and in vitro screening experiments.


                Current Opinion in Drug Discovery and Development .[Pubmed].



Creating reference datasets for Systems Biology applications using text mining.

   Krallinger, M., Rojas, A.M, Valencia, A.

High-throughput experimental techniques are generating large data collections with the aim of identifying novel entities involved in fundamental cellular processes as well as drawing a systematic picture of the relationships between individual components. Determining the accuracy of the resulting data and the selection of a subset of targets for more careful characterizations often requires relying on information provided by manually annotated data repositories. These repositories are incomplete and cover only a small fraction of the knowledge contained in the literature. We propose in this paper the use of text-mining technologies to extract, organize, and present information relevant for a particular biological topic. The aims of the resulting approach are (1) to enable topic-centric biological literature navigation, (2) to assist in the construction of manually revised data repositories, (3) to provide prioritization of biological entities for experimental studies, and (4) to enable human interpretation of large-scale experiments by providing direct links of bio-entities to relevant descriptions in the literature.


                Annals of the New York Academy of Sciences .[Pubmed].



EcID. A database for the inference of functional interactions in E. coli.

   Eduardo Andrés León, Ezkurdia, Iakes, García, Beatriz, Valencia, Alfonso, Juan, David.

The EcID database (Escherichia coli Interaction Database) provides a framework for the integration of information on functional interactions extracted from the following sources: EcoCyc (metabolic pathways, protein complexes and regulatory information), KEGG (metabolic pathways), MINT and IntAct (protein interactions). It also includes information on protein complexes from the two E. coli high-throughput pull-down experiments and potential interactions extracted from the literature using the web services associated to the iHOP text-mining system. Additionally, EcID incorporates results of various prediction methods, including two protein interaction prediction methods based on genomic information (Phylogenetic Profiles and Gene Neighbourhoods) and three methods based on the analysis of co-evolution (Mirror Tree, In Silico 2 Hybrid and Context Mirror). EcID associates to each prediction a specifically developed confidence score. The two main features that make EcID different from other systems are the combination of co-evolution-based predictions with the experimental data, and the introduction of E. coli-specific information, such as gene regulation information from EcoCyc. The possibilities offered by the combination of the EcID database information are illustrated with a prediction of potential functions for a group of poorly characterized genes related to yeaG. EcID is available online at http://ecid.bioinfo.cnio.es.


                Nucleic Acids Research .[Pubmed].



A novel patatin-like protein from cotton plant, GhPat1, is co-expresed with GhLox1 during Xanthomonas campestris-mediated hypersensitive cell death.

   Cacas, J, Marmey, P., Montillet, JL., Sayegh-Alhamdia,M.,Jalloul, Rojas, A.M, AM.,Cleviret, A., Michel,N.

In cotton plant, Xanthomonas-induced hypersensitive response (HR) is accompanied by a lipid peroxidation process involving a 9-lipoxygenase (LOX), GhLox1. Initiation of this oxidative metabolism implies the release of the LOX substrates, or polyunsaturated fatty acids. Since patatin-like proteins (PLPs) are likely candidates for mediating the latter step, we searched for genes encoding such enzymes, identified and cloned one of them that we named GhPat1. Biochemical and molecular studies showed that GhPat1 expression was up-regulated during the incompatible interaction, prior to the onset of the corresponding galactolipase activity and cell death symptoms in tissues. Protein sequence analysis and modelling also revealed that GhPat1 catalytic amino acids and fold were conserved across plant PLPs. Based on these results and our previous work (Jalloul et al. in Plant J 32:1-12, 2002), a role for GhPat1, in synergy with GhLox1, during HR-specific lipid peroxidation is discussed.


                Plant Cell Reports .[Pubmed].



2008 :

The Nod-like Receptor (NLR) Family: a tale of similarities and differences.

   Proell,M., Riedl, S.J., Fritz, JH., Rojas, A.M, Schwarzenbacher, R.

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.


                PLOS ONE .[Pubmed].



KCTD5, a putative substrate adaptor for cullin3 ubiquitin ligases.

   Bayon Y, Trinidad AG, de la Puerta ML, Del Carmen Rodriguez M, Bogetz J, Rojas, A.M, De Pereda JM, Rahmouni S, Williams S, Matsuzawa SI, Reed JC, Crespo MS, Mustelin T, Alonso A.

Potassium channel tetramerization domain (KCTD) proteins contain a bric-a-brac, tramtrak and broad complex (BTB) domain that is most similar to the tetramerization domain (T1) of voltage-gated potassium channels. Some BTB-domain-containing proteins have been shown recently to participate as substrate-specific adaptors in multimeric cullin E3 ligase reactions by recruiting proteins for ubiquitination and subsequent degradation by the proteasome. Twenty-two KCTD proteins have been found in the human genome, but their functions are largely unknown. In this study, we have characterized KCTD5, a new KCTD protein found in the cytosol of cultured cell lines. The expression of KCTD5 was upregulated post-transcriptionally in peripheral blood lymphocytes stimulated through the T-cell receptor. KCTD5 interacted specifically with cullin3, bound ubiquitinated proteins, and formed oligomers through its BTB domain. Analysis of the interaction with cullin3 showed that, in addition to the BTB domain, some amino acids in the N-terminus of KCTD5 are required for binding to cullin3. These findings suggest that KCTD5 is a substrate-specific adaptor for cullin3-based E3 ligases.


                FEBS .[Pubmed].



Prediction of protein interaction based on similarity of phylogenetic trees.

   Pazos F, Juan D, Izarzugaza JM, Eduardo Andrés León, Valencia A.

Computational methods for predicting protein interaction partners are becoming increasingly popular. Many of them are mature enough to be widely used by molecular biologists who can look for proteins related to the protein of interest in order to infer information about its context in the cell. In this chapter we describe the use of the mirrortree set of programs and related software for predicting protein interactions. They are all based on the idea that interacting or functionally related proteins tend to show similar phylogenetic trees due to coevolution. The basic mirrortree program can be used to calculate the similarity between the phylogenetic trees implicit in the multiple sequence alignments of two protein families. The ECID database contains protein interactions and relationships from different computational and experimental sources for the model organism Escherichia coli, including the ones generated with mirrortree. Finally, the TSEMA server uses the concept of tree similarity between interacting families to look for the best mapping between two families of interacting proteins: which member in one family interacts with which member in the other.


                Methods of Molecular Biology .[Pubmed].



Exploring the FL-160-CRP gene family through sequence variability of the complement regulatory protein (CRP) expressed by the trypomastigote stage ofTrypanosoma cruzi.

   Mathieu-Daude F, Lafay B, Touzet O, Lelivre J, Parrado F, Bosseno MF, Rojas, A.M, Fatha S, Ouaissi A, Brenire SF.

The complement regulatory protein (CRP) of Trypanosoma cruzi is a surface glycoprotein which confers to the infectious trypomastigote forms a protection against the lytic activity of the host complement. CRP belongs to the large family of the trans-sialidase-like proteins and its sequence is highly similar to those of the flagellar FL-160 and chronic exoantigen proteins, encoded by a multigene family. To further define the gene family encoding the CRP, we investigated the protein diversity among several strains of T. cruzi through the sequencing of trypomastigote transcripts, and used a phylogenetic analysis based on the multiple alignment of these proteins with the top scoring sequences detected by a database sequence homology search. Intrastrain variations in CRP sequences revealed the existence of several copies per strain. The interstrain variability of CRP was consistent with the genetic subdivisions of T. cruzi into lineages and discrete typing units. The phylogenetic analysis based on a 227 amino acid alignment of CRP sequences with the 200 putative proteins retrieved from the protein databases (including the sequences from the T. cruzi genome project) revealed that the CRP sequences clustered with the FL-160 proteins into a monophyletic group characterized by the presence of the 12 amino acid mimicry epitope that mimics nervous tissues. The phylogeny did not differentiate between the CRP and the FL-160 proteins. The identification of this group of CRP-like proteins and the high sequence similarity observed within it open up new prospects for the exploration of the localization, structure and function of these proteins and a better understanding of their involvement in key aspects of host-parasite interactions, such as the resistance to the complement. This work provides also information for the T. cruzi genome annotation of the trans-sialidase-like putative proteins.


                Infect Genet Evo .[Pubmed].



2007 : 

Are Promyelocytic Leukaemia Protein Nuclear Bodies a scaffold for caspase-2 programmed cell death?.

   Sanchez-Pulido, L, Valencia A., Rojas, A.M,

Promyelocytic leukaemia protein nuclear bodies (PML-NBs) are nuclear structures whose function is still poorly understood. They are implicated in various biological functions, such as viral infection, cellular transformation, innate immunity and growth control, and they might be dynamic hubs sensing stress and DNA damage. Data from PML(-/-) mice suggest that PML-NBs are involved in apoptosis via caspase-independent mechanisms, probably involving p53-dependent and independent pathways. However, the recently demonstrated co-localization of caspase-2 within the PML-NB nuclear structures presents a new paradigm for nuclear cell death. Here, we show that these nuclear structures have a protein known as SP100 that could contain a caspase recruitment domain (CARD). If verified experimentally, this discovery will suggest a mechanism by which caspase-2 could be recruited into the complex and ultimately lead to apoptosis.


                Trends in Biochemical Sciences .[Pubmed].



Filamin-A regulates actin-dependent clustering of HIV receptors.

   Jiménez-Baranda, S, Gómez-Moutón, C., Rojas, A.M, Martínez-Prats, L., Mira, E., Lacalle, R.A., Valencia, A., Dimitrov, D.S., Viola, A., Delgado, R., Martínez-A, C., Mañes, S.

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.


                Nature Cell Biology .[Pubmed].



Assessment of Predictions Submitted for the CASP7 Function Prediction Category.

   López, G., Rojas, A.M, Tress, M., Valencia, A.

Here we present a full overview of the Critical Assessment of Protein Structure Prediction (CASP7) function prediction category. Predictions were submitted for Gene Ontology molecular function terms, Enzyme Commission numbers, and ligand binding site residues. The first two categories were difficult to assess because very little new functional information becomes available after the experiment. The majority of the known Gene Ontology terms and all the Enzyme Commission numbers were available a priori to predictors before the experiment, so prediction for these two categories was not blind. Nevertheless, for Gene Ontology terms we were able to demonstrate that some groups made better predictions than others. In the binding residue category, the predictors did not know in advance which ligands were bound and therefore blind evaluation was possible, but there were disappointingly few predictions in this category. After CASP 6 and 7 the need to organize a more effective blind function prediction category is obvious, even if it means focusing on binding site prediction as the only category that can be truly assessed in the CASP spirit.


                Proteins .[Pubmed].



CARGO: a web portal to integrate customised biological information.

   Cases, I., Pisano, D., Eduardo Andrés, León, , Carro, A., Fernández, J.M., Vera, J., Rodríguez, J.M., Gómez- Lopez, G., Valencia, A., Rojas, A.M,

There is a huge quantity of information generated in Life Sciences, and it is dispersed in many databases and repositories. Despite the broad availability of the information, there is a great demand for methods that are able to look for, gather and display distributed data in a standardized and friendly way. CARGO (Cancer And Related Genes Online) is a configurable biological web portal designed as a tool to facilitate, integrate and visualize results from Internet resources, independently of their native format or access method. Through the use of small agents, called widgets, supported by a Rich Internet Application (RIA) paradigm based on AJAX, CARGO provides pieces of minimal, relevant and descriptive biological information. The tool is designed to be used by experimental biologists with no training in bioinformatics. In the current state, the system presents a list of human cancer genes.


                Nucleic Acids Research .[Pubmed].



The structure of a tandem pair of spectrin repeats of plectin reveals a modular organization of the plakin domain.

   Sonnenberg, A., Rojas, A.M, de Pereda, J.

Plectin is a large and versatile cytoskeletal linker and member of the plakin protein family. Plakins share a conserved region called the plakin domain located near their N terminus. We have determined the crystal structure of an N-terminal fragment of the plakin domain of plectin to 2.05 A resolution. This region is adjacent to the actin-binding domain and is required for efficient binding to the integrin alpha6beta4 in hemidesmosomes. The structure is formed by two spectrin repeats connected by an alpha-helix that spans these two repeats. While the first repeat is very similar to other known structures, the second repeat is structurally different with a hydrophobic core, narrower than that in canonical spectrin repeats. Sequence analysis of the plakin domain revealed the presence of up to nine consecutive spectrin repeats organized in an array of tandem modules, and a Src-homology 3 domain inserted in the central spectrin repeat. The structure of the plakin domain is reminiscent of the modular organization of members of the spectrin family. The architecture of the plakin domain suggests that it forms an elongated and flexible structure, and provides a novel molecular explanation for the contribution of plectin and other plakins to the elasticity and stability of tissues subjected to mechanical stress, such as the skin and striated muscle.


                Journal of Molecular Biology .[Pubmed].



Nocht regulates morphogenetic signals required for ventricular trabeculation.

   Grego-Bessa, J., Luna-Zurita, L., del Monte, G., Bolos, V., Melgar, P., Ballestar, E., Esteller, M., Rojas, A.M, Perez-Pomares, JM., de la Pompa, JL.

Ventricular chamber morphogenesis, first manifested by trabeculae formation, is crucial for cardiac function and embryonic viability and depends on cellular interactions between the endocardium and myocardium. We show that ventricular Notch1 activity is highest at presumptive trabecular endocardium. RBPJk and Notch1 mutants show impaired trabeculation and marker expression, attenuated EphrinB2, NRG1, and BMP10 expression and signaling, and decreased myocardial proliferation. Functional and molecular analyses show that Notch inhibition prevents EphrinB2 expression, and that EphrinB2 is a direct Notch target acting upstream of NRG1 in the ventricles. However, BMP10 levels are found to be independent of both EphrinB2 and NRG1 during trabeculation. Accordingly, exogenous BMP10 rescues the myocardial proliferative defect of in vitro-cultured RBPJk mutants, while exogenous NRG1 rescues differentiation in parallel. We suggest that during trabeculation Notch independently regulates cardiomyocyte proliferation and differentiation, two exquisitely balanced processes whose perturbation may result in congenital heart disease.


                Dev Cell .[Pubmed].



2006 : 

TreeDet: a web server to explore sequence space.

   Carro, A., Tress, M., de Juan, D.,Pazos,. F., Lopez-romero,P., del Sol, A.,Valencia, A., Rojas, A.M.

The TreeDet (Tree Determinant) Server is the first release of a system designed to integrate results from methods that predict functional sites in protein families. These methods take into account the relation between sequence conservation and evolutionary importance. TreeDet fully analyses the space of protein sequences in either user-uploaded or automatically generated multiple sequence alignments. The methods implemented in the server represent three main classes of methods for the detection of family-dependent conserved positions, a tree-based method, a correlation based method and a method that employs a principal component analyses coupled to a cluster algorithm. An additional method is provided to highlight the reliability of the position in the alignments.


                Nucleic Acids Research .[Pubmed].



Gas1 is related to the glial cell-derived neurotrophic factor family receptors alpha and regulates Ret signaling.

   Cabrera JR, Sanchez-Pulido L, Rojas, A.MValencia A, Mañes S, Naranjo JR, Mellström B.

The growth arrest-specific gene 1 (Gas1) protein has been proposed to function during development as an inhibitor of growth and a mediator of cell death and is also re-expressed in adult neurons during excitotoxic insult. Here we have demonstrated that the Gas1 protein shows high structural similarity to the glial cell-derived neurotrophic factor (GDNF) family receptors alpha, which mediate GDNF responses through the receptor tyrosine kinase Ret. We found that Gas1 binds Ret in a ligand-independent manner and sequesters Ret in lipid rafts. Signaling downstream of Ret is thus modified through a mechanism that involves the adaptor protein Shc as well as ERK, eventually blocking Akt activation. Consequently, when Gas1 is induced, Ret-mediated GDNF-dependent survival effects are compromised.


                Journal of Molecular Biology .[Pubmed].



Intelligent client for integrating bioinformatics services.

   Navas-Delgado I, Rojano-Muñoz Mdel M, Ramírez S, Pérez AJ, Eduardo Andrés León, Aldana-Montes JF, Trelles O.

In addition to existing bioinformatics software, a lot of new tools are being developed world wide to supply services for an ever growing, widely dispersed and heterogeneous collection of biological data. The integration of these resources under a common platform is a challenging task. To this end, several groups are developing integration technologies, in which services are usually registered in some sort of catalogue to allow novel discovering and accessing mechanisms to be implemented. However, each service demands specific interfaces to accommodate their parameters and it is a complicated task linking the different service inputs and outputs to solve a biological problem.


                Bioinformatics [Pubmed].



2005 :


Death inducer obliterator protein 1 in the context of DNA regulation. Sequence analyses of distant homologues point to a novel functional role.

   Rojas, A.M, Sanchez-Pulido L, Fütterer A, van Wely KH, Martinez-A C, Valencia A.

Death inducer obliterator protein 1 [DIDO1; also termed DIO-1 and death-associated transcription factor 1 (DATF-1)] is encoded by a gene thus far described only in higher vertebrates. Current gene ontology descriptions for this gene assign its function to an apoptosis-related process. The protein presents distinct splice variants and is distributed ubiquitously. Exhaustive sequence analyses of all DIDO variants identify distant homologues in yeast and other organisms. These homologues have a role in DNA regulation and chromatin stability, and form part of higher complexes linked to active chromatin. Further domain composition analyses performed in the context of related homologues suggest that DIDO-induced apoptosis is a secondary effect. Gene-targeted mice show alterations that include lagging chromosomes, and overexpression of the gene generates asymmetric nuclear divisions. Here we describe the analysis of these eukaryote-restricted proteins and propose a novel, DNA regulatory function for the DIDO protein in mammals.


                FEBS .[Pubmed].



A framework for computational and experimental methods: Identifying dimerization residues in CCR chemokine receptors.

   de Juan D, Mellado M, Rodriguez-Frade JM, Hernanz-Falcon P, Serrano A, Del Sol A, Valencia A, Martinez-A C., Rojas, A.M.

Solving relevant biological problems requires answering complex questions. Addressing such questions traditionally implied the design of time-consuming experimental procedures which most of the time are not accessible to average-sized laboratories. The current trend is to move towards a multidisciplinary approach integrating both theoretical knowledge and experimental work. This combination creates a powerful tool for shedding light on biological problems. To illustrate this concept, we present here a descriptive example of where computational methods were shown to be a key aspect in detecting crucial players in an important biological problem: the dimerization of chemokine receptors. Using evolutionary based sequence analysis in combination with structural predictions two CCR5 residues were selected as important for dimerization and further validated experimentally. The experimental validation of computational procedures demonstrated here provides a wealth of valuable information not obtainable by any of the individual approaches alone.


                Bioinformatics .[Pubmed].



Text mining for metabolic pathways, signaling cascades, and protein networks.

   Hoffmann R, Krallinger M, Eduardo Andrés LeónTamames J, Blaschke C, Valencia A.

The complexity of the information stored in databases and publications on metabolic and signaling pathways, the high throughput of experimental data, and the growing number of publications make it imperative to provide systems to help the researcher navigate through these interrelated information resources. Text-mining methods have started to play a key role in the creation and maintenance of links between the information stored in biological databases and its original sources in the literature. These links will be extremely useful for database updating and curation, especially if a number of technical problems can be solved satisfactorily, including the identification of protein and gene names (entities in general) and the characterization of their types of interactions. The first generation of openly accessible text-mining systems, such as iHOP (Information Hyperlinked over Proteins), provides additional functions to facilitate the reconstruction of protein interaction networks, combine database and text information, and support the scientist in the formulation of novel hypotheses. The next challenge is the generation of comprehensive information regarding the general function of signaling pathways and protein interaction networks.


                Science Signaling .[Pubmed].



Characterization of the protease domain of Rice tungro bacilliform virus responsible for the processing of the capsid protein from the polyprotein.

   Marmey P, Rojas, A.M., de Kochko A, Beachy RN, Fauquet CM.

Rice tungro bacilliform virus (RTBV) is a pararetrovirus, and a member of the family Caulimoviridae in the genus Badnavirus. RTBV has a long open reading frame that encodes a large polyprotein (P3). Pararetroviruses show similarities with retroviruses in molecular organization and replication. P3 contains a putative movement protein (MP), the capsid protein (CP), the aspartate protease (PR) and the reverse transcriptase (RT) with a ribonuclease H activity. PR is a member of the cluster of retroviral proteases and serves to proteolytically process P3. Previous work established the N- and C-terminal amino acid sequences of CP and RT, processing of RT by PR, and estimated the molecular mass of PR by western blot assays.


                Virol J. [Pubmed].



Evaluation of BioCreAtIvE assessment of task 2.

   Blaschke C, Eduardo Andrés León, Krallinger M, Valencia A.

Molecular Biology accumulated substantial amounts of data concerning functions of genes and proteins. Information relating to functional descriptions is generally extracted manually from textual data and stored in biological databases to build up annotations for large collections of gene products. Those annotation databases are crucial for the interpretation of large scale analysis approaches using bioinformatics or experimental techniques. Due to the growing accumulation of functional descriptions in biomedical literature the need for text mining tools to facilitate the extraction of such annotations is urgent. In order to make text mining tools useable in real world scenarios, for instance to assist database curators during annotation of protein function, comparisons and evaluations of different approaches on full text articles are needed.


                BMC Bioinformatics [Pubmed].






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